-
Notifications
You must be signed in to change notification settings - Fork 1
/
Copy path152538.html
410 lines (410 loc) · 143 KB
/
152538.html
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
<?xml version="1.0" encoding="UTF-8"?>
<!DOCTYPE article PUBLIC "-//NLM//DTD Journal Publishing DTD v2.3 20070202//EN" "journalpublishing.dtd">
<article article-type="research-article" dtd-version="2.3" xml:lang="EN" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:mml="http://www.w3.org/1998/Math/MathML">
<front>
<journal-meta>
<journal-id journal-id-type="hwp">genetics</journal-id>
<journal-id journal-id-type="pmc">genetics</journal-id>
<journal-id journal-id-type="publisher-id">genetics</journal-id>
<journal-title>Genetics</journal-title>
<abbrev-journal-title abbrev-type="full">Genetics</abbrev-journal-title>
<issn pub-type="ppub">0016-6731</issn>
<issn pub-type="epub">1943-2361</issn>
<publisher><publisher-name>Genetics Society of America</publisher-name></publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="publisher-id">GEN_152538</article-id>
<article-id pub-id-type="doi">10.1534/genetics.113.152538</article-id>
<article-categories><subj-group><subject>Investigation</subject></subj-group>
</article-categories>
<title-group>
<article-title>The Jaw of the Worm: GTPase-activating Protein EAT-17 Regulates Grinder Formation in <italic>Caenorhabditis elegans</italic></article-title>
<?RFL: S. Straud <italic>et al.</italic>?>
<?RFR: EAT-17 is a RabGAP for RAB-6.2?>
</title-group>
<contrib-group>
<contrib contrib-type="author"><name><surname>Straud</surname><given-names>Sarah</given-names></name><xref ref-type="aff" rid="aff1">*</xref><xref ref-type="fn" rid="afn2"><sup>1</sup></xref><xref ref-type="fn" rid="afn3"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Inhwan</given-names></name><xref ref-type="aff" rid="aff2"><sup>†</sup></xref><xref ref-type="fn" rid="afn2"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Song</surname><given-names>Bomi</given-names></name><xref ref-type="aff" rid="aff1">*</xref></contrib><contrib contrib-type="author"><name><surname>Avery</surname><given-names>Leon</given-names></name><xref ref-type="aff" rid="aff3"><sup>‡</sup></xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>You</surname><given-names>Young-Jai</given-names></name><xref ref-type="aff" rid="aff2"><sup>†</sup></xref><xref ref-type="corresp" rid="cor1"><sup>3</sup></xref></contrib>
<aff id="aff1"><label>*</label>Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148</aff>
<aff id="aff2"><label>†</label>Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia 23298-0614</aff>
<aff id="aff3"><label>‡</label>Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia 23298-0551</aff>
</contrib-group>
<author-notes><fn id="afn1"><p>Supporting information is available online at <ext-link ext-link-type="uri" xlink:href="http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.152538/-/DC1">http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.152538/-/DC1</ext-link>.</p></fn>
<fn id="afn2" fn-type="equal"><label>1</label><p>These authors contributed equally to this work.</p></fn>
<fn id="afn3" fn-type="present-address"><label>2</label><p>Present address: Department of Biological and Physical Sciences, Saint Augustine’s University, Raleigh, NC 27610-2298.</p></fn>
<corresp id="cor1"><label>3</label>Corresponding author: P. O. Box 980614, 1220 E. Broad St., MMRB Bldg, Room 2046, Richmond, VA 23298. E-mail: <email>[email protected]</email></corresp></author-notes>
<pub-date pub-type="epub-ppub"><month>09</month><year>2013</year></pub-date>
<volume>195</volume>
<issue>1</issue>
<fpage>0</fpage>
<lpage>0</lpage>
<permissions>
<copyright-statement>Copyright © 2013 by the Genetics Society of America</copyright-statement>
<copyright-year>2013</copyright-year>
</permissions>
<self-uri xlink:title="pdf" xlink:href="GEN_152538.pdf"/>
<abstract>
<p style="background-color:#FFFFFF">Constitutive transport of cellular materials is essential for cell survival<!-- Q1 --><!-- Q2 --><!-- Q3 --><!-- Q4 -->. Although multiple small GTPase Rab proteins are required for the process, few regulators of Rabs are known. Here we report that <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-3">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-3')"><sup><img src="/gsa/img/minus.png"/></sup></a>, a novel GTPase-activating protein (GAP), regulates <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-2">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-2')"><sup><img src="/gsa/img/minus.png"/></sup></a> function in grinder formation in <italic>Caenorhabditis elegans</italic>. We identified <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-4">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-4')"><sup><img src="/gsa/img/minus.png"/></sup></a> as a novel RabGAP that interacts with <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-3">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-3')"><sup><img src="/gsa/img/minus.png"/></sup></a>, a protein that presumably regulates vesicle trafficking between Golgi, the endoplasmic reticulum, and plasma membrane to form a functional grinder. <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-5">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-5')"><sup><img src="/gsa/img/minus.png"/></sup></a> has a canonical GAP domain that is critical for its function. RNA interference against 25 confirmed and/or predicted RABs in <italic>C. elegans</italic> shows that RNAi against <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-1">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-1')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> produces a phenotype identical to <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-1">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-1')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>. A directed yeast two-hybrid screen using <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-6">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-6')"><sup><img src="/gsa/img/minus.png"/></sup></a> as bait and each of the 25 RAB proteins as prey identifies <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-4">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-4')"><sup><img src="/gsa/img/minus.png"/></sup></a> as the interacting partner of <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-7">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-7')"><sup><img src="/gsa/img/minus.png"/></sup></a>, confirming that <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-5">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-5')"><sup><img src="/gsa/img/minus.png"/></sup></a> is a specific substrate of <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-8">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-8')"><sup><img src="/gsa/img/minus.png"/></sup></a>. Additionally, deletion mutants of <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-2">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-2')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> show grinder defects identical to those of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-2">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-2')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> loss-of-function mutants, and both <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-6">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-6')"><sup><img src="/gsa/img/minus.png"/></sup></a> and <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-9">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-9')"><sup><img src="/gsa/img/minus.png"/></sup></a> are expressed in the terminal bulb of the pharynx where the grinder is located. Collectively, these results suggest that <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-10">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-10')"><sup><img src="/gsa/img/minus.png"/></sup></a> is a specific GTPase-activating protein for <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-7">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-7')"><sup><img src="/gsa/img/minus.png"/></sup></a>. Based on the conserved function of Rab6 in vesicular transport, we propose that <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-11">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-11')"><sup><img src="/gsa/img/minus.png"/></sup></a> regulates the turnover rate of <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-8">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-8')"><sup><img src="/gsa/img/minus.png"/></sup></a> activity in cargo trafficking for grinder formation.</p>
</abstract>
<kwd-group>
<kwd>RAB</kwd><kwd>RABGAP</kwd><kwd>Golgi-endosome trafficking</kwd><kwd>grinder formation</kwd>
</kwd-group>
<counts><page-count count="0"/></counts>
<custom-meta-wrap>
<custom-meta><meta-name> DJS Export </meta-name><meta-value>v1</meta-value></custom-meta></custom-meta-wrap>
<!--*Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148, <sup>†</sup>Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia 23298-0614, and <sup>‡</sup>Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia 23298-0551-->
</article-meta>
</front>
<body>
<p style="background-color:#FFFFFF">CELLS constitutively transport newly synthesized proteins, lipids, and other molecules to their periphery through vesicle trafficking. As the process is conserved from yeast to humans, it is essential for the function and survival of cells. Rab GTPases are small GTP binding proteins that regulate the transport of vesicles between different compartments of the cell (<xref ref-type="bibr" rid="bib38 bib18 bib13">Zerial and McBride 2001; Jordens <italic>et al.</italic> 2005; Grosshans <italic>et al.</italic> 2006</xref>)<!-- Q5 -->. Rab6’s are localized to Golgi membranes to mark and target both anterograde cargos from Golgi to post-Golgi compartments (such as the plasma membrane) and retrograde cargos from early/recycling endosomes to Golgi and the endoplasmic reticulum (<xref ref-type="bibr" rid="bib17">Jasmin <italic>et al.</italic> 1992</xref>; <xref ref-type="bibr" rid="bib21 bib22">Martinez <italic>et al.</italic> 1994, 1997</xref>; <xref ref-type="bibr" rid="bib12">Girod <italic>et al.</italic> 1999</xref>; <xref ref-type="bibr" rid="bib24">Opdam <italic>et al.</italic> 2000;</xref> Del Nery <italic>et al.</italic> 2006). For fast turnover, Rabs require guanine nucleotide exchange factors (Rab GEFs) for activation and GTPase-activating proteins (Rab GAPs) to turn off activity (<xref ref-type="bibr" rid="bib13">Grosshans <italic>et al.</italic> 2006</xref>). While the role of Rab6 in membrane trafficking is well established, very few GAPs or GEFs for Rab6 have been identified; thus their physiological importance is largely unknown. The physiological roles of RabGAPs were addressed only recently in flies (<xref ref-type="bibr" rid="bib16">Houalla <italic>et al.</italic> 2010</xref>; <xref ref-type="bibr" rid="bib34">Uytterhoeven <italic>et al.</italic> 2011</xref>) and worms (<xref ref-type="bibr" rid="bib6">Chotard <italic>et al.</italic> 2010</xref>). In those cases, removing the function of the RabGAPs produced phenotypes almost identical to those of removing the relevant Rabs, showing the fundamental roles of RabGAPs in modulating Rab functions. Currently, the only identified GAP for Rab6 is GAPCenA in humans, which is associated with centrosomes and regulates Golgi dynamics in dividing cells (<xref ref-type="bibr" rid="bib8">Cuif <italic>et al.</italic> 1999</xref>). Nonetheless, misregulation of GAPs for other small G proteins, such as Ras, directly relates to diseases such as cancer, implicating the essential roles of GAPs in controlling the kinetics of G protein activity (<xref ref-type="bibr" rid="bib33">Tanabe <italic>et al.</italic> 2006</xref>; <xref ref-type="bibr" rid="bib11">Durkin <italic>et al.</italic> 2007</xref>; <xref ref-type="bibr" rid="bib25">Pamonsinlapatham <italic>et al.</italic> 2009</xref>).</p>
<p style="background-color:#FFFFFF">The <italic>Caenorhabditis elegans</italic> grinder is a complex structure required for proper grinding of food (bacteria) before it is passed to the intestine. When food is abundant, worms feed at an average rate of 200 pumps per minute (ppm) based on counts of pharyngeal pumping and grinding motions (Avery and You 2012; <xref ref-type="bibr" rid="bib26">Raizen <italic>et al.</italic> 2012</xref>). This high frequency of grinding throughout the worm’s life suggests possible mechanical erosion of the structure and thus suggests that constitutive transport of grinder components is essential to maintain the structure. Considering the location of this structure in the <italic>C. elegans</italic> pharynx, grinder components are likely produced by terminal bulb muscles pm6 and pm7 and trafficked to their apical surfaces (<xref ref-type="bibr" rid="bib1">Albertson and Thomson 1976</xref>). The exact components of the grinder and molecular mechanisms regulating their transport, however, remain unknown.</p>
<p style="background-color:#FFFFFF">Here we report that <italic>C. elegans</italic> gene <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-3">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-3')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> encodes a Rab GTPase-activating protein (Rab GAP) specific for <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-9">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-9')"><sup><img src="/gsa/img/minus.png"/></sup></a>, a Rab6 homolog in <italic>C. elegans</italic>. <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-4">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-4')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> loss-of-function and <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-3">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-3')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> deletion mutants show similar phenotypes, namely disorganized, poorly formed grinders. RNAi against <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-4">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-4')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> produces similar defects. A directed yeast two-hybrid screen using <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-12">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-12')"><sup><img src="/gsa/img/minus.png"/></sup></a> as bait identified <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-10">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-10')"><sup><img src="/gsa/img/minus.png"/></sup></a> as its interacting partner. Furthermore, transgenes with mutations in the conserved catalytic arginine residue required for the GAP activity of <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-13">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-13')"><sup><img src="/gsa/img/minus.png"/></sup></a> failed to rescue grinder defects when injected into <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-5">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-5')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants. Based on these results and the conserved function of Rab6 in vesicle transport, we propose that <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-11">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-11')"><sup><img src="/gsa/img/minus.png"/></sup></a> and <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-14">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-14')"><sup><img src="/gsa/img/minus.png"/></sup></a> cooperate in the transport of grinder components to the apical surfaces of the terminal bulb muscles to form a functional grinder.</p>
<sec id="s1" sec-type="materials|methods">
<title>Materials and Methods</title>
<sec id="s2">
<title>Worm culture</title>
