Control of rapsyn stability by the CUL-3-containing E3 ligase complex.
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.
Nam
Seunghee
S
Research Center for Cellulomics, Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National University, 151-742 Seoul, Korea.
Min
Kyoengwoo
K
Hwang
Hyejin
H
Lee
Hae-Ock
HO
Lee
Jung Hwa
JH
Yoon
Jongbok
J
Lee
Hyunsook
H
Park
Sungsu
S
Lee
Junho
J
D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D006801:Humans; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D020294:Myasthenic Syndromes, Congenital / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology*
D029742:Caenorhabditis elegans Proteins; D044842:Cullin Proteins; C510975:Kel-8 protein, C elegans; D009124:Muscle Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011978:Receptors, Nicotinic; C529480:UNC-29 protein, C elegans; C051609:peripheral membrane protein 43K; D044763:Ubiquitin-Conjugating Enzymes; C489359:ubc-1 protein, C elegans; C510976:CUL-3 protein, C elegans