Control of Rapsyn Stability by the CUL-3-containing E3 Ligase Complex
2009; Elsevier BV; Volume: 284; Issue: 12 Linguagem: Inglês
10.1074/jbc.m808230200
ISSN1083-351X
AutoresSeung‐Hee Nam, Kyoengwoo Min, Hye‐Jin Hwang, Hae-ock Lee, Jung Hwa Lee, Jong‐Bok Yoon, Hyun‐Sook Lee, Sungsu Park, Junho Lee,
Tópico(s)ATP Synthase and ATPases Research
ResumoRapsyn is a postsynaptic protein required for clustering of nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction. Here we report the mechanism for posttranslational control of rapsyn protein stability. We confirmed that C18H9.7-encoded RPY-1 is a rapsyn homolog in Caenorhabditis elegans by showing that human rapsyn rescued rpy-1 mutant phenotypes in nematodes, as determined by levamisole assays and micropost array behavioral assays. We found that RPY-1 was degraded in the absence of functional UNC-29, a non-α subunit of the receptor, in an allele-specific manner, but not in the absence of other receptor subunits. The cytoplasmic loop of UNC-29 was found to be critical for RPY-1 stability. Through RNA interference screening, we found that UBC-1, UBC-12, NEDD-8, and RBX-1 were required for degradation of RPY-1. We identified cullin (CUL)-3 as a component of E3 ligase and KEL-8 as the substrate adaptor of RPY-1. Mammalian rapsyn was ubiquitinated by the CUL3/KLHL8-containing E3 ligase in vitro, and the knockdown of KLHL-8, a mammalian KEL-8 homolog, inhibited rapsyn ubiquitination in vivo, implying evolutionary conservation of the rapsyn stability control machinery. kel-8 suppression and rpy-1 overexpression in C. elegans produced a phenotype similar to that of a loss-of-function mutation of rpy-1, suggesting that control of rapsyn abundance is important for proper function of the receptor. Our results suggest a link between the control of rapsyn abundance and congenital myasthenic syndromes. Rapsyn is a postsynaptic protein required for clustering of nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction. Here we report the mechanism for posttranslational control of rapsyn protein stability. We confirmed that C18H9.7-encoded RPY-1 is a rapsyn homolog in Caenorhabditis elegans by showing that human rapsyn rescued rpy-1 mutant phenotypes in nematodes, as determined by levamisole assays and micropost array behavioral assays. We found that RPY-1 was degraded in the absence of functional UNC-29, a non-α subunit of the receptor, in an allele-specific manner, but not in the absence of other receptor subunits. The cytoplasmic loop of UNC-29 was found to be critical for RPY-1 stability. Through RNA interference screening, we found that UBC-1, UBC-12, NEDD-8, and RBX-1 were required for degradation of RPY-1. We identified cullin (CUL)-3 as a component of E3 ligase and KEL-8 as the substrate adaptor of RPY-1. Mammalian rapsyn was ubiquitinated by the CUL3/KLHL8-containing E3 ligase in vitro, and the knockdown of KLHL-8, a mammalian KEL-8 homolog, inhibited rapsyn ubiquitination in vivo, implying evolutionary conservation of the rapsyn stability control machinery. kel-8 suppression and rpy-1 overexpression in C. elegans produced a phenotype similar to that of a loss-of-function mutation of rpy-1, suggesting that control of rapsyn abundance is important for proper function of the receptor. Our results suggest a link between the control of rapsyn abundance and congenital myasthenic syndromes. Neuromuscular junctions, which are composed of a presynaptic terminal, basal lamina, and a postsynaptic terminal, are highly specialized structures through which neuronal signals are transmitted to muscles (1Burden S.J. Genes Dev. 1998; 12: 133-148Crossref PubMed Scopus (170) Google Scholar). Acetylcholine (ACh) 5The abbreviations used are: Ach, acetylcholine; nAChR, nicotinic acetylcholine receptor; MUSK, muscle-specific kinase; CL, cytoplasmic loop; CMS, congenital myasthenic syndrome; RNAi, RNA interference; CUL-3, cullin 3; WT, wild type; GFP, green fluorescent protein; PI, proteasome inhibitor; HA, hemagglutinin; Ub, ubiquitin. 