<p style="background-color:#FFFFFF">Worms were cultured and handled as described (<xref ref-type="bibr" rid="bib32">Sulston and Hodgkin 1988</xref>) with the following modifications. First, they were routinely grown on NGMSR plates (<xref ref-type="bibr" rid="bib3">Avery 1993</xref>)<!-- Q6 -->. In addition, all worms were maintained at 20° on <italic>Escherichia coli</italic> <a href="http://www.wormbase.org/db/get?name=HB101;class=Strain" id="HB101-1">HB101</a><a href="javascript:removeLinkAfterConfirm('HB101-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> unless indicated otherwise. The wild-type strains are Bristol <a href="http://www.wormbase.org/db/get?name=N2;class=Strain" id="N2-1">N2</a><a href="javascript:removeLinkAfterConfirm('N2-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> and Hawaiian <a href="http://www.wormbase.org/db/get?name=CB4856;class=Strain" id="CB4856-1">CB4856</a><a href="javascript:removeLinkAfterConfirm('CB4856-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>. <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-6">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-6')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=ad707;class=Variation" id="ad707-1">ad707</a><a href="javascript:removeLinkAfterConfirm('ad707-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic> <underline>and <a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-5">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-5')"><sup><img src="/gsa/img/minus.png"/></sup></a></underline><italic><underline>(</underline></italic><italic><a href="http://www.wormbase.org/db/get?name=ok2254;class=Variation" id="ok2254-1">ok2254</a><a href="javascript:removeLinkAfterConfirm('ok2254-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic> were used throughout this study after being outcrossed twice to <a href="http://www.wormbase.org/db/get?name=N2;class=Strain" id="N2-2">N2</a><a href="javascript:removeLinkAfterConfirm('N2-2')"><sup><img src="/gsa/img/minus.png"/></sup></a><!-- Q7 -->. Other strains used in this study are DA2120 <italic>adEx2120[<a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17p-1">eat-17p</a><a href="javascript:removeLinkAfterConfirm('eat-17p-1')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>::<italic>GFP <a href="http://www.wormbase.org/db/get?name=rol-6;class=Gene" id="rol-6-1">rol-6</a><a href="javascript:removeLinkAfterConfirm('rol-6-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>(sd)]</italic>, DA773 <italic><a href="http://www.wormbase.org/db/get?name=unc-93;class=Gene" id="unc-93-1">unc-93</a><a href="javascript:removeLinkAfterConfirm('unc-93-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=e1500;class=Variation" id="e1500sd-1">e1500sd</a><a href="javascript:removeLinkAfterConfirm('e1500sd-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>; <italic><a href="http://www.wormbase.org/db/get?name=lin-15;class=Gene" id="lin-15-1">lin-15</a><a href="javascript:removeLinkAfterConfirm('lin-15-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=n309;class=Variation" id="n309-1">n309</a><a href="javascript:removeLinkAfterConfirm('n309-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>) <a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-7">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-7')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=ad707;class=Variation" id="ad707-2">ad707</a><a href="javascript:removeLinkAfterConfirm('ad707-2')"><sup><img src="/gsa/img/minus.png"/></sup></a>) <a href="http://www.wormbase.org/db/get?name=sup-10;class=Gene" id="sup-10-1">sup-10</a><a href="javascript:removeLinkAfterConfirm('sup-10-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=n183;class=Variation" id="n183-1">n183</a><a href="javascript:removeLinkAfterConfirm('n183-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>, <a href="http://www.wormbase.org/db/get?name=DA1814;class=Strain" id="DA1814-1">DA1814</a><a href="javascript:removeLinkAfterConfirm('DA1814-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> <italic><a href="http://www.wormbase.org/db/get?name=ser-1;class=Gene" id="ser-1-1">ser-1</a><a href="javascript:removeLinkAfterConfirm('ser-1-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=ok345;class=Variation" id="ok345-1">ok345</a><a href="javascript:removeLinkAfterConfirm('ok345-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>, <a href="http://www.wormbase.org/db/get?name=RB758;class=Strain" id="RB758-1">RB758</a><a href="javascript:removeLinkAfterConfirm('RB758-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> <italic><a href="http://www.wormbase.org/db/get?name=hda-4;class=Gene" id="hda-4-1">hda-4</a><a href="javascript:removeLinkAfterConfirm('hda-4-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=ok518;class=Variation" id="ok518-1">ok518</a><a href="javascript:removeLinkAfterConfirm('ok518-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>, <a href="http://www.wormbase.org/db/get?name=RB787;class=Strain" id="RB787-1">RB787</a><a href="javascript:removeLinkAfterConfirm('RB787-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> <italic><a href="http://www.wormbase.org/db/get?name=T27A8.2;class=Gene" id="T27A8.2-1">T27A8.2</a><a href="javascript:removeLinkAfterConfirm('T27A8.2-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=ok570;class=Variation" id="ok570-1">ok570</a><a href="javascript:removeLinkAfterConfirm('ok570-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>, <a href="http://www.wormbase.org/db/get?name=DA707;class=Strain" id="DA707-1">DA707</a><a href="javascript:removeLinkAfterConfirm('DA707-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-8">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-8')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=ad707;class=Variation" id="ad707-3">ad707</a><a href="javascript:removeLinkAfterConfirm('ad707-3')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>, <a href="http://www.wormbase.org/db/get?name=NL2099;class=Strain" id="NL2099-1">NL2099</a><a href="javascript:removeLinkAfterConfirm('NL2099-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> <italic><a href="http://www.wormbase.org/db/get?name=rrf-3;class=Gene" id="rrf-3-1">rrf-3</a><a href="javascript:removeLinkAfterConfirm('rrf-3-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=pk1426;class=Variation" id="pk1426-1">pk1426</a><a href="javascript:removeLinkAfterConfirm('pk1426-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>, DA1983 <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-9">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-9')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=ad707;class=Variation" id="ad707-4">ad707</a><a href="javascript:removeLinkAfterConfirm('ad707-4')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>; <italic>adEx1983[<a href="http://www.wormbase.org/db/get?name=F01G12;class=Clone" id="F01G12-1">F01G12</a><a href="javascript:removeLinkAfterConfirm('F01G12-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> <a href="http://www.wormbase.org/db/get?name=T24D11;class=Clone" id="T24D11-1">T24D11</a><a href="javascript:removeLinkAfterConfirm('T24D11-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> <a href="http://www.wormbase.org/db/get?name=rol-6;class=Gene" id="rol-6-2">rol-6</a><a href="javascript:removeLinkAfterConfirm('rol-6-2')"><sup><img src="/gsa/img/minus.png"/></sup></a>(d)]</italic>, DA1982 <italic><a href="http://www.wormbase.org/db/get?name=rrf-3;class=Gene" id="rrf-3-2">rrf-3</a><a href="javascript:removeLinkAfterConfirm('rrf-3-2')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=pk1426;class=Variation" id="pk1426-2">pk1426</a><a href="javascript:removeLinkAfterConfirm('pk1426-2')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>; <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-10">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-10')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=ad707;class=Variation" id="ad707-5">ad707</a><a href="javascript:removeLinkAfterConfirm('ad707-5')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>, DA2035 <italic>adEx2035[<a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2p-1">rab-6.2p</a><a href="javascript:removeLinkAfterConfirm('rab-6.2p-1')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>::<italic>GFP <a href="http://www.wormbase.org/db/get?name=rol-6;class=Gene" id="rol-6-3">rol-6</a><a href="javascript:removeLinkAfterConfirm('rol-6-3')"><sup><img src="/gsa/img/minus.png"/></sup></a>(d)]</italic>, DA2033 <italic><a href="http://www.wormbase.org/db/get?name=eat-5;class=Gene" id="eat-5-1">eat-5</a><a href="javascript:removeLinkAfterConfirm('eat-5-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=ad1402;class=Variation" id="ad1402-1">ad1402</a><a href="javascript:removeLinkAfterConfirm('ad1402-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>; <italic>adEx2033[<a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-12">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-12')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>::<italic>GFP <a href="http://www.wormbase.org/db/get?name=unc-122;class=Gene" id="unc-122-1">unc-122</a><a href="javascript:removeLinkAfterConfirm('unc-122-1')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>::<italic>RFP <a href="http://www.wormbase.org/db/get?name=eat-5;class=Gene" id="eat-5-2">eat-5</a><a href="javascript:removeLinkAfterConfirm('eat-5-2')"><sup><img src="/gsa/img/minus.png"/></sup></a>(+)]</italic>, YJ89 <underline><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-6">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-6')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=ok2254;class=Variation" id="ok2254-2">ok2254</a><a href="javascript:removeLinkAfterConfirm('ok2254-2')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</underline>, MT993 <italic><a href="http://www.wormbase.org/db/get?name=lin-10;class=Gene" id="lin-10-1">lin-10</a><a href="javascript:removeLinkAfterConfirm('lin-10-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=e1439;class=Variation" id="e1439-1">e1439</a><a href="javascript:removeLinkAfterConfirm('e1439-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>; <italic><a href="http://www.wormbase.org/db/get?name=him-5;class=Gene" id="him-5-1">him-5</a><a href="javascript:removeLinkAfterConfirm('him-5-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=e1467;class=Variation" id="e1467ts-1">e1467ts</a><a href="javascript:removeLinkAfterConfirm('e1467ts-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>.</p>
</sec>
<sec id="s3">
<title>SNP mapping</title>
<p style="background-color:#FFFFFF"><a href="http://www.wormbase.org/db/get?name=CB4856;class=Strain" id="CB4856-2">CB4856</a><a href="javascript:removeLinkAfterConfirm('CB4856-2')"><sup><img src="/gsa/img/minus.png"/></sup></a> males were crossed to DA773 hermaphrodites. <italic><a href="http://www.wormbase.org/db/get?name=unc-93;class=Gene" id="unc-93-2">unc-93</a><a href="javascript:removeLinkAfterConfirm('unc-93-2')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>; <italic><a href="http://www.wormbase.org/db/get?name=lin-15;class=Gene" id="lin-15-2">lin-15</a><a href="javascript:removeLinkAfterConfirm('lin-15-2')"><sup><img src="/gsa/img/minus.png"/></sup></a> <a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-11">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-11')"><sup><img src="/gsa/img/minus.png"/></sup></a> <a href="http://www.wormbase.org/db/get?name=sup-10;class=Gene" id="sup-10-2">sup-10</a><a href="javascript:removeLinkAfterConfirm('sup-10-2')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>/<a href="http://www.wormbase.org/db/get?name=CB4856;class=Strain" id="CB4856-3">CB4856</a><a href="javascript:removeLinkAfterConfirm('CB4856-3')"><sup><img src="/gsa/img/minus.png"/></sup></a>(+) worms were isolated in the F<sub>2</sub> generation by their ability to produce 25% <a href="http://www.wormbase.org/db/get?name=WBPhenotype%3A0000700;class=Phenotype" id="Muv-1">Muv</a><a href="javascript:removeLinkAfterConfirm('Muv-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> non-<a href="http://www.wormbase.org/db/get?name=WBPhenotype%3A0000643;class=Phenotype" id="Unc-1">Unc</a><a href="javascript:removeLinkAfterConfirm('Unc-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> self-progeny and the near absence of non-<a href="http://www.wormbase.org/db/get?name=WBPhenotype%3A0000700;class=Phenotype" id="Muv-2">Muv</a><a href="javascript:removeLinkAfterConfirm('Muv-2')"><sup><img src="/gsa/img/minus.png"/></sup></a> non-<a href="http://www.wormbase.org/db/get?name=WBPhenotype%3A0000643;class=Phenotype" id="Unc-2">Unc</a><a href="javascript:removeLinkAfterConfirm('Unc-2')"><sup><img src="/gsa/img/minus.png"/></sup></a> self-progeny. Sup non-<a href="http://www.wormbase.org/db/get?name=WBPhenotype%3A0000700;class=Phenotype" id="Muv-3">Muv</a><a href="javascript:removeLinkAfterConfirm('Muv-3')"><sup><img src="/gsa/img/minus.png"/></sup></a> recombinants were isolated in the F<sub>3</sub> generation. These were scored for the <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-12">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-12')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> feeding defect, and their progeny were analyzed by PCR and subsequent restriction digestion for SNPs located between <italic><a href="http://www.wormbase.org/db/get?name=lin-15;class=Gene" id="lin-15-3">lin-15</a><a href="javascript:removeLinkAfterConfirm('lin-15-3')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> and <italic><a href="http://www.wormbase.org/db/get?name=sup-10;class=Gene" id="sup-10-3">sup-10</a><a href="javascript:removeLinkAfterConfirm('sup-10-3')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>. SNPs examined and primers used for their amplification are shown in <ext-link ext-link-type="uri" xlink:href="http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.152538/-/DC1/genetics.113.152538-1.pdf">Supporting Information</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.152538/-/DC1/genetics.113.152538-5.pdf">Table S1</ext-link>.</p>
<p style="background-color:#FFFFFF">Deletion mutants of <italic><a href="http://www.wormbase.org/db/get?name=ser-1;class=Gene" id="ser-1-2">ser-1</a><a href="javascript:removeLinkAfterConfirm('ser-1-2')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=ok345;class=Variation" id="ok345-2">ok345</a><a href="javascript:removeLinkAfterConfirm('ok345-2')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>, <italic><a href="http://www.wormbase.org/db/get?name=hda-4;class=Gene" id="hda-4-2">hda-4</a><a href="javascript:removeLinkAfterConfirm('hda-4-2')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=ok518;class=Variation" id="ok518-2">ok518</a><a href="javascript:removeLinkAfterConfirm('ok518-2')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>, and <italic><a href="http://www.wormbase.org/db/get?name=T27A8.2;class=Gene" id="T27A8.2-2">T27A8.2</a><a href="javascript:removeLinkAfterConfirm('T27A8.2-2')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=ok570;class=Variation" id="ok570-2">ok570</a><a href="javascript:removeLinkAfterConfirm('ok570-2')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic> were mapped by scoring F<sub>4</sub> progeny for deletion detected by PCR. Primers used for detecting the deletions are shown in <ext-link ext-link-type="uri" xlink:href="http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.152538/-/DC1/genetics.113.152538-4.pdf">Table S2</ext-link>.</p>
</sec>
<sec id="s4">
<title>Analysis of <italic>eat-17</italic> gene structure and identification of splice variants</title>
<p style="background-color:#FFFFFF">To determine the intron and exon structure of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-14">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-14')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>, primers were designed based on Genefinder predictions shown in WormBase to amplify partial and full-length transcripts by RT–PCR. First-strand poly(A)<sup>+</sup> cDNA derived from mixed stage <a href="http://www.wormbase.org/db/get?name=N2;class=Strain" id="N2-3">N2</a><a href="javascript:removeLinkAfterConfirm('N2-3')"><sup><img src="/gsa/img/minus.png"/></sup></a> hermaphrodites was used as template in all reactions. Trizol (Invitrogen) was used to isolate RNA and the First-Strand cDNA Synthesis kit (Roche) was used to generate cDNA. Primers used to amplify PCR fragments are shown in <ext-link ext-link-type="uri" xlink:href="http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.152538/-/DC1/genetics.113.152538-2.pdf">Table S3</ext-link>. All PCR reactions yielded a single product, and all exons except the first appeared to be correct. To assess the number and abundance of different splice variants, full-length <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-15">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-15')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> cDNAs were cloned into the pGEM-T Easy vector (Promega) and sequenced.</p>
</sec>
<sec id="s5">
<title>SL1 <italic>trans</italic>-splicing</title>
<p style="background-color:#FFFFFF">To identify the first exon of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-16">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-16')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>, a forward primer recognizing the 22 nucleotide-SL1-splice leader sequence (5′-ggtttaattacccaagtttgag -3′) and two nested reverse primers recognizing sequences in exon 2 [1050: 5′-tgttcagctgctccatcttg-3′(outside primer)/1051: 5′-cgacttcatttacgcatactg-3′(inside primer)] were used. Products were purified (Qiaquick Gel Extraction kit), cloned into pGEM-T Easy vectors and sequenced. The resulting sequences were BLASTed (blastn) against <italic>C. elegans</italic> ESTs and genomic DNA using WormBase.</p>
