Abstract
Damaged and misfolded proteins that are no longer functional in the cell need to be eliminated. Failure to do so might lead to their accumulation and aggregation, a hallmark of many neurodegenerative diseases. Protein quality control pathways play a major role in the degradation of these proteins, which is mediated mainly by the ubiquitin proteasome system. Despite significant focus on identifying ubiquitin ligases involved in these pathways, along with their substrates, a systems-level understanding of these pathways has been lacking. For instance, as misfolded proteins are rapidly ubiquitylated, unconjugated ubiquitin is rapidly depleted from the cell upon misfolding stress; yet it is unknown whether certain targets compete more efficiently to be ubiquitylated. Using a system-wide approach, we applied statistical and computational methods to identify characteristics enriched among proteins that are further ubiquitylated after heat shock. We discovered that distinct populations of structured and, surprisingly, intrinsically disordered proteins are prone to ubiquitylation. Proteomic analysis revealed that abundant and highly structured proteins constitute the bulk of proteins in the low-solubility fraction after heat shock, but only a portion is ubiquitylated. In contrast, ubiquitylated, intrinsically disordered proteins are enriched in the low-solubility fraction after heat shock. These proteins have a very low abundance in the cell, are rarely encoded by essential genes, and are enriched in binding motifs. In additional experiments, we confirmed that several of the identified intrinsically disordered proteins were ubiquitylated after heat shock and demonstrated for two of them that their disordered regions are important for ubiquitylation after heat shock. We propose that intrinsically disordered regions may be recognized by the protein quality control machinery and thereby facilitate the ubiquitylation of proteins after heat shock.
Highlights
From the ‡Department of Biochemistry & Molecular Biology and Centre for High-Throughput Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
We previously identified which proteins in Saccharomyces cerevisiae were ubiquitylated or further ubiquitylated upon Heat shock (HS) using quantitative mass spectrometry (Fig. 1A) [11]
HS Ubiquitylated Proteins Are Enriched for Intrinsically Disordered Proteins—In order to understand how the ubiquitinproteasome system rations unconjugated ubiquitin among different misfolded proteins, we aimed to identify characteristics that are enriched among proteins that are ubiquitylated after misfolding stress
Summary
From the ‡Department of Biochemistry & Molecular Biology and Centre for High-Throughput Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada. Because of the importance of maintaining protein homeostasis, numerous ubiquitin ligases in different cellular compartments function in protein quality control pathways to target misfolded or damaged proteins for degradation via the proteasome. The conserved Hrd ubiquitin ligase is involved in the endoplasmic-reticulum-associated degradation pathway that targets endoplasmic reticulum proteins for retro-translocation to the cytoplasm and proteasome degradation [7]. Many studies investigating degradation protein quality control have employed model substrates (e.g. mutated proteins that misfold) to reveal which components are involved in a given quality control machinery. These approaches do not typically reveal the whole spectrum of substrates for Molecular & Cellular Proteomics 12.9. We discovered an unexpected susceptibility of intrinsically disordered proteins for ubiquitylation after misfolding stress
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