5The abbreviations used are: Ach, acetylcholine; nAChR, nicotinic acetylcholine receptor; MUSK, muscle-specific kinase; CL, cytoplasmic loop; CMS, congenital myasthenic syndrome; RNAi, RNA interference; CUL-3, cullin 3; WT, wild type; GFP, green fluorescent protein; PI, proteasome inhibitor; HA, hemagglutinin; Ub, ubiquitin. is a major excitatory neurotransmitter at the neuromuscular junctions in various organisms, including nematodes and vertebrates (2Rand J.B. Worm Book.doi/10.1895/wormbook.1.131.1Date: 2007Google Scholar, 3Littleton J.T. Ganetzky B. 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Nature. 1995; 377: 232-236Crossref PubMed Scopus (472) Google Scholar). In addition, it is well known that rapsyn is a causal gene for congenital myasthenic syndrome (CMS) (25Ohno K. Engel A.G. Shen X.M. Selcen D. Brengman J. Harper C.M. Tsujino A. Milone M. Am. J. Hum. Genet. 2002; 70: 875-885Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 26Dunne V. Maselli R.A. J. Hum. Genet. 2003; 48: 204-207Crossref PubMed Scopus (35) Google Scholar). Control of rapsyn abundance has been known to be important for proper AChR clustering (27LaRochelle W.J. Froehner S.C. J. Biol. Chem. 1986; 261: 5270-5274Abstract Full Text PDF PubMed Google Scholar). Inhibition of AChR clustering by down-regulation or overexpression of rapsyn implies that tight regulation is necessary for homeostatic postsynaptic AChR clustering (28Yoshihara C.M. Hall Z.W. J. Cell Biol. 1993; 122: 169-179Crossref PubMed Scopus (32) Google Scholar, 29Han H. Noakes P.G. Phillips W.D. J. Neurocytol. 1999; 28: 763-775Crossref PubMed Scopus (21) Google Scholar). Such regulation can be accomplished by transcription factors, such as kaiso and ∂-catenin (30Rodova M. Kelly K.F. VanSaun M. Daniel J.M. Werle M.J. Mol. Cell. Biol. 2004; 24: 7188-7196Crossref PubMed Scopus (88) Google Scholar). The balance between positive and negative regulation of protein degradation also affects protein levels. The abundance of postsynaptic proteins, such as PSD-95, Shank, GKAP, AKAP79/150, and glutamate receptors is controlled by ubiquitination and proteasome-mediated turnover (31Colledge M. Snyder E.M. Crozier R.A. Soderling J.A. Jin Y. Langeberg L.K. Lu H. Bear M.F. Scott J.D. Neuron. 2003; 40: 595-607Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 32Ehlers M.D. Nat. Neurosci. 2003; 6: 231-242Crossref PubMed Scopus (817) Google Scholar, 33Burbea M. Dreier L. Dittman J.S. Grunwald M.E. Kaplan J.M. 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Levamisole-sensitive nAChRs in the muscles of C. elegans are composed of LEV-1, UNC-29, UNC-38, and UNC-63, and resemble the mammalian nAChR in structure and function (2Rand J.B. Worm Book.doi/10.1895/wormbook.1.131.1Date: 2007Google Scholar). Although no clear homolog of neural agrin or MUSK has been found (37Hrus A. Lau G. Hutter H. Schenk S. Ferralli J. Brown-Luedi M. Chiquet-Ehrismann R. Canevascini S. PLoS ONE. 2007; 2: e731Crossref PubMed Scopus (16) Google Scholar, 38Forrester W.C. Dell M. Perens E. Garriga G. Nature. 1999; 400: 881-885Crossref PubMed Scopus (136) Google Scholar), rpy-1 is a putative homolog of rapsyn. We first show that rpy-1 of C. elegans is a structural and functional homolog of mammalian rapsyn. Through levamisole resistance assays and recently developed post-microassays, we show that rpy-1 is required for AChR-mediated muscle function. We then elucidate a posttranslational mechanism for rapsyn stability control. We show that a functional reporter of muscular C. elegans RPY-1 is degraded by the ubiquitin-proteasome pathway in the absence of a non-α nAChR subunit, UNC-29. Through RNAi screening, we identified factors that are required for degradation of rapsyn. In particular, we identified cullin (CUL)-3 as a component of the E3 ligase complex and KEL-8 as the adaptor between CUL-3 and rapsyn; both proteins were conserved in mammals. We then show that the CUL-3-containing E3 ligase complex degraded rapsyn in vitro, and that knockdown of KLHL8, the mammalian KEL-8 homolog, inhibited ubiquitination of rapsyn in mammalian cells. Finally, we show that inhibition of RPY-1 degradation produced a phenotype that was similar to that seen with a loss-of-function mutation of the rpy-1 gene. We conclude with a discussion of the potential link between rapsyn abundance and CMS. Nematode Experiments-The C. elegans Bristol strain N2 was used as the WT strain (39Brenner S. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). The N2 and mutant animals were grown at 20 °C on NGM plates. The unc-29 (e193) and rpy-1 (ok145) mutant strains were generous gifts from Caenorhabditis Genetics Center (Minneapolis, MN). The rpy-1 (ok145) strain was backcrossed at least four times before being used in the experiments. Assays for rpy-1 Mutant Phenotypes-The effect of levamisole on muscle contraction and egg laying was examined. For the studies of muscle hypercontraction, adult animals were placed on 55-mm Petri dishes containing 50 μm levamisole. After 4 h, the animals were observed for hypercontraction, and photographs were taken using a stereomicroscope. To measure the sensitivity of egg laying to levamisole, levamisole-treated animals were examined for the numbers of eggs that they laid over 3 h in the presence and absence of levamisole. Microfabrication of Post-microarrays-We used photolithography (40Xia Y. Whitesides G.M. Angew. Chem. Int. Ed. 1998; 37: 550-575Crossref PubMed Google Scholar) to prepare masters containing arrays of posts of SU-8 photoresist (MicroChem, Newton, MA) with 300-μm diameter, 100-μm height, and a distance of 150 μm between the posts (41Park S. Hwang H. Nam S.W. Martinez F. Austin R.H. Ryu W.S. PLoS ONE. 2008; 3e2550Crossref PubMed Scopus (107) Google Scholar). The actual masters that were used for molding of the agar (positives) were replicas (negatives) of these photolithographically prepared masters in Sylgard 184 polydimethylsiloxane (Dow-Corning, Midland, MI). Bacto-agar (4%) in NGM buffer (50 mm NaCl, 1 mm CaCl2, 1 mm MgSO4, and 25 mm KH2PO4) was poured at 70 °C onto the polydimethylsiloxane mold in a Petri dish and left to cool at room temperature. Air bubbles trapped in the small features of the mold were released by scraping the surface of the agar with the edge of a razor blade. After 1 h, the agar was cut and lifted from the mold and placed structure-side up onto a Petri dish. Just before use, the agar grid structure was filled with NGM buffer. L4 and young adult worms were selected from growth plates with a platinum worm pick, washed in NGM buffer, and placed into the fluid-filled agar microstructure. Worms were confined to the structure by the surface tension of the liquid layer. NGM buffer was added to the structure to offset evaporation. Data Collection from Post-microarray Experiments-Movies of worms moving in the microstructures were taken using an SMZ 1500 stereomicroscope (Nikon Instruments, Japan) with a CCD camera (DS-Fi1; Nikon Instruments, Japan). The motion of the worms was analyzed using ImageJ (National Health Institutes, Bethesda, MD). Construction of the Reporter Genes and rpy-1 Derivatives-For examination of rpy-1 expression patterns, we constructed a reporter gene that encoded the full-length C. elegans rpy-1 gene fused to GFP and Discosoma sp. red reporter genes. To ensure avoidance of mosaicism, which is often caused by extrachromosomal arrays, we observed GFP patterns in at least five independent transgenic lines. To examine the role of the RING domain of RPY-1, we created a construct that contained a mutated RING domain in which two His histidine residues were changed to glutamines by an overlap PCR method using the indicated primer sets. PCR was first performed using primer sets 43-5/43-7 and 43-6/43-8; a second round of PCR was performed with the 43-5 and 43-8 primers and the initial PCR products as template. This mutant rpy-1 gene was then subcloned into the pPD95.75 (pJL525) plasmid. The presence of the mutations in pJL525 was confirmed by sequencing. To examine whether the human RAPSN gene could rescue the rpy-1 (ok145) mutation, we designed constructs in which the cDNAs of the representative RAPSN isoform (NM_032645) were fused to the GFP reporter gene under the control of a muscle-specific promoter, myo-3. As a positive control, we designed a construct