</sec>
<sec id="s6">
<title>Cosmid rescue</title>
<p style="background-color:#FFFFFF">Cosmids used in this study were obtained from Alan Coulson (Wellcome Trust, UK). <a href="http://www.wormbase.org/db/get?name=T24D11;class=Clone" id="T24D11-2">T24D11</a><a href="javascript:removeLinkAfterConfirm('T24D11-2')"><sup><img src="/gsa/img/minus.png"/></sup></a> and <a href="http://www.wormbase.org/db/get?name=F01G12;class=Clone" id="F01G12-2">F01G12</a><a href="javascript:removeLinkAfterConfirm('F01G12-2')"><sup><img src="/gsa/img/minus.png"/></sup></a> were isolated using the Qiagen Plasmid Mini kit and then co-injected into <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-17">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-17')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants (50 ng/μl). Plasmids pPD118.20 <italic><a href="http://www.wormbase.org/db/get?name=myo-3;class=Gene" id="myo-3-1">myo-3</a><a href="javascript:removeLinkAfterConfirm('myo-3-1')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>::GFP (from Andrew Fire) and pRAK3 <italic><a href="http://www.wormbase.org/db/get?name=rol-6;class=Gene" id="rol-6-4">rol-6</a><a href="javascript:removeLinkAfterConfirm('rol-6-4')"><sup><img src="/gsa/img/minus.png"/></sup></a>(d)</italic> were used as co-injection markers at a concentration of 10 ng/μl<!-- Q8 -->. Worms were fed <italic>Comamonas</italic> before injection to allow good growth (<xref ref-type="bibr" rid="bib4">Avery and Shtonda 2003</xref>). Transgenic worms were then fed <a href="http://www.wormbase.org/db/get?name=DA837;class=Strain" id="DA837-1">DA837</a><a href="javascript:removeLinkAfterConfirm('DA837-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> to enhance the feeding defect.</p>
<p style="background-color:#FFFFFF">Growth rates of transgenic and nontransgenic progeny were compared to assess rescue. Eggs from transgenic mothers were placed individually onto plates seeded with <a href="http://www.wormbase.org/db/get?name=DA837;class=Strain" id="DA837-2">DA837</a><a href="javascript:removeLinkAfterConfirm('DA837-2')"><sup><img src="/gsa/img/minus.png"/></sup></a> and then checked every 12 hr to stage the worms. Growth rate is defined as the inverse of the time required for worms to reach adulthood and produce progeny. Worms were scored as adults if they had laid at least one egg.</p>
</sec>
<sec id="s7">
<title>Generating the <italic>eat-17</italic> rescue construct</title>
<p style="background-color:#FFFFFF">Rescuing fragments containing the full-length <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-19">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-19')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> cDNA were generated by overlap extension PCR (<xref ref-type="bibr" rid="bib15">Ho <italic>et al.</italic> 1989</xref>). In first round PCR reactions, 5′ and 3′ <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-20">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-20')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> cDNA fragments were amplified using primers shown in <ext-link ext-link-type="uri" xlink:href="http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.152538/-/DC1/genetics.113.152538-7.pdf">Table S4</ext-link>. The resulting PCR fragments were purified and subjected to a second round of PCR using the overlap extension method using primers 1076 and 1085. These products were subjected to a third round of PCR to fuse the <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-21">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-21')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> promoter and <italic><a href="http://www.wormbase.org/db/get?name=unc-54;class=Gene" id="unc-54-1">unc-54</a><a href="javascript:removeLinkAfterConfirm('unc-54-1')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> 3′-UTR to each end. The resulting PCR products were purified and injected into <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-22">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-22')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> worms (25 ng/μl). The final injected product contained 5.6 kb of sequence located directly upstream of the <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-23">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-23')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> transcriptional start site. A 2.5-kb full-length <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-24">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-24')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> cDNA construct was fused to this “promoter region” and the heterologous <italic><a href="http://www.wormbase.org/db/get?name=unc-54;class=Gene" id="unc-54-2">unc-54</a><a href="javascript:removeLinkAfterConfirm('unc-54-2')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> 3′-UTR was added to promote stability. This construct lacked intron elements, including the large 4-kb first intron. <italic><a href="http://www.wormbase.org/db/get?name=let-858;class=Gene" id="let-858-1">let-858</a><a href="javascript:removeLinkAfterConfirm('let-858-1')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>::GFP plasmid DNA (18 ng/μl) was used as a co-injection marker.</p>
<p style="background-color:#FFFFFF">Two transgenic lines were isolated and examined. F<sub>2</sub> transgenic animals were identified using the SZX12GFP dissecting scope (Olympus). Because many transgenic embryos die, only animals that reached adulthood were scored for the grinder phenotype. For both transgenic lines, 100% of the adult transgenic animals were rescued for defects in grinder formation. Grinder morphology was observed using a Zeiss Axio A2 Imager at either ×630 or ×1000 magnification. Images were acquired using Zeiss Axiovision software.</p>
</sec>
<sec id="s8">
<title>Assay for EAT-17 GAP activity</title>
<p style="background-color:#FFFFFF">To determine the functional importance of the GAP activity of <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-16">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-16')"><sup><img src="/gsa/img/minus.png"/></sup></a>, rescuing fragments containing the catalytically inactive R116/119K and R116/119A mutations were generated using the overlap extension method as describe above. To generate the R116/119K mutation, however, primers 1142 and 1143 were used in the amplification of the 5′ and 3′ ends of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-25">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-25')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>, respectively. To generate the 116/119A mutation, primers 1144 and 1145 were used in first round reactions. Second and third round reactions are identical to those described above in the generation of the wild-type <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-26">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-26')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> construct. The resulting PCR products were gel purified and injected into <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-27">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-27')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> worms at a concentration of 25 ng/μl. <italic><a href="http://www.wormbase.org/db/get?name=let-858;class=Gene" id="let-858-2">let-858</a><a href="javascript:removeLinkAfterConfirm('let-858-2')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>::GFP plasmid DNA was used as a co-injection marker at a concentration of 100 ng/μl. Wild-type, R116/119K, and R116/119A injections were performed in parallel. To score for rescue of grinder defects, the F<sub>1</sub> gravid adult progeny of injected mothers were picked to 4% agar pads containing 10 mM sodium azide (Sigma, St. Louis). Defects in grinder formation were recorded and then the presence or absence of GFP expression was determined. Worms were observed using a Zeiss Axio A2 Imager with a ×100 objective lens. Images were acquired using Zeiss Axiovision software.</p>
</sec>
<sec id="s9">
<title>RNAi</title>
<p style="background-color:#FFFFFF">RNAi was performed as described with minor modifications (<xref ref-type="bibr" rid="bib19">Kamath and Ahringer 2003</xref>). cDNA sequences of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-28">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-28')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> and the 25 <italic>C</italic>. <italic>elegans</italic> Rabs were amplified by PCR using primers shown in <ext-link ext-link-type="uri" xlink:href="http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.152538/-/DC1/genetics.113.152538-6.pdf">Table S5</ext-link>. The HiScribe RNAi Transcription kit (New England Biolabs) was used to generate dsRNAs.</p>
</sec>
<sec id="s10">
<title>GFP fusions of EAT-17 and RAB-6.2</title>
<sec id="s11">
<title><italic>eat-17p</italic>::<italic>GFP:</italic></title>
<p style="background-color:#FFFFFF">A 5.6-kb <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-29">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-29')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> promoter was amplified from <a href="http://www.wormbase.org/db/get?name=N2;class=Strain" id="N2-4">N2</a><a href="javascript:removeLinkAfterConfirm('N2-4')"><sup><img src="/gsa/img/minus.png"/></sup></a> genomic DNA using the following primers: 1073, 5′-taggttacggtagttggtacg-3′ and 1167, 5′-gaaaagttcttctcctttactcatcggcggtgacaattgg-3′. In addition to the 5′ regulatory sequence, this DNA contains the first six codons of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-30">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-30')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>. GFP was amplified from pPD95.75 plasmid DNA (gift from A. Fire) using primers: 1166, 5′-ccaattgtcaccgccgatgagtaaaggagaagaacttttc-3′ and MS46, 5′-tttggtatattgggaatggtattctg-</p>
<p style="background-color:#FFFFFF">3′. DNAs were fused together by overlap extension PCR using the following nested</p>
<p style="background-color:#FFFFFF">primers: 1157, 5′-acggtagtgttttatcagtagtg-3′ and MS10, 5′-caaacccaaaccttcttccgatc-3′.</p>
<p style="background-color:#FFFFFF">The resulting product was purified and injected into <a href="http://www.wormbase.org/db/get?name=N2;class=Strain" id="N2-5">N2</a><a href="javascript:removeLinkAfterConfirm('N2-5')"><sup><img src="/gsa/img/minus.png"/></sup></a> adults with pRAK3 <italic><a href="http://www.wormbase.org/db/get?name=rol-6;class=Gene" id="rol-6-5">rol-6</a><a href="javascript:removeLinkAfterConfirm('rol-6-5')"><sup><img src="/gsa/img/minus.png"/></sup></a>(d)</italic> as a co-injection marker. The established transgenic lines were observed using a Zeiss Axio A2 Imager at either ×630 or ×1000 magnification. Images were acquired using Zeiss Axiovision software.</p>
</sec>
<sec id="s12">
<title><italic>rab-6.2p</italic>::<italic>GFP:</italic></title>
<p style="background-color:#FFFFFF">A 2.8-kb <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-7">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-7')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> promoter fragment was amplified from <a href="http://www.wormbase.org/db/get?name=N2;class=Strain" id="N2-6">N2</a><a href="javascript:removeLinkAfterConfirm('N2-6')"><sup><img src="/gsa/img/minus.png"/></sup></a> genomic DNA using the following primers: 1186, 5′-aatcgcacagcaggcctcc-3′ and 1180, 5′-gtgaaaagttcttctcctttactcggattaccaaagtccgacat-3′. In addition to the 5′ regulatory sequence, this DNA fragment contains the first seven codons of <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-8">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-8')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>. GFP was amplified from plasmid pPD95.75 using the following primers: 1175, 5′-atgtcggactttggtaatccgagtaaaggagaagaacttttcac-3′ and MS46, 5′-tttggtatattgggaatggtattctg-3′. Products were fused by overlap extension PCR using the following primers: 1188, 5′-tttgccgaacggaagagcc-3′ and MS10, 5′-caaacccaaaccttcttccgatc-3′. The final product was purified and injected into <a href="http://www.wormbase.org/db/get?name=N2;class=Strain" id="N2-7">N2</a><a href="javascript:removeLinkAfterConfirm('N2-7')"><sup><img src="/gsa/img/minus.png"/></sup></a> adults with <italic><a href="http://www.wormbase.org/db/get?name=rol-6;class=Gene" id="rol-6-6">rol-6</a><a href="javascript:removeLinkAfterConfirm('rol-6-6')"><sup><img src="/gsa/img/minus.png"/></sup></a>(d)</italic> as a co-injection marker. GFP expression was observed as described above.</p>
</sec>
</sec>
<sec id="s13">
<title>Yeast two-hybrid assay</title>
<p style="background-color:#FFFFFF">To generate first-strand cDNA, RNA was isolated from a well-fed, mixed stage <a href="http://www.wormbase.org/db/get?name=N2;class=Strain" id="N2-8">N2</a><a href="javascript:removeLinkAfterConfirm('N2-8')"><sup><img src="/gsa/img/minus.png"/></sup></a> population using Trizol. Poly(A)<sup>+</sup> first-strand cDNA was prepared using the First-Strand cDNA Synthesis kit for RT–PCR (Roche).</p>
<p style="background-color:#FFFFFF">Inserts used for preys were amplified by PCR using the Expand Long Template PCR kit (Roche). Primers to amplify the preys are shown in <ext-link ext-link-type="uri" xlink:href="http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.152538/-/DC1/genetics.113.152538-3.pdf">Table S6</ext-link>. PCR products were cloned into the pGEM-T Easy vector and sequenced. Constructs with wild-type sequence were subcloned into the pACT2 vector (gift from R. Lin), in frame with the GAL4 activation domain<!-- Q9 -->.</p>
<p style="background-color:#FFFFFF">Six truncated versions of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-31">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-31')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> were used as baits after amplification, sequencing and cloning into the pVJL11 vector (gift from M. Cobb), in frame with the LexA DNA binding domain<!-- Q10 -->. Primers are shown in <ext-link ext-link-type="uri" xlink:href="http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.152538/-/DC1/genetics.113.152538-9.pdf">Table S7</ext-link>. Mutant versions of the baits were generated using an <italic>in vitro</italic> mutagenesis kit (Invitrogen). Primers containing mutations are shown in <ext-link ext-link-type="uri" xlink:href="http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.152538/-/DC1/genetics.113.152538-7.pdf">Table S4</ext-link>.</p>
<p style="background-color:#FFFFFF">To test for interactions, combinations of baits and preys were transformed into the yeast strain L40 (<xref ref-type="bibr" rid="bib14">Hama <italic>et al.</italic> 1999</xref>). X-gal filter assays and quantitative ONPG assays were performed as described (Yeast Protocols Handbook, Clontech)<!-- Q11 -->. An average of two colonies was tested for each. For growth assays, colonies were streaked onto plates lacking histidine and supplemented with 5 mM 3-amino-1,2,4-triazole (3-AT). Miller units are calculated from OD<sub>420</sub>/<italic>t</italic> × <italic>V</italic> × OD<sub>600</sub>.</p>
</sec>
<sec id="s14">
<title>Chitin staining</title>
<p style="background-color:#FFFFFF">Chitin was stained as described (<xref ref-type="bibr" rid="bib27">Ruvkun and Finney 2005</xref>). Fluorescein-conjugated chitin-binding probe (P5211S, NEB) was used at a 1:100 dilution.</p>
</sec>
<sec id="s15">
<title>Grinder function assay</title>
<p style="background-color:#FFFFFF">Worms were fed mCherry-expressing <a href="http://www.wormbase.org/db/get?name=HB101;class=Strain" id="HB101-2">HB101</a><a href="javascript:removeLinkAfterConfirm('HB101-2')"><sup><img src="/gsa/img/minus.png"/></sup></a> as described (<xref ref-type="bibr" rid="bib37">You <italic>et al.</italic> 2008</xref>). Images were acquired using Zeiss Axiovision software.</p>
</sec>
</sec>
<sec id="s16" sec-type="results">
<title>Results</title>
<sec id="s17">
<title><italic>eat-17</italic> mutants are Eat due to defects in grinder formation</title>
