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

Seunghee Nam, Kyoengwoo Min, Hyejin Hwang, Hae Ock Lee, Jung Hwa Lee, Jongbok Yoon, Hyunsook Lee, Sungsu Park, Junho Lee

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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-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.

Original languageEnglish
Pages (from-to)8195-8206
Number of pages12
JournalJournal of Biological Chemistry
Volume284
Issue number12
DOIs
Publication statusPublished - 2009 Mar 20

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Nam, S., Min, K., Hwang, H., Lee, H. O., Lee, J. H., Yoon, J., Lee, H., Park, S., & Lee, J. (2009). Control of rapsyn stability by the CUL-3-containing E3 ligase complex. Journal of Biological Chemistry, 284(12), 8195-8206. https://doi.org/10.1074/jbc.M808230200