Control of rapsyn stability by the CUL-3-containing E3 ligase complex

Abstract

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-alpha 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.

FIGURE 1.

Analysis of rpy-1 mutant phenotype in C. elegans. A, the RPY-1::GFP reporter was functional in vivo. The rpy-1 mutant phenotypes were rescued by the expression of the rpy-1::gfp reporter or the human rapsyn gene under the control of both the rpy-1 promoter and muscle-specific promoter (Pmyo-3) in the levamisole assay. The upper panels show muscle hypercontraction, and the bottom panels show enhanced egg laying. The scale bars represent 100 (top row) and 50 μm (bottom row), respectively. Anterior is on the left and dorsal at the top. The arrows indicate the locations of the vulvae, and the arrowheads indicate two-cell stage eggs. B and C, the graphs summarize the results of the levamisole assay. B represents hypercontraction and C represents the egg-laying defect observed in A. D, the rescue of the rpy-1 mutant phenotypes by the rpy-1 expression using the rpy-1 promoter and the myo-3 promoter as analyzed by the micropost assay. The left graph represents post-array assay results and the right panels show the movements of the strains. ^*, p < 0.001. The numbers indicate individual worms. The scale bar represents 500 μm.

FIGURE 2.

The RPY-1 protein was unstable in the absence of UNC-29. A, the RPY-1 protein is expressed in muscles and neurons. The left panels show a 3-fold stage embryo; the middle and right panels are adults. The scale bars indicate 50 (middle image) and 100 μm (right panel). B, expression patterns of the RPY-1::GFP in N2, unc-29 RNAi, and mutant backgrounds. The arrows indicate the ventral nerve cord (VC). The scale bar represents 50 μm. C, GFP expression patterns of the RPY-1 protein in other nAChR subunit genes, unc-38, unc-63, and lev-1, RNAi, and mutant backgrounds, as indicated. The scale bar represents 50 μm. D, RPY-1 protein stability was dependent on the presence of the UNC-29 protein but not on the other nAChR subunits, UNC-38, UNC-63, or LEV-1 proteins. The arrows indicate the VC. The scale bar represents 50 μm. E, specific requirement of the cytoplasmic loop domain of UNC-29 for RPY-1 stability. The upper left panels show the effect of the deletion of two transmembrane domains of UNC-29 on the stability of RPY-1. The other panels show the effects of chimeric UNC-29 proteins, each of which contained the CL domain of UNC-38, UNC-63, or LEV-1, on the stability of RPY-1. The arrows indicate the VC. The scale bar represents 50 μm.

FIGURE 3.

RPY-1 is degraded by the ubiquitin-proteasome pathway. A, the RPY-1::GFP protein was stabilized in vivo by treatment with a proteasome inhibitor mixture (PI). The RPY-1::GFP protein is in the unc-29 background. The arrows indicate the ventral nerve cord. The scale bar represents 100 μm. B, PI treatment blocked RPY-1 protein degradation in the unc-29 mutant animals. A small amount of the RPY-1::GFP protein was present without PI treatment, probably due to its persistent expression in neurons (lanes 4 and 5). When treated with the PI, polyubiquitinated RPY-1::GFP proteins were visible (lane 6). The RPY-1::GFP protein was detected by Western blot analysis with anti-RPY-1 antibody. Western blot analysis using an anti-actin antibody was used as a loading control. C, RPY-1 was ubiquitinated in cultured cells. 293T cells were cotransfected with MYC-tagged WT RPY-1 and HA-tagged Ub. After 48 h, the cells were treated with 20 μm MG132 for 6 h (lane 5) or left untreated. RPY-1-Ub complexes were analyzed by immunoprecipitation (IP) with an anti-MYC antibody (9E10) followed by immunoblotting (IB) using monoclonal anti-HA antibodies. The same blot was reprobed with 9E10 to normalize RPY-1 immunoprecipitation. D, RPY-1 degradation depends on the ubiquitin pathway. The upper panels show representative results. The bottom graphs summarize the results of ts20 cell experiments. Each bar represents average value ± S.E. from 3 independent experiments. Actin was used as a loading control. CHX, cycloheximide; DMSO, dimethyl sulfoxide.