<p style="background-color:#FFFFFF"><italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-33">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-33')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants were isolated from a genetic screen for defects in feeding behavior. They are defective in trapping bacteria and have slightly asynchronous terminal bulb contractions during feeding (<xref ref-type="bibr" rid="bib3">Avery 1993</xref>). The most striking phenotype, however, is a defect in their grinders; wild-type grinders consist of highly refractile cuticle ridges that can be observed by DIC optics (<xref ref-type="fig" rid="fig1">Figure 1A</xref>, arrow). These ridges are completely disorganized in <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-34">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-34')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). In wild-type worms, the grinder is composed of three separate plates that are arranged in approximate triradial symmetry (<xref ref-type="bibr" rid="bib1">Albertson and Thomson 1976</xref>; von Lieven 2003). These plates are secreted from the apical surfaces of pharyngeal muscle cells pm6 and pm7 in the terminal bulb (<xref ref-type="bibr" rid="bib1">Albertson and Thomson 1976</xref>). During feeding, the plates rotate against one another, grinding any material that comes between them. Due to defects in the grinder, <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-35">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-35')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants cannot grind bacteria efficiently. As a result, unground bacteria pass into the intestine (<xref ref-type="fig" rid="fig1">Figure 1, C–F</xref>) contributing to the <a href="http://www.wormbase.org/db/get?name=WBPhenotype%3A0000659;class=Phenotype" id="Eat-2">Eat</a><a href="javascript:removeLinkAfterConfirm('Eat-2')"><sup><img src="/gsa/img/minus.png"/></sup></a> phenotype.</p>
<fig id="fig1" position="float" fig-type="figure"><label>Figure 1</label><caption><p><italic>eat-17</italic> mutants show defects in grinder formation. (A) DIC image of wild-type pharynx. The <italic>C. elegans</italic> grinder is composed of three plates arranged in approximate triradial symmetry. Two fully formed plates are visible in lateral sections (arrow). (B) DIC image of <italic>eat-17(ad707)</italic> mutant pharynx. Most of the material comprising the grinder plates is missing in <italic>eat-17</italic> mutants. (C) The intestine of wild-type worms fed mCherry-expressing <italic>E. coli</italic> shows smooth fluorescence due to fluorescence released from ground bacteria. (D) The intestine of <italic>eat-17</italic> mutants show many unground bacteria indicated by an arrow. (E and F) Corresponding DIC images of wild type (E) and <italic>eat-17</italic> mutant (F), respectively. In F, the arrow indicates DIC image of unground bacteria. For all images, anterior is shown to the left. Bars, 10 μm.</p></caption><graphic xlink:href="gen.152538fig1"></graphic></fig>
</sec>
<sec id="s18">
<title><italic>eat-17</italic> encodes a GTPase activating protein with coiled-coil domains at the C terminus</title>
<p style="background-color:#FFFFFF">Previous work placed <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-42">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-42')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> on chromosome X, between <italic><a href="http://www.wormbase.org/db/get?name=lin-15;class=Gene" id="lin-15-4">lin-15</a><a href="javascript:removeLinkAfterConfirm('lin-15-4')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> and <italic><a href="http://www.wormbase.org/db/get?name=sup-10;class=Gene" id="sup-10-4">sup-10</a><a href="javascript:removeLinkAfterConfirm('sup-10-4')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> (<xref ref-type="bibr" rid="bib3">Avery 1993</xref>). We further mapped <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-43">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-43')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> to a 47-kb region between two SNP markers, <italic><a href="http://www.wormbase.org/db/get?name=pkP6096;class=Variation" id="pkP6096-1">pkP6096</a><a href="javascript:removeLinkAfterConfirm('pkP6096-1')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> and <italic>snp-<a href="http://www.wormbase.org/db/get?name=F38E9.2;class=Gene" id="F38E9.2-1">F38E9.2</a><a href="javascript:removeLinkAfterConfirm('F38E9.2-1')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> (<xref ref-type="fig" rid="fig2">Figure 2A</xref>), using a multipoint SNP mapping strategy (<xref ref-type="bibr" rid="bib36">Wicks <italic>et al.</italic> 2001</xref>). Three genes were predicted in this interval (<xref ref-type="fig" rid="fig2">Figure 2B</xref>): <a href="http://www.wormbase.org/db/get?name=F01G12.1;class=Gene" id="F01G12.1-1">F01G12.1</a><a href="javascript:removeLinkAfterConfirm('F01G12.1-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> encodes a putative copper transporter, <a href="http://www.wormbase.org/db/get?name=T24D11.1;class=Gene" id="T24D11.1-1">T24D11.1</a><a href="javascript:removeLinkAfterConfirm('T24D11.1-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> (currently designated as <italic><a href="http://www.wormbase.org/db/get?name=tbc-4;class=Gene" id="tbc-4-1">tbc-4</a><a href="javascript:removeLinkAfterConfirm('tbc-4-1')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>) is predicted to encode a Rab GAP containing a TBC (Tre2/Bub/Cdc16 homology) domain—a canonical GAP domain—and <a href="http://www.wormbase.org/db/get?name=F01G12.6;class=Gene" id="F01G12.6-1">F01G12.6</a><a href="javascript:removeLinkAfterConfirm('F01G12.6-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> is predicted to encode a protein with several coiled-coil domains. Interestingly, both <a href="http://www.wormbase.org/db/get?name=T24D11.1;class=Gene" id="T24D11.1-2">T24D11.1</a><a href="javascript:removeLinkAfterConfirm('T24D11.1-2')"><sup><img src="/gsa/img/minus.png"/></sup></a> and <a href="http://www.wormbase.org/db/get?name=F01G12.6;class=Gene" id="F01G12.6-2">F01G12.6</a><a href="javascript:removeLinkAfterConfirm('F01G12.6-2')"><sup><img src="/gsa/img/minus.png"/></sup></a> share homology with the human protein Evi5 (<ext-link ext-link-type="uri" xlink:href="http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.152538/-/DC1/genetics.113.152538-8.pdf">Figure S1</ext-link>), a Rab11 GAP, whose mutation is associated with neuroblastoma (<xref ref-type="bibr" rid="bib20 bib9">Liao <italic>et al.</italic> 1997; Dabbeekeh <italic>et al.</italic> 2007</xref>). Because <a href="http://www.wormbase.org/db/get?name=T24D11.1;class=Gene" id="T24D11.1-3">T24D11.1</a><a href="javascript:removeLinkAfterConfirm('T24D11.1-3')"><sup><img src="/gsa/img/minus.png"/></sup></a> and <a href="http://www.wormbase.org/db/get?name=F01G12.6;class=Gene" id="F01G12.6-3">F01G12.6</a><a href="javascript:removeLinkAfterConfirm('F01G12.6-3')"><sup><img src="/gsa/img/minus.png"/></sup></a> are transcribed in the same direction (<xref ref-type="fig" rid="fig2">Figure 2B</xref>) and because we were unable to retrieve cDNAs coding individual genes through RT–PCR, we hypothesized that these two predicted genes comprised a single gene. RT–PCR using primers within the third predicted exon of <a href="http://www.wormbase.org/db/get?name=T24D11.1;class=Gene" id="T24D11.1-4">T24D11.1</a><a href="javascript:removeLinkAfterConfirm('T24D11.1-4')"><sup><img src="/gsa/img/minus.png"/></sup></a> and the fifth predicted exon of <a href="http://www.wormbase.org/db/get?name=F01G12.6;class=Gene" id="F01G12.6-4">F01G12.6</a><a href="javascript:removeLinkAfterConfirm('F01G12.6-4')"><sup><img src="/gsa/img/minus.png"/></sup></a> identified a single transcript (indicated with arrows in <xref ref-type="fig" rid="fig2">Figure 2B</xref>), confirming <a href="http://www.wormbase.org/db/get?name=T24D11.1;class=Gene" id="T24D11.1-5">T24D11.1</a><a href="javascript:removeLinkAfterConfirm('T24D11.1-5')"><sup><img src="/gsa/img/minus.png"/></sup></a> and <a href="http://www.wormbase.org/db/get?name=F01G12.6;class=Gene" id="F01G12.6-5">F01G12.6</a><a href="javascript:removeLinkAfterConfirm('F01G12.6-5')"><sup><img src="/gsa/img/minus.png"/></sup></a> are parts of the same gene. Furthermore, by injecting two cosmids, <a href="http://www.wormbase.org/db/get?name=T24D11;class=Clone" id="T24D11-3">T24D11</a><a href="javascript:removeLinkAfterConfirm('T24D11-3')"><sup><img src="/gsa/img/minus.png"/></sup></a> and <a href="http://www.wormbase.org/db/get?name=F01G12;class=Clone" id="F01G12-3">F01G12</a><a href="javascript:removeLinkAfterConfirm('F01G12-3')"><sup><img src="/gsa/img/minus.png"/></sup></a>, which completely cover this region, we rescued the growth, feeding, and grinder defects of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-44">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-44')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants. Injecting either alone did not rescue the phenotype (data not shown).</p>
<fig id="fig2" position="float" fig-type="figure"><label>Figure 2</label><caption><p><italic>eat-17</italic> gene structure. (A) Region in <italic>C. elegans</italic> genome where <italic>eat-17</italic> maps. <italic>eat-17</italic> was mapped to 47 kb between two SNP markers <italic>pkP6096</italic> and <italic>snp-F38E9.2</italic> using a multipoint SNP mapping strategy. (B) Genes predicted to be located in the interval and the gene structure of <italic>eat-17</italic>. Red arrows indicate the exons targeted to amplify and validate <italic>eat-17</italic> transcripts. (C) Exon usages of four possible splice forms deduced from SL1 and RT–PCR analysis. The red box indicates exon 4B. The two different splice forms <italic>eat-17a</italic> and <italic>eat-17b</italic> encode two proteins differing by three amino acids. (D) Predicted protein structure of EAT-17 and the location of the mutation in the <italic>ad707</italic> allele. An N-terminal Rab GAP domain is followed by three C-terminal predicted coiled-coil domains. The arrow indicates the position of the mutated amino acid in <italic>ad707</italic>.</p></caption><graphic xlink:href="gen.152538fig2"></graphic></fig>
<p style="background-color:#FFFFFF">To determine the gene structure of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-50">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-50')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>, we first tried to isolate full-length transcripts with the intron/exon structure predicted in WormBase (<ext-link ext-link-type="uri" xlink:href="http://www.wormbase.org">www.wormbase.org</ext-link>) but failed. Next we identified individual intron/exon boundaries and found that the first exon was incorrectly predicted. To identify the 5′ end of the transcript, we used SL1 <italic>trans</italic>-splicing analysis. In <italic>C. elegans</italic>, ∼60% of all transcripts are either SL1 or SL2 <italic>trans</italic>-spliced at their 5′ ends (Blumenthal 1995; <xref ref-type="bibr" rid="bib7">Conrad <italic>et al.</italic> 1995</xref>). The results show that the first exon of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-51">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-51')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> is located ∼4 kb upstream of exon 2 (indicated with * in <xref ref-type="fig" rid="fig2">Figure 2B</xref>). We also learned that this transcript contains ∼40 bp of 5′-UTR. Using a primer targeting the newly identified first exon and one targeting the last predicted exon of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-52">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-52')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>, we were able to amplify a single 2.5-kb fragment. To confirm that we had isolated the full-length transcript, we generated a rescuing transgene in which this 2.5-kb sequence was attached to an ∼5.6-kb region of sequence upstream of the predicted <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-53">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-53')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> transcriptional start site (<italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-54">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-54')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> promoter) at its 5′ end and to a 500-bp <italic><a href="http://www.wormbase.org/db/get?name=unc-54;class=Gene" id="unc-54-3">unc-54</a><a href="javascript:removeLinkAfterConfirm('unc-54-3')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> 3′-UTR. When this construct was injected into <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-55">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-55')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants, we found that 100% of surviving F<sub>2</sub> transgenic progeny were rescued for defects in their grinders (<xref ref-type="fig" rid="fig3">Figure 3A</xref>).</p>
<fig id="fig3" position="float" fig-type="figure"><label>Figure 3</label><caption><p>EAT-17 interacts with RAB-6.2. (A) <italic>eat-17</italic> rescue. Expressing a wild-type <italic>eat-17</italic> cDNA construct in mutant worms rescues defects in grinder formation. Grinder plates are of normal size (yellow arrow) and there are no unground bacteria in the intestine (red arrow). (B) Fluorescence image of A. Smooth fluorescence in the intestine indicates the bacteria were ground (red arrow). (C) <italic>rab-6.2(ok2254)</italic> mutants show a grinder defect. Grinder is formed but the size is small. (D) <italic>eat-17(ad707)</italic> grown at 15° contains large refractile body in the terminal bulb (arrow). (E) Yeast two-hybrid screen using EAT-17 as bait identified RAB-6.2 as the only interacting Rab protein. (F) A constitutively GTP bound form of RAB-6.1 enhances binding of EAT-17 to RAB-6.1. On the other hand, a constitutively GDP bound form of RAB-6.2 (6.2T) abolishes binding of EAT-17 to RAB-6.2. The EAT-17 construct used was a truncated protein (amino acids 1–460) containing only first coiled coil domain. The truncated protein binds to RAB-6.2 very efficiently, suggesting the first coiled-coil domain is sufficient for the binding. Abbreviations are as follows: 6.1, RAB-6.1 full-length wild type; 6.1Q, RAB-6.1 with Q70L mutation; 6.1T, RAB-6.1 with T27N mutation; 6.2T, RAB-6.2 with T27N mutation; 6.2, RAB-6.2 full-length wild type; 6.2Q, RAB-6.2 with Q69L mutation. <italic>*P</italic> < 0.05; NS, not statistically significant by Student <italic>t</italic>-test. Miller units calculated as OD<sub>420</sub>/<italic>t</italic> × <italic>V</italic> × OD<sub>600</sub>. Bars, 10 μm.</p></caption><graphic xlink:href="gen.152538fig3"></graphic></fig>
<p style="background-color:#FFFFFF">To determine the exact splicing pattern(s) of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-59">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-59')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>, we cloned the amplified PCR fragments into vectors and sequenced the inserts (see <italic>Materials and Methods</italic>). We named the possible splice products <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-24">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-24')"><sup><img src="/gsa/img/minus.png"/></sup></a> a, b, c, and d (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). EAT-17a has intron/exon boundaries consistent with WormBase/GeneFinder predictions. EAT-17b uses an alternative splice acceptor site at the exon 3/4 boundary (exon 4A, labeled in red, <xref ref-type="fig" rid="fig2">Figure 2, B and C</xref>). For both of these, predicted exon 5 of <a href="http://www.wormbase.org/db/get?name=T24D11.1;class=Gene" id="T24D11.1-6">T24D11.1</a><a href="javascript:removeLinkAfterConfirm('T24D11.1-6')"><sup><img src="/gsa/img/minus.png"/></sup></a> was truncated by 180 nucleotides and spliced directly to predicted exon 2 of <a href="http://www.wormbase.org/db/get?name=F01G12.6;class=Gene" id="F01G12.6-6">F01G12.6</a><a href="javascript:removeLinkAfterConfirm('F01G12.6-6')"><sup><img src="/gsa/img/minus.png"/></sup></a> in WormBase. Neither splice form contained the predicted first exon of <a href="http://www.wormbase.org/db/get?name=F01G12.6;class=Gene" id="F01G12.6-7">F01G12.6</a><a href="javascript:removeLinkAfterConfirm('F01G12.6-7')"><sup><img src="/gsa/img/minus.png"/></sup></a>. The proteins encoded by EAT-17a and EAT-17b differ by only 3 amino acids. When we designed a set of primers to amplify sequences between exon 1 and exon 6 (corresponding to the predicted first exon of <a href="http://www.wormbase.org/db/get?name=F01G12.6;class=Gene" id="F01G12.6-8">F01G12.6</a><a href="javascript:removeLinkAfterConfirm('F01G12.6-8')"><sup><img src="/gsa/img/minus.png"/></sup></a>), we could isolate transcripts that included exon 6. We named the predicted transcripts that include exon 6 EAT-17c and EAT-17d (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). GeneFinder predictions suggested a splicing pattern that would create a stop codon at the junction between exon 5 and exon 6. This transcript would encode a truncated protein lacking most of the C terminus. It seems most likely that the a and b splice forms encoding full-length proteins are the functional ones.</p>