that contained the C. elegans rpy-1 cDNA fused to the GFP reporter gene. Each cDNA was amplified by PCR using the primer sets 43-132/43-133 (for C. elegans rpy-1 cDNA) and hRAPSN-1/hRAPSN-2 (for human RAPSN cDNA) and subcloned into the Pmyo-3::gfp vector (modified pPD114.108). The constructs were confirmed by sequencing. The sequences of the primers are listed in supplemental Table S3. All reporter vectors were a gift from Dr. Andrew Fire (Stanford University, CA). The Construction of unc-29 Reporter Genes and Chimeric Derivatives-To examine the relationship between RPY-1 and UNC-29, we constructed the full-length C. elegans unc-29 gene by PCR with primer sets unc-29-9/unc-29-10. To test the role of the cytoplasmic loop region of UNC-29, we created a construct with this region deleted (unc-29 ΔCL), as well as constructs with deletions of transmembrane domain 1, 2 (unc-29 ΔM1, 2), by PCR using the primer sets unc-29-29/unc-29-37, unc-29-38/unc-29-33, unc-29-9/unc-29-13, and unc-29-14/unc-29-10, respectively. We made unc-29 chimeric constructs in which each cytoplasmic loop of UNC-38, UNC-63, and LEV-1 was independently fused to the unc-29 ΔCL construct. These constructs were confirmed by sequencing. Germ Line Transformation-Germ line transformations were carried out using standard protocols (42Mello C.C. Kramer J.M. Stinchcomb D. Ambros V. EMBO J. 1991; 10: 3959-3970Crossref PubMed Scopus (2426) Google Scholar). The coinjection marker used was the dominant rol-6 DNA pRF4 and sur-5 gene. At least three independent lines were established and used for experiments. Proteasome Inhibitor Treatment and Analysis of RPY-1 in C. elegans-To test whether RPY-1 is degraded by the proteasome pathway, we treated the animals with a commonly used proteasome inhibitor mixture (PI) containing MG132 and lactacystin (Cayman Chemical, Ann Arbor, MI). The PI experiments were performed as previously described, with some modifications (43Bush K.T. Goldberg A.L. Nigam S.K. J. Biol. Chem. 1997; 272: 9086-9092Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 44Courbard J.R. Fiore F. Adelaide J. Borg J.P. Birnbaum D. Ollendorff V. J. Biol. Chem. 2002; 277: 45267-45275Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 45Rocca A. Lamaze C. Subtil A. Dautry-Varsat A. Mol. Biol. Cell. 2001; 12: 1293-1301Crossref PubMed Scopus (98) Google Scholar). Briefly, the wild-type and unc-29 mutant animals containing the rpy-1::gfp reporter construct were grown in liquid culture (wormbook.org/chapters/www_strainmaintain), purified by sucrose flotation, and collected into a 15-ml tube. PI was added at a final concentration of 50 μm, after which the animals were incubated for 6 h at 20 °C on a Nutator mixer before being washed with M9. To extract protein from PI-treated animals, collected animals were frozen at -80 °C and then ground in liquid nitrogen and resuspended in lysis buffer (50 mm HEPES/KOH (pH 7.6), 1 mm EDTA, 150 mm NaCl, 0.1% Nonidet P-40, 10% glycerol, 1 mm dithiothreitol, 1 mm PMSF, protease inhibitor mixture (Calbiochem, Darmstadt, Germany), 10 mm N-ethylmaleimide (Sigma)). This lysis buffer was mixed as described (wormbook.org/chapters/www_intromethodscellbiology), with some modifications. The extract was centrifuged at 18,000 × g for 1 h and pre-cleared with protein G-agarose (Upstate, Charlottesville, VA) for 1-2 h at 4 °C and then incubated with anti-GFP tagagarose (MBL, Nagoya, Japan) overnight at 4 °C. After washing with the wash buffer (50 mm HEPES/KOH, pH 7.6, 1 mm EDTA, 150 mm NaCl, 0.1% Nonidet P-40, 1 mm dithiothreitol, 1 mm PMSF, protease inhibitor mixture), bound proteins were eluted in the reducing SDS-PAGE sample buffer. Samples were then centrifuged briefly and the supernatant loaded onto a 6% SDS-PAGE gel. The proteins were transferred onto a nitrocellulose transfer membrane. The blots were first incubated with primary antibodies for 1 h and then with the secondary antibodies (horseradish peroxidase-conjugated anti-rabbit antibodies, diluted 1:5000; Amersham Biosciences) for 40 min in TBST. The RPY-1::GFP proteins were detected using an anti-RPY-1 antibody. An