A Pro-Cathepsin L Mutant Is a Luminal Substrate for Endoplasmic-Reticulum-Associated Degradation in C. elegans

Mark T Miedel,Kate E Stephen,Nathan J Graf,Olivia S Long,Gary A Silverman,Stephen C Pak,David H Perlmutter,Cliff J Luke,Todd Lamitina

doi:10.1371/journal.pone.0040145

Abstract

Endoplasmic-reticulum associated degradation (ERAD) is a major cellular misfolded protein disposal pathway that is well conserved from yeast to mammals. In yeast, a mutant of carboxypeptidase Y (CPY*) was found to be a luminal ER substrate and has served as a useful marker to help identify modifiers of the ERAD pathway. Due to its ease of genetic manipulation and the ability to conduct a genome wide screen for modifiers of molecular pathways, C. elegans has become one of the preferred metazoans for studying cell biological processes, such as ERAD. However, a marker of ERAD activity comparable to CPY* has not been developed for this model system. We describe a mutant of pro-cathepsin L fused to YFP that no longer targets to the lysosome, but is efficiently eliminated by the ERAD pathway. Using this mutant pro-cathepsin L, we found that components of the mammalian ERAD system that participate in the degradation of ER luminal substrates were conserved in C. elegans. This transgenic line will facilitate high-throughput genetic or pharmacological screens for ERAD modifiers using widefield epifluorescence microscopy.

Highlights

Biological pathways governing protein transcription, synthesis, folding, modification, trafficking and degradation maintain cellular protein homeostasis [1]
Its usefulness as a model for studying ERAD has been hindered by the lack of well-characterized luminal substrates that permit the process to be tracked biochemically, or microscopically in real-time
We determined whether mutated CPL-1 was a luminal ERAD substrate in C. elegans

Summary

Introduction

Biological pathways governing protein transcription, synthesis, folding, modification, trafficking and degradation maintain cellular protein homeostasis (proteostasis) [1]. Protein degradative pathways are important, as they are the definitive step for removing toxic accumulations of misfolded or aggregated proteins generated by mutations or environmental stressors. Failure to eliminate these proteins can trigger cellular dysfunction or death that is characteristic of several neurodegenerative disorders, the serpinopathies and some inborn errors of metabolism [1,2,3]. While many of the molecular components of yeast ERAD are conserved in metazoans, significant differences exist [10,11,12]. The C. elegans system has not been fully exploited due to the absence of well-defined luminal substrates that permit the visual, biochemical or genetic assessment of putative ERAD modifier genes

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