<p style="background-color:#FFFFFF">Analysis of the predicted protein sequence revealed two conserved domains in <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-25">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-25')"><sup><img src="/gsa/img/minus.png"/></sup></a>: a Rab GTPase activating (Rab GAP) domain and three coiled-coil domains (CC1–3, <xref ref-type="fig" rid="fig2">Figure 2D</xref>). The Rab GAP domain implicates <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-26">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-26')"><sup><img src="/gsa/img/minus.png"/></sup></a> in vesicle trafficking, a role consistent with the grinder defects in <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-60">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-60')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants. To identify the specific mutation in <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-61">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-61')"><sup><img src="/gsa/img/minus.png"/></sup></a>(<a href="http://www.wormbase.org/db/get?name=ad707;class=Variation" id="ad707-10">ad707</a><a href="javascript:removeLinkAfterConfirm('ad707-10')"><sup><img src="/gsa/img/minus.png"/></sup></a>)</italic>, we sequenced the predicted <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-62">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-62')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> coding region (based on our RT–PCR results) and found a C-to-T base-pair transition at nucleotide 607 in exon 5 of the <italic><a href="http://www.wormbase.org/db/get?name=ad707;class=Variation" id="ad707-11">ad707</a><a href="javascript:removeLinkAfterConfirm('ad707-11')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutant allele, creating a stop codon at amino acid 203 (<xref ref-type="fig" rid="fig2">Figure 2D</xref>). This mutation would generate a protein with a truncated RabGAP domain, likely rendering it nonfunctional.</p>
<p style="background-color:#FFFFFF">To determine whether <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-27">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-27')"><sup><img src="/gsa/img/minus.png"/></sup></a> contains GAP activity, we mutated the catalytic arginine residue (R116) to either lysine (K) or alanine (A) to abolish the GAP activity and expressed these mutated constructs in <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-63">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-63')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants (see <italic>Materials and Methods</italic>). A total of 21% of F<sub>1</sub>’s carrying a wild-type copy of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-64">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-64')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> were rescued for defects in grinder formation (<xref ref-type="fig" rid="fig3">Figure 3, A and B</xref>), while only 4.1 and 1.6% of mutants expressing either the R116K or R116A mutations were rescued (<xref ref-type="table" rid="t1">Table 1</xref>). Collectively, these data show that the catalytic activity of <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-28">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-28')"><sup><img src="/gsa/img/minus.png"/></sup></a> is critical for its function and that <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-29">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-29')"><sup><img src="/gsa/img/minus.png"/></sup></a> is a GAP protein.</p>
<table-wrap id="t1" position="float">
<label>Table 1</label><caption><title>GAP activity is required for full EAT-17 function</title></caption>
<table frame="above">
<col width="29.01%"/>
<col width="20.12%"/>
<col width="30.96%"/>
<col width="19.91%"/>
<thead>
<tr>
<th valign="top" align="left" scope="col"></th>
<th valign="top" align="center" scope="col">No. rescued</th>
<th valign="top" align="center" scope="col">Total no. examined</th>
<th valign="top" align="center" scope="col">% rescue</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="left" scope="row">Wild-type <italic>eat-17</italic> construct</td>
<td valign="top" align="char" char=".">16</td>
<td valign="top" align="char" char=".">76</td>
<td valign="top" align="char" char=".">21</td>
</tr>
<tr>
<td valign="top" align="left" scope="row">R116K</td>
<td valign="top" align="char" char=".">2</td>
<td valign="top" align="char" char=".">49</td>
<td valign="top" align="char" char=".">4.1</td>
</tr>
<tr>
<td valign="top" align="left" scope="row">R116A</td>
<td valign="top" align="char" char=".">1</td>
<td valign="top" align="char" char=".">64</td>
<td valign="top" align="char" char=".">1.6</td>
</tr>
</tbody>
</table><table-wrap-foot>
<fn><p><italic>eat-17</italic> mutants were injected with DNA constructs encoding either wild type or one of two mutant versions (R116K or R116A) of the EAT-17 protein (see <italic>Materials and Methods</italic>). F<sub>1</sub> transgenic progeny were blindly assayed for rescue of grinder defects. GFP was used to identify transgenic progeny afterward.</p></fn>
</table-wrap-foot></table-wrap>
</sec>
<sec id="s19">
<title>EAT-17 and RAB-6.2 interact to regulate grinder formation</title>
<p style="background-color:#FFFFFF">To identify the substrates of the GAP activity of <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-33">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-33')"><sup><img src="/gsa/img/minus.png"/></sup></a>, we performed RNA interference against 25 of the 29 predicted Rab genes in the <italic>C. elegans</italic> genome (<xref ref-type="bibr" rid="bib2">Audhya <italic>et al.</italic> 2007</xref>) and looked for a grinder phenotype similar to that of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-67">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-67')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants. RNAi was performed in worms mutant for <italic><a href="http://www.wormbase.org/db/get?name=rrf-3;class=Gene" id="rrf-3-3">rrf-3</a><a href="javascript:removeLinkAfterConfirm('rrf-3-3')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>, a hypersensitive background for RNAi (<xref ref-type="bibr" rid="bib30">Simmer <italic>et al.</italic> 2002</xref>). Among the 25 Rab genes tested, RNAi against <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-10">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-10')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> alone produced a phenotype similar to <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-68">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-68')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> (<xref ref-type="table" rid="t2">Table 2</xref>), whereas <italic><a href="http://www.wormbase.org/db/get?name=rrf-3;class=Gene" id="rrf-3-4">rrf-3</a><a href="javascript:removeLinkAfterConfirm('rrf-3-4')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants show no obvious defects in grinder formation (data not shown). When we examined <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-11">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-11')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> deletion mutants, the defects in the size and organization of the grinder plates were identical to those of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-69">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-69')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants (<xref ref-type="fig" rid="fig1">Figures 1B</xref> and <xref ref-type="fig" rid="fig3">3C</xref>), confirming our RNAi results.</p>
<table-wrap id="t2" position="float">
<label>Table 2</label><caption><title>RNAi of <italic>rab-6.2</italic> phenocopies <italic>eat-17</italic> mutants</title></caption>
<table frame="above">
<col width="26.69%"/>
<col width="73.31%"/>
<thead>
<tr>
<th valign="top" align="left" scope="col">dsRNA injected</th>
<th valign="top" align="center" scope="col">Phenotype</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="left" scope="row">None</td>
<td valign="top" align="center">Wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-1</italic></td>
<td valign="top" align="center">Embryonic lethal</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-2</italic></td>
<td valign="top" align="center">Slight growth delay</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-3</italic></td>
<td valign="top" align="center">Wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-5</italic></td>
<td valign="top" align="center">Embryonic lethal</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-6.1</italic></td>
<td valign="top" align="center">Wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-6.2</italic></td>
<td valign="top" align="center">Growth delay with abnormal grinder</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-7</italic></td>
<td valign="top" align="center">Wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-8</italic></td>
<td valign="top" align="center">Loopy movement, exaggerated body bends</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-10</italic></td>
<td valign="top" align="center">ND<xref ref-type="table-fn" rid="t2n1"><italic><sup>a</sup></italic></xref></td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-11.1</italic></td>
<td valign="top" align="center">Embryonic lethal</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-11.2</italic></td>
<td valign="top" align="center">Arrested as L1s</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-14</italic></td>
<td valign="top" align="center">Some show growth delay, Dpy<xref ref-type="table-fn" rid="t2n2"><italic><sup>b</sup></italic></xref></td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-18</italic></td>
<td valign="top" align="center">Growth delay, infrequent pumping but normal grinder</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-19</italic></td>
<td valign="top" align="center">Wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-21</italic></td>
<td valign="top" align="center">Sick, starved, and strong Dpy</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-27</italic></td>
<td valign="top" align="center">Wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-28</italic></td>
<td valign="top" align="center">Wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-30</italic></td>
<td valign="top" align="center">Wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-33</italic></td>
<td valign="top" align="center">Slightly Unc<xref ref-type="table-fn" rid="t2n3"><italic><sup>c</sup></italic></xref>, otherwise wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-35</italic></td>
<td valign="top" align="center">Wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-37</italic></td>
<td valign="top" align="center">Loopy movement, otherwise wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>rab-39</italic></td>
<td valign="top" align="center">Wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>4R79.2</italic></td>
<td valign="top" align="center">Loopy movement, Unc, otherwise wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>K02E10.1</italic></td>
<td valign="top" align="center">ND</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>F11A5.4</italic></td>
<td valign="top" align="center">Wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>F11A5.3</italic></td>
<td valign="top" align="center">Wild type</td>
</tr>
<tr>
<td valign="top" align="left" scope="row"><italic>C56E6.2</italic></td>
<td valign="top" align="center">Loopy movement, Unc</td>
</tr>
</tbody>
</table><table-wrap-foot>
<fn><p>Among 25 Rabs tested, RNAi of <italic>rab-6.2</italic> shows an identical grinder phenotype to <italic>eat-17</italic>. Full-length dsRNAs for each Rab listed were injected into <italic>rrf-3</italic> RNAi hypersensitive mutants. Worms were grown at 15° to enhance the phenotype.</p></fn>
<fn id="t2n1"><label>a</label><p>ND, Not determined.</p></fn>
<fn id="t2n2"><label>b</label><p>Dpy, dumpy phenotype, worms are shorter than wild type.</p></fn>
<fn id="t2n3"><label>c</label><p>Unc, uncoordinated movement phenotype, worms move abnormally.</p></fn>
</table-wrap-foot></table-wrap>
<p style="background-color:#FFFFFF">To examine whether <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-34">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-34')"><sup><img src="/gsa/img/minus.png"/></sup></a> and <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-23">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-23')"><sup><img src="/gsa/img/minus.png"/></sup></a> directly bind to each other and whether the binding is specific, we performed directed yeast two-hybrid assays with <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-35">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-35')"><sup><img src="/gsa/img/minus.png"/></sup></a> and each of the 25 RAB proteins. Among all the RABs, only <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-24">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-24')"><sup><img src="/gsa/img/minus.png"/></sup></a> interacted with <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-36">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-36')"><sup><img src="/gsa/img/minus.png"/></sup></a> (<xref ref-type="fig" rid="fig3">Figure 3E</xref>, data not shown). To examine if the guanylate nucleotide binding status of <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-25">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-25')"><sup><img src="/gsa/img/minus.png"/></sup></a> is critical, we introduce two types of mutations into <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-26">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-26')"><sup><img src="/gsa/img/minus.png"/></sup></a>: (1) glutamine (Q69) to leucine (L) to produce a constitutively GTP bound form and (2) threonine (T27) to asparagine (N) to produce a constitutively GDP bound form (<xref ref-type="bibr" rid="bib21 bib22">Martinez <italic>et al.</italic> 1994, 1997</xref>). The interaction between <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-37">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-37')"><sup><img src="/gsa/img/minus.png"/></sup></a> and <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-27">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-27')"><sup><img src="/gsa/img/minus.png"/></sup></a> was completely abolished when the T27N mutation was introduced (<xref ref-type="fig" rid="fig3">Figure 3E</xref>, 6.2T), but it was not further enhanced with the Q69L mutation (<xref ref-type="fig" rid="fig3">Figure 3C</xref>, 6.2Q), as compared to wild-type <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-28">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-28')"><sup><img src="/gsa/img/minus.png"/></sup></a> (<xref ref-type="fig" rid="fig3">Figure 3F</xref>, 6.2). This suggests that the reaction reached maximum binding with wild-type <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-29">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-29')"><sup><img src="/gsa/img/minus.png"/></sup></a> and that we could not enhance binding any further under the conditions in which we performed the assay.</p>
<p style="background-color:#FFFFFF"><a href="http://www.wormbase.org/db/get?name=RAB-6.1;class=Gene" id="RAB-6.1-6">RAB-6.1</a><a href="javascript:removeLinkAfterConfirm('RAB-6.1-6')"><sup><img src="/gsa/img/minus.png"/></sup></a>, the only other Rab6 in the <italic>C. elegans</italic> genome, shares 81% amino acid sequence identity with <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-30">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-30')"><sup><img src="/gsa/img/minus.png"/></sup></a>. Because of the high similarity between the two Rab6’s, we tested whether <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-38">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-38')"><sup><img src="/gsa/img/minus.png"/></sup></a> could bind to <a href="http://www.wormbase.org/db/get?name=RAB-6.1;class=Gene" id="RAB-6.1-7">RAB-6.1</a><a href="javascript:removeLinkAfterConfirm('RAB-6.1-7')"><sup><img src="/gsa/img/minus.png"/></sup></a> as well. Although we did not detect any binding between wild-type <a href="http://www.wormbase.org/db/get?name=RAB-6.1;class=Gene" id="RAB-6.1-8">RAB-6.1</a><a href="javascript:removeLinkAfterConfirm('RAB-6.1-8')"><sup><img src="/gsa/img/minus.png"/></sup></a> and <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-39">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-39')"><sup><img src="/gsa/img/minus.png"/></sup></a> in our assay, <a href="http://www.wormbase.org/db/get?name=RAB-6.1;class=Gene" id="RAB-6.1-9">RAB-6.1</a><a href="javascript:removeLinkAfterConfirm('RAB-6.1-9')"><sup><img src="/gsa/img/minus.png"/></sup></a>(Q70L) (equivalent to Q69L in <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-31">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-31')"><sup><img src="/gsa/img/minus.png"/></sup></a>) showed enhanced binding to <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-40">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-40')"><sup><img src="/gsa/img/minus.png"/></sup></a>, suggesting that <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-41">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-41')"><sup><img src="/gsa/img/minus.png"/></sup></a> binds to RAB-6’s through the conserved guanylate nucleotide binding site. Taken together, these data strongly support the hypothesis that <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-32">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-32')"><sup><img src="/gsa/img/minus.png"/></sup></a> is a specific substrate of <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-42">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-42')"><sup><img src="/gsa/img/minus.png"/></sup></a> and that <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-43">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-43')"><sup><img src="/gsa/img/minus.png"/></sup></a> promotes hydrolysis of GTP by binding the active (GTP bound) form of RAB-6.</p>