FIGURE 4.

The identification of factors involved in RPY-1 degradation. A and B, identification of UBC-1, UBC-12, NED-8, RBX-1, and CUL-3. The graph (A) summarizes the results of RNAi assays with genes involved in the degradation of RPY-1. The y axis represents the percentage of animals with stabilized RPY-1::GFP in the muscles after each RNAi experiment. B, the panels show representative effects of RNAi on RPY-1 stability in the wild-type background. The arrowheads indicate stable RPY-1::GFP expression in the muscles. The scale bar represents 100 μm. C and D, KEL-8 is the substrate adaptor for RPY-1 ubiquitination. C, the physical interaction of KEL-8 with rapsyn and CUL-3 as determined by yeast two-hybrid assays. KEL-8 interacts with CUL-3 and RPY-1. KEL-8 did not interact with RPY-1[mRING, H395, 398Q-RING]::GFP, which carries two His to Gln mutations in the RING domain. D, the degradation of RPY-1 required KEL-8 and the RPY-1 RING domain. The upper panels show the expression of RPY-1::GFP in wild-type animals with or without kel-8 RNAi. The arrowhead indicates the expression of RPY-1 GFP in the muscles. The scale bar represents 100 μm. The bottom panels show unc-29 (e193) animals carrying either a wild-type rpy-1 transgene (left panel) or the rpy-1[mRING]::gfp reporter (right panel). The arrowhead indicates the muscles. The scale bar represents 25 μm. E, the RING domain was required for the function of RPY-1. The left panel shows that muscle hypercontraction was absent after levamisole treatment; the right panel shows that enhanced egg laying was absent after levamisole treatment. The scale bars represent 100 (left panel) and 25 μm (right panel).

FIGURE 5.

The in vitro ubiquitination of mammalian rapsyn by CUL3-containing E3 ligase and the effect of KLHL8 knockdown on the ubiquitination of rapsyn. A, the physical interaction between KLHL8, CUL3, and rapsyn. 293T cells transiently expressing epitope-tagged KEAP1, KLHL8 (KLHL8 F.L.), the BTB domain of KLHL8 (KLHL8 N332 (1-332 amino acids), and KLHL8 N178 (1-178 amino acids)), and the Kelch domain of KLHL8 (KLHL8 C462 (159-620 amino acids) or KLHL8 C309 (312-620 amino acids)) were used. The BTB domain alone can interact with CUL3 (lanes 3 and 4, middle panel); the Kelch domain alone can interact with rapsyn (lanes 5 and 6, bottom panel). B, the in vitro ubiquitination of rapsyn by CUL3-containing E3 ligase. Purified recombinant human rapsyn was incubated with immunoprecipitated E3 ligase complexes containing the BTB domain of KLHL8 alone (lane 2), full-length KEAP1 (lane 3), or full-length KLHL8 (lane 4) in the presence of purified E1, E2, and Ub proteins. The reaction mixture in lane 1 did not contain the E3 ligase complex, and lane 5 contained the purified rapsyn protein alone. The asterisk indicates a nonspecific product. C, the knockdown of KLHL8 inhibited the ubiquitination of rapsyn in 3T3 cells. IB, immunoblot; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 6.

Suppression of kel-8 and overexpression of RPY-1 cause phenotypes similar to that of the rpy-1 mutation. A and B, muscle hypercontraction (A) and enhanced egg laying (B) in the kel-8 RNAi and RPY-1::GFP-overexpressing animals. N2 and rpy-1 (ok145) animals were used as controls. C shows representative images of the phenotypes from B and C. The upper panels show muscle hypercontraction, and the bottom panels show enhanced egg laying after levamisole treatment. The arrows indicate the locations of the vulvae. The arrowhead indicates a two-cell stage egg. The scale bars represent 100 (top row) and 25 μm (bottom row).