anti-actin antibody was used as a loading control. RNAi Experiments-To identify genes responsible for rapsyn degradation by the proteasome pathway, we performed RNAi experiments. We investigated all available UBC genes by using the previously described feeding method (46Jones D. Crowe E. Stevens T.A. Candido E.P. Genome Biol. 2002; 3RESEARCH0002PubMed Google Scholar). We used an RNAi library purchased from MRC (Cambridge, UK). In addition, we created the ubc-3, cul-2, and cul-3 constructs for RNAi because these genes were not covered by the library. The cul-2, cul-3, and ubc-3 cDNAs were amplified by reverse transcriptase-PCR using primer sets cul-2-1/cul-2-2, cul-3-1/cul-3-2, and ubc-3-1/ubc-3-2, respectively, and subcloned into an L4440 feeding vector (pPD129.36). These constructs were confirmed by sequencing. We transformed the obtained constructs into HT115 (DE3) bacterial cells and performed RNAi feeding. For genes whose RNAi-mediated knockdown caused embryonic lethality, we fed L1 larvae with RNAi bacteria and observed the phenotypes when they reached adulthood. The sequences of the primers are listed in supplemental Table S3. Yeast Two-hybrid Assay-To determine whether rapsyn, KEL-8, and CUL-3 interact with one another, we performed yeast two-hybrid assays. The bait plasmids, cul-1::pAS2-1, cul-3::pAS2-1, WT rpy-1::pAS2-1, and mRING rpy-1:: pAS2-1, were constructed by subcloning cDNAs amplified by PCR using the primer sets cul-1-1/cul-1-2, cul-3-3/cul-3-2, and 43-114/43-18, respectively, into the pAS2-1 vector. We designed a prey plasmid (kel-8::pACTII) by subcloning kel-8 cDNA, which was amplified by reverse transcriptase-PCR using the primer set kel-8-10/kel-11, into the pACTII vector. These constructs were confirmed by sequencing. Each construct was transformed into yeast strain cells (Clontech, Mountain View, CA), which were then cultured on Trp-Leu- or Trp- Leu-His- plates. The sequences of the primers are listed in supplemental Table S3. Antibody Production-The rpy-1 cDNA fragment encoding amino acids 453-597 of the RPY-1 protein was amplified by PCR using primer set 43-152/153 and then inserted into pRSETA using BamHI and HindIII (Invitrogen). This recombinant His6::RPY-1 protein was expressed in BL21 Escherichia coli and purified according to the QIAexpressionist protocol (Qiagen). Antibody was produced by PEPTRON Inc. (Daejeon, Korea). Cell Culture Experiments-We purchased human rapsyn cDNA from Invitrogen. Full-length cDNAs of KLHL8 (KIAA1328) and KEAP1 (KIAA0132) were generously provided by the Kazusa DNA Research Institute. To construct plasmids for the expression of the N-terminal FLAG-, MYC-, or HA-tagged proteins, cDNAs were amplified by PCR with the appropriate primers and ligated into the pcDNA3.1(+) vector (Invitrogen). For expression of C-terminal FLAG-tagged proteins, the PCR products, which were generated with appropriate primers, were ligated into the p3XFLAG-CMV-14 vector (Sigma). The constructs for the expression of CUL3 and RBX1 were previously described (47Min K.W. Hwang J.W. Lee J.S. Park Y. Tamura T.A. Yoon J.B. J. Biol. Chem. 2003; 278: 15905-15910Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). All protein processing was performed in accordance with standard protocols. ts20 Cell Experiments-ts20 cells were transfected with MYC::RPY-1 at 34 °C. 24 h later, cells were split and maintained at either 34 or 40 °C for an additional 12 h, and then treated with cycloheximide (20 μg/ml). Cells were harvested at the indicated time points and Western blot analysis was performed for anti-MYC and anti-actin monoclonal antibody. The relative levels of MYC::RPY-1 and actin were obtained by densitometry using Multi Gause version 3.0 (Fujifilm). MYC::RPY-1/actin levels were arbitrarily set as 1 for the 0-h samples for each experimental group. In Vitro Ubiquitination-The constructs expressing HA-CUL3, HA-RBX1, and MYC-KLHL8 (or MYC-KEAP1) were co-transfected into a HeLa cell line. MYC-KLHL8 is a chimeric gene that contains the BTB domain of KEAP1 and the Kelch domains of KLHL8. This was done because the full-length KLHL8 protein was difficult to obtain as a soluble protein. The HA-CUL3/RBX1/MYC-KLHL8 complex (E3) was purified using HA-conjugated agarose beads (Sigma). Recombinant FLAG-RAPSYN was expressed in SF21 cells and purified by Newgex Inc. (Seoul, Korea). The Ub ligation reaction mixtures were incubated at 37 °C for 1 h and proteins were analyzed by Western blotting. Short Hairpin RNA Experiments-The short hairpin RNA expression lentiviral vectors used for targeting of the KLHL8 gene and a control vector were purchased from Macrogen Inc. (Seoul, Korea). 