<p style="background-color:#FFFFFF">Our studies identified <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-44">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-44')"><sup><img src="/gsa/img/minus.png"/></sup></a> as a GAP protein with three coiled-coil domains. Coiled-coil domains are generally involved in protein–protein interactions, and GAPCenA, a mammalian RabGAP, interacts with Rab6 through a coiled-coil domain (<xref ref-type="bibr" rid="bib8">Cuif <italic>et al.</italic> 1999</xref>). When we tested several truncated <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-45">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-45')"><sup><img src="/gsa/img/minus.png"/></sup></a> constructs, <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-46">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-46')"><sup><img src="/gsa/img/minus.png"/></sup></a> (aa 1–316), <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-47">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-47')"><sup><img src="/gsa/img/minus.png"/></sup></a> (aa 1–364), and <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-48">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-48')"><sup><img src="/gsa/img/minus.png"/></sup></a> (aa 1–405), they did not interact with <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-33">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-33')"><sup><img src="/gsa/img/minus.png"/></sup></a>, showing that the GAP domain alone is not sufficient for this interaction (data not shown). A construct of <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-49">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-49')"><sup><img src="/gsa/img/minus.png"/></sup></a> containing the first coiled-coil domain [<a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-50">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-50')"><sup><img src="/gsa/img/minus.png"/></sup></a> (aa 1–460)], however, shows significant interaction with <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-34">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-34')"><sup><img src="/gsa/img/minus.png"/></sup></a> (<xref ref-type="fig" rid="fig3">Figure 3E</xref>). Collectively, our results show that the GAP domain of <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-51">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-51')"><sup><img src="/gsa/img/minus.png"/></sup></a> mediates its catalytic activity and the first coiled-coil domain mediates its interaction with <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-35">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-35')"><sup><img src="/gsa/img/minus.png"/></sup></a>. Presumably, the coiled-coil domains are important to provide <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-52">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-52')"><sup><img src="/gsa/img/minus.png"/></sup></a> with specificity toward <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-36">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-36')"><sup><img src="/gsa/img/minus.png"/></sup></a>. Altogether, these data suggest a conserved structural mechanism of interaction between Rab6’s and their RabGAP proteins through the coiled-coil domain (<xref ref-type="bibr" rid="bib8">Cuif <italic>et al.</italic> 1999</xref>).</p>
</sec>
<sec id="s20">
<title>EAT-17 and RAB-6.2 are colocalized to terminal bulb muscle, the site of grinder secretion</title>
<p style="background-color:#FFFFFF">To determine whether <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-54">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-54')"><sup><img src="/gsa/img/minus.png"/></sup></a> and <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-38">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-38')"><sup><img src="/gsa/img/minus.png"/></sup></a> function together in the terminal bulb, we examined the expression patterns of transgenic lines that carry the promoters of each gene fused with GFP (see <italic>Materials and Methods</italic>). Both <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-55">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-55')"><sup><img src="/gsa/img/minus.png"/></sup></a> and <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-39">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-39')"><sup><img src="/gsa/img/minus.png"/></sup></a> are strongly expressed in the terminal bulb muscles that secrete the components of the grinder (<xref ref-type="fig" rid="fig4">Figure 4, A–D</xref>, see <italic>Materials and Methods</italic>). Our construct also shows <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-56">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-56')"><sup><img src="/gsa/img/minus.png"/></sup></a> expression outside of the pharynx, such as in intestine and vulva (<xref ref-type="fig" rid="fig4">Figure 4, E and F</xref>).</p>
<fig id="fig4" position="float" fig-type="figure"><label>Figure 4</label><caption><p>EAT-17 is expressed in pharyngeal muscle, intestine, and vulva. (A and B) Corresponding GFP and DIC images of EAT-17::GFP. (C and D) Corresponding GFP and DIC images of RAB-6.2::GFP. Both EAT-17 and RAB-6.2 are highly expressed in the pharyngeal muscle. RAB-6-2 is also highly expressed in neurons. (E and F) Corresponding GFP and DIC images of EAT-17::GFP in the pharynx (red arrows), intestine (white arrows), and vulva (yellow arrows). For <xref ref-type="fig" rid="fig4">Figure 4, A–D</xref>, bars, 10 μm. For Figure 4, E and F, bars, 30 μm.</p></caption><graphic xlink:href="gen.152538fig4"></graphic></fig>
<p style="background-color:#FFFFFF">This tissue expression pattern of <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-61">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-61')"><sup><img src="/gsa/img/minus.png"/></sup></a> overlaps largely with that previously reported for <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-42">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-42')"><sup><img src="/gsa/img/minus.png"/></sup></a> (<xref ref-type="bibr" rid="bib39">Zhang <italic>et al.</italic> 2012</xref>), supporting the inference that <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-62">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-62')"><sup><img src="/gsa/img/minus.png"/></sup></a> is a specific GAP to regulate <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-43">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-43')"><sup><img src="/gsa/img/minus.png"/></sup></a> activity in those tissues.</p>
</sec>
<sec id="s21">
<title>Chitin deposition is superficially intact in <italic>eat-17</italic> and <italic>rab-6.2</italic> mutants</title>
<p style="background-color:#FFFFFF">Chitin provides mechanical support to exoskeletons in many invertebrate animals. In yeast, proper chitin deposition is essential and strictly regulated by controlling the trafficking of its synthase, chs3p (<xref ref-type="bibr" rid="bib28">Schorr <italic>et al.</italic> 2001</xref>). Because chitin is one of the components of the grinder and its synthase is regulated by trafficking, we stained the grinders of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-73">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-73')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> and <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-16">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-16')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants for chitin to examine if chitin deposition is misregulated in these mutants. Despite the global disorganization of the grinder, the overall chitin deposition was indistinguishable among <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-74">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-74')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>, <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-17">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-17')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>, and wild type (<xref ref-type="fig" rid="fig5">Figure 5, A–F</xref>). However, when grown at a low temperature, <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-75">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-75')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants occasionally have large refractory bodies in the terminal bulbs that secrete the components of the grinder (<xref ref-type="fig" rid="fig3">Figure 3D</xref>). Interestingly, at least in appearance, those bodies share similar refractile characteristics with that of grinder. These results may suggest that the materials trafficked by <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-44">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-44')"><sup><img src="/gsa/img/minus.png"/></sup></a> from terminal bulb could be the components of the grinder and that the refractile bodies in <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-76">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-76')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> could be these components, accumulating in the muscle because of the trafficking defect.</p>
<fig id="fig5" position="float" fig-type="figure"><label>Figure 5</label><caption><p>Chitin deposition in the grinders of <italic>eat-17</italic> and <italic>rab-6.2</italic> mutants is normal. (A and B) Chitin staining of grinder from wild type and corresponding DIC image. (C and D) From <italic>eat-17</italic> mutants and corresponding DIC image. (E and F) From <italic>rab-6.2</italic> mutants and corresponding DIC image. Arrowheads indicate the position of the grinder. Bars, 10 μm.</p></caption><graphic xlink:href="gen.152538fig5"></graphic></fig>
</sec>
</sec>
<sec id="s22" sec-type="discussion">
<title>Discussion</title>
<p style="background-color:#FFFFFF">In this study we mapped and characterized <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-79">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-79')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>, a new GTPase-activating protein gene in <italic>C. elegans</italic>. Using two different approaches, RNA interference and yeast two hybrid, we showed that <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-63">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-63')"><sup><img src="/gsa/img/minus.png"/></sup></a> interacts with <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-45">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-45')"><sup><img src="/gsa/img/minus.png"/></sup></a>, a conserved small G protein regulating membrane trafficking from Golgi (Del Nery <italic>et al.</italic> 2006; <xref ref-type="bibr" rid="bib12">Girod <italic>et al.</italic> 1999</xref>). The fact that both approaches identify only <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-46">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-46')"><sup><img src="/gsa/img/minus.png"/></sup></a> as a substrate of <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-64">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-64')"><sup><img src="/gsa/img/minus.png"/></sup></a> among 25 tested Rabs strongly suggests <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-65">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-65')"><sup><img src="/gsa/img/minus.png"/></sup></a> is a specific GAP for <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-47">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-47')"><sup><img src="/gsa/img/minus.png"/></sup></a>. Both <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-80">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-80')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> and <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-20">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-20')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants show identical defects in their grinders, the part of the feeding organ that crushes food, and which, therefore, is essential for rapid growth and normal development. The new grinder replaces the old one during every molt, implicating that the grinder components needed to be transported during every molt to build a new one<!-- Q12 -->. Our observations that a mutation in <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-21">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-21')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>, which functions in cargo trafficking, and a mutation in its GAP protein <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-66">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-66')"><sup><img src="/gsa/img/minus.png"/></sup></a> show their most striking phenotypes in the grinder, suggest that precise regulation of trafficking of grinder components is critical to build a functional grinder.</p>
<p style="background-color:#FFFFFF">Although we do not know the identities of the cargos in <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-48">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-48')"><sup><img src="/gsa/img/minus.png"/></sup></a> targeted vesicles, we found that <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-81">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-81')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants grown at 15° have refractory bodies in the terminal bulbs. The bodies appear similar (<italic>i.e.</italic>, highly refractile) to the grinder. This suggests that those bodies may be grinder components accumulating in the muscle because of slow trafficking at low temperature. In fact, <xref ref-type="bibr" rid="bib6">Chotard <italic>et al.</italic> (2010)</xref> showed that the mutant of <italic><a href="http://www.wormbase.org/db/get?name=tbc-2;class=Gene" id="tbc-2-1">tbc-2</a><a href="javascript:removeLinkAfterConfirm('tbc-2-1')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic>, which encodes another RabGAP in <italic>C. elegans</italic>, accumulates enormous <a href="http://www.wormbase.org/db/get?name=RAB-7;class=Gene" id="RAB-7-1">RAB-7</a><a href="javascript:removeLinkAfterConfirm('RAB-7-1')"><sup><img src="/gsa/img/minus.png"/></sup></a>-positive late endosome in the intestine containing refractile materials. These results strongly suggest that the refractile bodies can be a common phenotype in endosomal trafficking mutants and that <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-67">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-67')"><sup><img src="/gsa/img/minus.png"/></sup></a> regulates endosomal trafficking.</p>
<p style="background-color:#FFFFFF">Small G proteins are substrates of GAPs for their fast turnover. The importance of GAPs is demonstrated in many examples such as the oncogene Evi5, which is homologous to <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-68">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-68')"><sup><img src="/gsa/img/minus.png"/></sup></a> (<xref ref-type="bibr" rid="bib20 bib9">Liao <italic>et al.</italic> 1997; Dabbeekeh <italic>et al.</italic> 2007</xref>). Only one other GAP for Rab6 has been identified so far, GAPCenA, which also interacts with Rab6 through a coiled-coil domain. The requirement of the coiled-coil domain for interaction appears to be unique for the Rab6 GAPs. Studies have shown that a class of Rab6 effecter, Golgins, interacts with Rab6’s through their coiled-coil domains to direct vesicle capture and sorting within the Golgi. It is possible that Rab6’s bind to their GAPs and effectors through a similar mechanism mediated by coiled-coil domains (<xref ref-type="bibr" rid="bib31">Sinka <italic>et al.</italic> 2008</xref>).</p>
<p style="background-color:#FFFFFF">Our discovery of <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-69">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-69')"><sup><img src="/gsa/img/minus.png"/></sup></a> and its specificity toward <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-49">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-49')"><sup><img src="/gsa/img/minus.png"/></sup></a> strongly suggest that there are specific GAPs yet to be discovered for <a href="http://www.wormbase.org/db/get?name=RAB-6.1;class=Gene" id="RAB-6.1-10">RAB-6.1</a><a href="javascript:removeLinkAfterConfirm('RAB-6.1-10')"><sup><img src="/gsa/img/minus.png"/></sup></a>, the only other Rab6 in <italic>C. elegans</italic>, sharing 81% amino acid sequence identity with <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-50">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-50')"><sup><img src="/gsa/img/minus.png"/></sup></a>. <italic><a href="http://www.wormbase.org/db/get?name=rab-6.1;class=Gene" id="rab-6.1-2">rab-6.1</a><a href="javascript:removeLinkAfterConfirm('rab-6.1-2')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> and <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-22">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-22')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> are partially redundant; <italic><a href="http://www.wormbase.org/db/get?name=rab-6.1;class=Gene" id="rab-6.1-3">rab-6.1</a><a href="javascript:removeLinkAfterConfirm('rab-6.1-3')"><sup><img src="/gsa/img/minus.png"/></sup></a>(RNAi)</italic> worms are superficially normal, and <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-23">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-23')"><sup><img src="/gsa/img/minus.png"/></sup></a>(RNAi)</italic> worms have defects only in the grinder. Knockout of both genes, however, produces gross defects in molting and growth. The fact that <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-70">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-70')"><sup><img src="/gsa/img/minus.png"/></sup></a> does not interact with <a href="http://www.wormbase.org/db/get?name=RAB-6.1;class=Gene" id="RAB-6.1-11">RAB-6.1</a><a href="javascript:removeLinkAfterConfirm('RAB-6.1-11')"><sup><img src="/gsa/img/minus.png"/></sup></a> to regulate grinder formation and the similarity of the <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-82">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-82')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> phenotype to that of the <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-24">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-24')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> single mutant or <italic><a href="http://www.wormbase.org/db/get?name=rab-6.2;class=Gene" id="rab-6.2-25">rab-6.2</a><a href="javascript:removeLinkAfterConfirm('rab-6.2-25')"><sup><img src="/gsa/img/minus.png"/></sup></a>(RNAi)</italic> is consistent with the proposed <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-71">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-71')"><sup><img src="/gsa/img/minus.png"/></sup></a> specificity toward <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-51">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-51')"><sup><img src="/gsa/img/minus.png"/></sup></a>.</p>
<p style="background-color:#FFFFFF">Recently <xref ref-type="bibr" rid="bib39">Zhang <italic>et al.</italic> (2012</xref>) identified <a href="http://www.wormbase.org/db/get?name=LIN-10;class=Gene" id="LIN-10-1">LIN-10</a><a href="javascript:removeLinkAfterConfirm('LIN-10-1')"><sup><img src="/gsa/img/minus.png"/></sup></a> as an <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-52">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-52')"><sup><img src="/gsa/img/minus.png"/></sup></a> effector to regulate glutamate receptor recycling in <italic>C. elegans</italic> neurons. However, we did not see any grinder defect in <italic><a href="http://www.wormbase.org/db/get?name=lin-10;class=Gene" id="lin-10-2">lin-10</a><a href="javascript:removeLinkAfterConfirm('lin-10-2')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants (data not shown). In addition, we could not detect <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-72">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-72')"><sup><img src="/gsa/img/minus.png"/></sup></a> expression in neurons in which <a href="http://www.wormbase.org/db/get?name=LIN-10;class=Gene" id="LIN-10-2">LIN-10</a><a href="javascript:removeLinkAfterConfirm('LIN-10-2')"><sup><img src="/gsa/img/minus.png"/></sup></a> interacts with <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-53">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-53')"><sup><img src="/gsa/img/minus.png"/></sup></a>. Although this does not exclude the possibility that <a href="http://www.wormbase.org/db/get?name=LIN-10;class=Gene" id="LIN-10-3">LIN-10</a><a href="javascript:removeLinkAfterConfirm('LIN-10-3')"><sup><img src="/gsa/img/minus.png"/></sup></a> functions as an effector for <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-54">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-54')"><sup><img src="/gsa/img/minus.png"/></sup></a> and <a href="http://www.wormbase.org/db/get?name=EAT-17;class=Gene" id="EAT-17-73">EAT-17</a><a href="javascript:removeLinkAfterConfirm('EAT-17-73')"><sup><img src="/gsa/img/minus.png"/></sup></a> pathway in other places, <a href="http://www.wormbase.org/db/get?name=LIN-10;class=Gene" id="LIN-10-4">LIN-10</a><a href="javascript:removeLinkAfterConfirm('LIN-10-4')"><sup><img src="/gsa/img/minus.png"/></sup></a> does not obviously function in grinder formation. This suggests <a href="http://www.wormbase.org/db/get?name=LIN-10;class=Gene" id="LIN-10-5">LIN-10</a><a href="javascript:removeLinkAfterConfirm('LIN-10-5')"><sup><img src="/gsa/img/minus.png"/></sup></a> affects <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-55">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-55')"><sup><img src="/gsa/img/minus.png"/></sup></a> function probably specifically in neurons, supporting the notion that differential expression of specific GEFs and GAPs in different places and at different times provides spatial and temporal specificity to the G proteins (<xref ref-type="bibr" rid="bib29">Segev and Kahn 2012</xref>).</p>
<p style="background-color:#FFFFFF">That mutations in a Rab and one of its GAPs cause an identical phenotype demonstrates how critical the precise kinetics of the Rab provided by their GAPs are. Furthermore, this implies that we could identify Rab interactors such as their GAPs by looking for mutants exhibiting phenotypes similar to those of Rab mutants. Among 20 TBC GAP domain-containing proteins, only two have been characterized (<xref ref-type="bibr" rid="bib23 bib6">Mukhopadhyay <italic>et al.</italic> 2007; Chotard <italic>et al.</italic> 2010</xref>). Our finding provides an insight that may be useful for discovering more GAPs for these Rabs. In addition, using the <a href="http://www.wormbase.org/db/get?name=WBPhenotype%3A0000659;class=Phenotype" id="Eat-3">Eat</a><a href="javascript:removeLinkAfterConfirm('Eat-3')"><sup><img src="/gsa/img/minus.png"/></sup></a> phenotype caused by the grinder defect in <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-83">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-83')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants, we could identify other interactors such as <a href="http://www.wormbase.org/db/get?name=RAB-6.2;class=Gene" id="RAB-6.2-56">RAB-6.2</a><a href="javascript:removeLinkAfterConfirm('RAB-6.2-56')"><sup><img src="/gsa/img/minus.png"/></sup></a> effectors through genetic screens to isolate suppressors or enhancers of <italic><a href="http://www.wormbase.org/db/get?name=eat-17;class=Gene" id="eat-17-84">eat-17</a><a href="javascript:removeLinkAfterConfirm('eat-17-84')"><sup><img src="/gsa/img/minus.png"/></sup></a></italic> mutants.</p>
</sec>
<sec id="s23">
<title></title>
</sec>
</body>
<back>
<ack>
<title>Acknowledgments</title>
<p style="background-color:#FFFFFF">Several strains used in these studies were provided by the <italic>Caenorhabditis</italic> Genetics Center, which is funded by National Institutes of Health (NIH) Office of Research Infrastructure Programs (P40 OD010440). We thank Melanie Cobb and Rueyling Lin for DNA constructs and reagents and David Raizen, Jim McKay, Scott Cameron, and Pamela Marshall for invaluable discussions. This work was supported by NIH grant HL46154 (L.A.) and 09SDG2150070 from the American Heart Association (Y.-J.Y.).</p>
</ack>
<fn-group><fn id="fn1"><p>Manuscript received April 25, 2013; accepted for publication June 12, 2013<italic>Communicating editor: D. I. Greenstein</italic></p></fn>
</fn-group>
<fn-group>
<title>AUTHOR QUERIES</title>
<fn id="Q1" fn-type="other"><p>Please verify all supporting information links in the article.</p></fn>
<fn id="Q2" fn-type="other"><p>Please check use of italics throughout article. NOTE: Journal style includes gene names and alleles set in italics.</p></fn>
<fn id="Q3" fn-type="other"><p>Please check the WormBase links throughout. A list of entities linked in this article is available at http://textpresso-dev.caltech.edu/gsa/worm/entity_link_tables/152538.html.</p></fn>
<fn id="Q4" fn-type="other"><p>Please verify all author affiliations and zip codes. The two Virginia Commonwealth University affiliations have been grouped together. Okay?</p></fn>
<fn id="Q5" fn-type="other"><p>Please note that Genetics style requires that when in-text references are grouped, they be listed by earliest date first, not alphabetically.</p></fn>
<fn id="Q6" fn-type="other"><p>NGM is a standard abbreviation, but NGMSR is not. Please check and add the additional meaning of “SR” to NGM (nematode growth medium) or otherwise amend as needed at first mention in “Worm culture” section.</p></fn>
<fn id="Q7" fn-type="other"><p>Please compare the use of italics/roman in <underline>rab-6.2</underline><italic><underline>(</underline></italic><italic>ok2254)</italic> and below <underline>rab-6.2(</underline>ok2254) and make consistent unless the appearance of each needs to be as they appear.</p></fn>
<fn id="Q8" fn-type="other"><p>Please provide an affiliation for Andrew Fire as a gift provider in Materials and Methods.</p></fn>
<fn id="Q9" fn-type="other"><p>Please provide an affiliation for R. Lin as a gift provider in Materials and Methods.</p></fn>
<fn id="Q10" fn-type="other"><p>Please provide an affiliation for M. Cobb as a gift provider in Materials and Methods.</p></fn>
<fn id="Q11" fn-type="other"><p>Please replace ONPG with its definition in Materials and Methods.</p></fn>
<fn id="Q12" fn-type="other"><p>Please check the sentence: “The new grinder replaces the old one during every molt . . .” and amend for clarity.</p></fn>
<fn id="Q13" fn-type="other"><p>Unable to find Raizen et al. 2012 with internet search in order to add the correct format for the citation. Please amend the reference with complete information as needed. Thanks!</p></fn>
<fn id="Q14" fn-type="other"><p><italic>Please</italic> provide page numbers for Ruykun 2005 reference.</p></fn></fn-group>
<ref-list>
<title>Literature Cited</title>
<ref id="bib1"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Albertson</surname><given-names>D. G.</given-names></name><name><surname>Thomson</surname><given-names>J. N.</given-names></name></person-group>, <year>1976</year> <article-title>The pharynx of Caenorhabditis elegans.</article-title> <source>Philos. Trans. R. Soc. Lond. B Biol. Sci.</source> <volume>275</volume>: <fpage>299</fpage>–<lpage>325</lpage>.</citation></ref>
<ref id="bib2"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Audhya</surname><given-names>A.</given-names></name><name><surname>Desai</surname><given-names>A.</given-names></name><name><surname>Oegema</surname><given-names>K.</given-names></name></person-group>, <year>2007</year> <article-title>A role for Rab5 in structuring the endoplasmic reticulum.</article-title> <source>J. Cell Biol.</source> <volume>178</volume>: <fpage>43</fpage>–<lpage>56</lpage>.</citation></ref>
<ref id="bib3"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Avery</surname><given-names>L.</given-names></name></person-group>, <year>1993</year> <article-title>The genetics of feeding in Caenorhabditis elegans.</article-title> <source>Genetics</source> <volume>133</volume>: <fpage>897</fpage>–<lpage>917</lpage>.</citation></ref>
<ref id="bib4"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Avery</surname><given-names>L.</given-names></name><name><surname>Shtonda</surname><given-names>B. B.</given-names></name></person-group>, <year>2003</year> <article-title>Food transport in the C. elegans pharynx.</article-title> <source>J. Exp. Biol.</source> <volume>206</volume>: <fpage>2441</fpage>–<lpage>2457</lpage>.</citation></ref>
<ref id="bib5"><citation citation-type="journal" id="a">Avery, L. and You, Y.J. <italic>C. elegans</italic> feeding (May 21, 2012), <italic>WormBook</italic>, ed. The <italic>C. elegans</italic> Research Community, WormBook, doi/10.1895/wormbook.1.150.1, http://www .wormbook.org.</citation>
<citation citation-type="journal" id="b"><person-group person-group-type="author"><name><surname>Blumenthal</surname><given-names>T.</given-names></name></person-group>, <year>1995</year> <article-title>Trans-splicing and polycistronic transcription in Caenorhabditis elegans.</article-title> <source>Trends Genet.</source> <volume>11</volume>: <fpage>132</fpage>–<lpage>136</lpage>.</citation></ref>
<ref id="bib6"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Chotard</surname><given-names>L.</given-names></name><name><surname>Mishra</surname><given-names>A. K.</given-names></name><name><surname>Sylvain</surname><given-names>M. A.</given-names></name><name><surname>Tuck</surname><given-names>S.</given-names></name><name><surname>Lambright</surname><given-names>D. G.</given-names></name><etal/></person-group>, <year>2010</year> <article-title>TBC-2 regulates RAB-5/RAB-7-mediated endosomal trafficking in Caenorhabditis elegans.</article-title> <source>Mol. Biol. Cell</source> <volume>21</volume>: <fpage>2285</fpage>–<lpage>2296</lpage>.</citation></ref>
<ref id="bib7"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Conrad</surname><given-names>R.</given-names></name><name><surname>Lea</surname><given-names>K.</given-names></name><name><surname>Blumenthal</surname><given-names>T.</given-names></name></person-group>, <year>1995</year> <article-title>SL1 trans-splicing specified by AU-rich synthetic RNA inserted at the 5′ end of Caenorhabditis elegans pre-mRNA.</article-title> <source>RNA</source> <volume>1</volume>: <fpage>164</fpage>–<lpage>170</lpage>.</citation></ref>
<ref id="bib8"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Cuif</surname><given-names>M. H.</given-names></name><name><surname>Possmayer</surname><given-names>F.</given-names></name><name><surname>Zander</surname><given-names>H.</given-names></name><name><surname>Bordes</surname><given-names>N.</given-names></name><name><surname>Jollivet</surname><given-names>F.</given-names></name><etal/></person-group>, <year>1999</year> <article-title>Characterization of GAPCenA, a GTPase activating protein for Rab6, part of which associates with the centrosome.</article-title> <source>EMBO J.</source> <volume>18</volume>: <fpage>1772</fpage>–<lpage>1782</lpage>.</citation></ref>
<ref id="bib9"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Dabbeekeh</surname><given-names>J. T.</given-names></name><name><surname>Faitar</surname><given-names>S. L.</given-names></name><name><surname>Dufresne</surname><given-names>C. P.</given-names></name><name><surname>Cowell</surname><given-names>J. K.</given-names></name></person-group>, <year>2007</year> <article-title>The EVI5 TBC domain provides the GTPase-activating protein motif for RAB11.</article-title> <source>Oncogene</source> <volume>26</volume>: <fpage>2804</fpage>–<lpage>2808</lpage>.</citation></ref>
<ref id="bib10"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Del Nery</surname><given-names>E.</given-names></name><name><surname>Miserey-Lenkei</surname><given-names>S.</given-names></name><name><surname>Falguieres</surname><given-names>T.</given-names></name><name><surname>Nizak</surname><given-names>C.</given-names></name><name><surname>Johannes</surname><given-names>L.</given-names></name><etal/></person-group>, <year>2006</year> <article-title>Rab6A and Rab6A’ GTPases play non-overlapping roles in membrane trafficking.</article-title> <source>Traffic</source> <volume>7</volume>: <fpage>394</fpage>–<lpage>407</lpage>.</citation></ref>