3T3 cells were infected with 35 ml of 10-fold concentrated virus for 8 h and then incubated for a further 48 h. Stable lines expressing short hairpin-red fluorescent protein were selected and used for further experiments. RPY-1 Is a Homolog of Rapsyn in C. elegans-The C. elegans RPY-1 protein, a putative rapsyn homolog encoded by C18H9.7, contains eight TPR domains and a RING domain. These domains are conserved in rapsyn proteins of other species (supplemental Fig. S1, A and C). We first examined the phenotype of a deletion mutation in the rpy-1 gene. The examined mutation was rpy-1 (ok145), which contains a deletion of exons 4 through 10 that results in a severely truncated protein (supplemental Fig. S1B). Similar to animals that carry mutations in nAChR genes, rpy-1 (ok145) mutant animals were resistant to levamisole, an agonist of nAChRs (48Lewis J.A. Wu C.H. Berg H. Levine J.H. Genetics. 1980; 95: 905-928Crossref PubMed Google Scholar, 49Lewis J.A. Wu C.H. Levine J.H. Berg H. Neuroscience. 1980; 5: 967-989Crossref PubMed Scopus (191) Google Scholar); they did not hypercontract or show increased egg laying after treatment (Fig. 1, A-C). Although rpy-1 mutant animals clearly showed levamisole resistance typical of nAChR mutations, they did not show significant defective motility on media plates in the absence of levamisole treatment (Fig. 1D). For increased sensitivity, we applied the post-microarray assay, which is a recently developed sensitive behavioral assay (41Park S. Hwang H. Nam S.W. Martinez F. Austin R.H. Ryu W.S. PLoS ONE. 2008; 3e2550Crossref PubMed Scopus (107) Google Scholar). With this method, we were able to detect a significant motility defect in rpy-1 (ok145) mutant animals (Fig. 1D). We then tested whether a GFP reporter gene harboring the rpy-1 gene (pJL524) could rescue rpy-1 mutation phenotypic characteristics. Muscle hypercontraction and increased egg laying caused by this mutation were rescued by injection of the rpy-1::gfp construct at a concentration of 75 μg/ml (Fig. 1, A-C; supplemental Table S1). The motility defect detected by the post-microassay was also rescued (Fig. 1D, supplemental Movies S1-S7). We then found that the expression of rpy-1 in muscles using the myo-3 promoter rescued the rpy-1 mutant phenotypes (Fig. 1, A-C; supplemental Table S1). Furthermore, we found that expression of the human rapsyn gene in the muscles of the nematode rpy-1 mutant rescued levamisole resistance (Fig. 1, A-C, supplemental Table S2) and motility defects (Fig. 1D, supplemental Movie S7). Although it is formally possible that RPY-1 and mammalian rapsyn may have distinct functions, the most probable interpretation of the data presented above is that RPY-1 is the functional homolog of rapsyn in the nematode. RPY-1 Is Expressed in Muscles and Neurons-Next, we examined the expression pattern of rpy-1 using a reporter gene construct that contained the putative promoter region and full-length coding region of rpy-1. RPY-1 was first detected at 260-270 min in embryonic development (data not shown) and persisted throughout development (Fig. 2A). In adult animals, RPY-1 was expressed in body-wall muscles, head muscles, and sex-specific muscles of hermaphrodites. The rpy-1::gfp reporter gene was also expressed in neurons (Fig. 2A). Muscular RPY-1 Is Destabilized in the Absence of UNC-29, but Not Other nAChR Subunits-Knowing that rpy-1 is a rapsyn homolog, we started to investigate the unsolved question of whether and how RPY-1 stability is controlled. Proteins that ar
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