<ref id="bib11"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Durkin</surname><given-names>M. E.</given-names></name><name><surname>Yuan</surname><given-names>B. Z.</given-names></name><name><surname>Zhou</surname><given-names>X.</given-names></name><name><surname>Zimonjic</surname><given-names>D. B.</given-names></name><name><surname>Lowy</surname><given-names>D. R.</given-names></name><etal/></person-group>, <year>2007</year> <article-title>DLC-1:a Rho GTPase-activating protein and tumour suppressor.</article-title> <source>J. Cell. Mol. Med.</source> <volume>11</volume>: <fpage>1185</fpage>–<lpage>1207</lpage>.</citation></ref>
<ref id="bib12"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Girod</surname><given-names>A.</given-names></name><name><surname>Storrie</surname><given-names>B.</given-names></name><name><surname>Simpson</surname><given-names>J. C.</given-names></name><name><surname>Johannes</surname><given-names>L.</given-names></name><name><surname>Goud</surname><given-names>B.</given-names></name><etal/></person-group>, <year>1999</year> <article-title>Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum.</article-title> <source>Nat. Cell Biol.</source> <volume>1</volume>: <fpage>423</fpage>–<lpage>430</lpage>.</citation></ref>
<ref id="bib13"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Grosshans</surname><given-names>B. L.</given-names></name><name><surname>Ortiz</surname><given-names>D.</given-names></name><name><surname>Novick</surname><given-names>P.</given-names></name></person-group>, <year>2006</year> <article-title>Rabs and their effectors: achieving specificity in membrane traffic.</article-title> <source>Proc. Natl. Acad. Sci. USA</source> <volume>103</volume>: <fpage>11821</fpage>–<lpage>11827</lpage>.</citation></ref>
<ref id="bib14"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Hama</surname><given-names>H.</given-names></name><name><surname>Tall</surname><given-names>G. G.</given-names></name><name><surname>Horazdovsky</surname><given-names>B. F.</given-names></name></person-group>, <year>1999</year> <article-title>Vps9p is a guanine nucleotide exchange factor involved in vesicle-mediated vacuolar protein transport.</article-title> <source>J. Biol. Chem.</source> <volume>274</volume>: <fpage>15284</fpage>–<lpage>15291</lpage>.</citation></ref>
<ref id="bib15"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Ho</surname><given-names>S. N.</given-names></name><name><surname>Hunt</surname><given-names>H. D.</given-names></name><name><surname>Horton</surname><given-names>R. M.</given-names></name><name><surname>Pullen</surname><given-names>J. K.</given-names></name><name><surname>Pease</surname><given-names>L. R.</given-names></name></person-group>, <year>1989</year> <article-title>Site-directed mutagenesis by overlap extension using the polymerase chain reaction.</article-title> <source>Gene</source> <volume>77</volume>: <fpage>51</fpage>–<lpage>59</lpage>.</citation></ref>
<ref id="bib16"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Houalla</surname><given-names>T.</given-names></name><name><surname>Shi</surname><given-names>L.</given-names></name><name><surname>van Meyel</surname><given-names>D. J.</given-names></name><name><surname>Rao</surname><given-names>Y.</given-names></name></person-group>, <year>2010</year> <article-title>Rab-mediated vesicular transport is required for neuronal positioning in the developing Drosophila visual system.</article-title> <source>Mol. Brain</source> <volume>3</volume>: <fpage>19</fpage>.</citation></ref>
<ref id="bib17"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Jasmin</surname><given-names>B. J.</given-names></name><name><surname>Goud</surname><given-names>B.</given-names></name><name><surname>Camus</surname><given-names>G.</given-names></name><name><surname>Cartaud</surname><given-names>J.</given-names></name></person-group>, <year>1992</year> <article-title>The low molecular weight guanosine triphosphate-binding protein Rab6p associates with distinct post-Golgi vesicles in Torpedo marmorata electrocytes.</article-title> <source>Neuroscience</source> <volume>49</volume>: <fpage>849</fpage>–<lpage>855</lpage>.</citation></ref>
<ref id="bib18"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Jordens</surname><given-names>I.</given-names></name><name><surname>Marsman</surname><given-names>M.</given-names></name><name><surname>Kuijl</surname><given-names>C.</given-names></name><name><surname>Neefjes</surname><given-names>J.</given-names></name></person-group>, <year>2005</year> <article-title>Rab proteins, connecting transport and vesicle fusion.</article-title> <source>Traffic</source> <volume>6</volume>: <fpage>1070</fpage>–<lpage>1077</lpage>.</citation></ref>
<ref id="bib19"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Kamath</surname><given-names>R. S.</given-names></name><name><surname>Ahringer</surname><given-names>J.</given-names></name></person-group>, <year>2003</year> <article-title>Genome-wide RNAi screening in Caenorhabditis elegans.</article-title> <source>Methods</source> <volume>30</volume>: <fpage>313</fpage>–<lpage>321</lpage>.</citation></ref>
<ref id="bib20"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Liao</surname><given-names>X.</given-names></name><name><surname>Du</surname><given-names>Y.</given-names></name><name><surname>Morse</surname><given-names>H. C.</given-names><suffix>3rd</suffix></name><name><surname>Jenkins</surname><given-names>N. A.</given-names></name><name><surname>Copeland</surname><given-names>N. G.</given-names></name></person-group>, <year>1997</year> <article-title>Proviral integrations at the Evi5 locus disrupt a novel 90 kDa protein with homology to the Tre2 oncogene and cell-cycle regulatory proteins.</article-title> <source>Oncogene</source> <volume>14</volume>: <fpage>1023</fpage>–<lpage>1029</lpage>.</citation></ref>
<ref id="bib21"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Martinez</surname><given-names>O.</given-names></name><name><surname>Schmidt</surname><given-names>A.</given-names></name><name><surname>Salamero</surname><given-names>J.</given-names></name><name><surname>Hoflack</surname><given-names>B.</given-names></name><name><surname>Roa</surname><given-names>M.</given-names></name><etal/></person-group>, <year>1994</year> <article-title>The small GTP-binding protein rab6 functions in intra-Golgi transport.</article-title> <source>J. Cell Biol.</source> <volume>127</volume>: <fpage>1575</fpage>–<lpage>1588</lpage>.</citation></ref>
<ref id="bib22"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Martinez</surname><given-names>O.</given-names></name><name><surname>Antony</surname><given-names>C.</given-names></name><name><surname>Pehau-Arnaudet</surname><given-names>G.</given-names></name><name><surname>Berger</surname><given-names>E. G.</given-names></name><name><surname>Salamero</surname><given-names>J.</given-names></name><etal/></person-group>, <year>1997</year> <article-title>GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum.</article-title> <source>Proc. Natl. Acad. Sci. USA</source> <volume>94</volume>: <fpage>1828</fpage>–<lpage>1833</lpage>.</citation></ref>
<ref id="bib23"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Mukhopadhyay</surname><given-names>A.</given-names></name><name><surname>Pan</surname><given-names>X.</given-names></name><name><surname>Lambright</surname><given-names>D. G.</given-names></name><name><surname>Tissenbaum</surname><given-names>H. A.</given-names></name></person-group>, <year>2007</year> <article-title>An endocytic pathway as a target of tubby for regulation of fat storage.</article-title> <source>EMBO Rep.</source> <volume>8</volume>: <fpage>931</fpage>–<lpage>938</lpage>.</citation></ref>
<ref id="bib24"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Opdam</surname><given-names>F. J.</given-names></name><name><surname>Echard</surname><given-names>A.</given-names></name><name><surname>Croes</surname><given-names>H. J.</given-names></name><name><surname>van den Hurk</surname><given-names>J. A.</given-names></name><name><surname>van de Vorstenbosch</surname><given-names>R. A.</given-names></name><etal/></person-group>, <year>2000</year> <article-title>The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus.</article-title> <source>J. Cell Sci.</source> <volume>113</volume>(<issue>Pt 15</issue>): <fpage>2725</fpage>–<lpage>2735</lpage>.</citation></ref>
<ref id="bib25"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Pamonsinlapatham</surname><given-names>P.</given-names></name><name><surname>Hadj-Slimane</surname><given-names>R.</given-names></name><name><surname>Lepelletier</surname><given-names>Y.</given-names></name><name><surname>Allain</surname><given-names>B.</given-names></name><name><surname>Toccafondi</surname><given-names>M.</given-names></name><etal/></person-group>, <year>2009</year> <article-title>P120-Ras GTPase activating protein (RasGAP): a multi-interacting protein in downstream signaling.</article-title> <source>Biochimie</source> <volume>91</volume>: <fpage>320</fpage>–<lpage>328</lpage>.</citation></ref>
<ref id="bib26"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Raizen</surname><given-names>D.</given-names></name><name><surname>Song</surname><given-names>B. M.</given-names></name><name><surname>Trojanowski</surname><given-names>N.</given-names></name><name><surname>You</surname><given-names>Y. J.</given-names></name></person-group>, <year>2012</year> <article-title>Methods for measuring pharyngeal behaviors.</article-title> <source>WormBook</source> <volume>•••</volume>: <fpage>1</fpage>–<lpage>13</lpage>.</citation></ref><!-- Q13 --><ref id="bib27"><citation citation-type="web">Ruvkun, G., and M. Finney, 2005 Antibody staining of formaldehyde-fixed animals, pp. XXX <ext-link ext-link-type="uri" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.wormatlas.org/EMmethods/Antibodystaining.htm">http://www.wormatlas.org/EMmethods/Antibodystaining.htm</ext-link> in <italic>Wormatlas</italic>, edited by D. Hall. WormBook.</citation></ref><!-- Q14 --><ref id="bib28"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Schorr</surname><given-names>M.</given-names></name><name><surname>Then</surname><given-names>A.</given-names></name><name><surname>Tahirovic</surname><given-names>S.</given-names></name><name><surname>Hug</surname><given-names>N.</given-names></name><name><surname>Mayinger</surname><given-names>P.</given-names></name></person-group>, <year>2001</year> <article-title>The phosphoinositide phosphatase Sac1p controls trafficking of the yeast Chs3p chitin synthase.</article-title> <source>Curr. Biol.</source> <volume>11</volume>: <fpage>1421</fpage>–<lpage>1426</lpage>.</citation></ref>
<ref id="bib29"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Segev</surname><given-names>N.</given-names></name><name><surname>Kahn</surname><given-names>R. A.</given-names></name></person-group>, <year>2012</year> <article-title>Kinetic and cell-based analyses of GTPase regulators.</article-title> <source>Cell. Logist.</source> <volume>2</volume>: <fpage>138</fpage>–<lpage>139</lpage>.</citation></ref>
<ref id="bib30"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Simmer</surname><given-names>F.</given-names></name><name><surname>Tijsterman</surname><given-names>M.</given-names></name><name><surname>Parrish</surname><given-names>S.</given-names></name><name><surname>Koushika</surname><given-names>S. P.</given-names></name><name><surname>Nonet</surname><given-names>M. L.</given-names></name><etal/></person-group>, <year>2002</year> <article-title>Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi.</article-title> <source>Curr. Biol.</source> <volume>12</volume>: <fpage>1317</fpage>–<lpage>1319</lpage>.</citation></ref>
<ref id="bib31"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Sinka</surname><given-names>R.</given-names></name><name><surname>Gillingham</surname><given-names>A. K.</given-names></name><name><surname>Kondylis</surname><given-names>V.</given-names></name><name><surname>Munro</surname><given-names>S.</given-names></name></person-group>, <year>2008</year> <article-title>Golgi coiled-coil proteins contain multiple binding sites for Rab family G proteins.</article-title> <source>J. Cell Biol.</source> <volume>183</volume>: <fpage>607</fpage>–<lpage>615</lpage>.</citation></ref>
<ref id="bib32"><citation citation-type="book"><person-group person-group-type="author"><name><surname>Sulston</surname><given-names>J. E.</given-names></name><name><surname>Hodgkin</surname><given-names>J. G.</given-names></name></person-group>, <year>1988</year> <article-title>Methods</article-title>, pp. <fpage>587</fpage>–<lpage>606</lpage> in <source>The Nematode Caenorhabditis elegans</source>, edited by <person-group person-group-type="editor"><name><surname>Wood</surname><given-names>W. B.</given-names></name></person-group> <publisher-name>Cold Spring Harbor Laboratory Press</publisher-name>, <publisher-loc>Cold Spring Harbor, NY</publisher-loc>.</citation></ref>
<ref id="bib33"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Tanabe</surname><given-names>K.</given-names></name><name><surname>Kon</surname><given-names>S.</given-names></name><name><surname>Natsume</surname><given-names>W.</given-names></name><name><surname>Torii</surname><given-names>T.</given-names></name><name><surname>Watanabe</surname><given-names>T.</given-names></name><etal/></person-group>, <year>2006</year> <article-title>Involvement of a novel ADP-ribosylation factor GTPase-activating protein, SMAP, in membrane trafficking: implications in cancer cell biology.</article-title> <source>Cancer Sci.</source> <volume>97</volume>: <fpage>801</fpage>–<lpage>806</lpage>.</citation></ref>
<ref id="bib34"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Uytterhoeven</surname><given-names>V.</given-names></name><name><surname>Kuenen</surname><given-names>S.</given-names></name><name><surname>Kasprowicz</surname><given-names>J.</given-names></name><name><surname>Miskiewicz</surname><given-names>K.</given-names></name><name><surname>Verstreken</surname><given-names>P.</given-names></name></person-group>, <year>2011</year> <article-title>Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins.</article-title> <source>Cell</source> <volume>145</volume>: <fpage>117</fpage>–<lpage>132</lpage>.</citation></ref>
<ref id="bib35"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>von Lieven</surname><given-names>A. F.</given-names></name></person-group>, <year>2003</year> <article-title>Functional morphology and evolutionary origin of the three-part pharynx in nematodes.</article-title> <source>Zoology (Jena)</source> <volume>106</volume>: <fpage>183</fpage>–<lpage>201</lpage>.</citation></ref>
<ref id="bib36"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Wicks</surname><given-names>S. R.</given-names></name><name><surname>Yeh</surname><given-names>R. T.</given-names></name><name><surname>Gish</surname><given-names>W. R.</given-names></name><name><surname>Waterston</surname><given-names>R. H.</given-names></name><name><surname>Plasterk</surname><given-names>R. H.</given-names></name></person-group>, <year>2001</year> <article-title>Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map.</article-title> <source>Nat. Genet.</source> <volume>28</volume>: <fpage>160</fpage>–<lpage>164</lpage>.</citation></ref>
<ref id="bib37"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>You</surname><given-names>Y. J.</given-names></name><name><surname>Kim</surname><given-names>J.</given-names></name><name><surname>Raizen</surname><given-names>D. M.</given-names></name><name><surname>Avery</surname><given-names>L.</given-names></name></person-group>, <year>2008</year> <article-title>Insulin, cGMP, and TGF-beta signals regulate food intake and quiescence in C. elegans: a model for satiety.</article-title> <source>Cell Metab.</source> <volume>7</volume>: <fpage>249</fpage>–<lpage>257</lpage>.</citation></ref>
<ref id="bib38"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Zerial</surname><given-names>M.</given-names></name><name><surname>McBride</surname><given-names>H.</given-names></name></person-group>, <year>2001</year> <article-title>Rab proteins as membrane organizers.</article-title> <source>Nat. Rev. Mol. Cell Biol.</source> <volume>2</volume>: <fpage>107</fpage>–<lpage>117</lpage>.</citation></ref>
<ref id="bib39"><citation citation-type="journal"><person-group person-group-type="author"><name><surname>Zhang</surname><given-names>D.</given-names></name><name><surname>Isack</surname><given-names>N. R.</given-names></name><name><surname>Glodowski</surname><given-names>D. R.</given-names></name><name><surname>Liu</surname><given-names>J.</given-names></name><name><surname>Chen</surname><given-names>C. C.</given-names></name><etal/></person-group>, <year>2012</year> <article-title>RAB-6.2 and the retromer regulate glutamate receptor recycling through a retrograde pathway.</article-title> <source>J. Cell Biol.</source> <volume>196</volume>: <fpage>85</fpage>–<lpage>101</lpage>.</citation></ref>
</ref-list></back>
</article>