Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility

摘要

Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations. Author Summary Most polypeptides by necessity must fold into three-dimensional structures in order to become functional proteins. Misfolding, either during or subsequent to initial folding, can result in toxic protein aggregation. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. One cause of misfolding is the presence of missense mutations, which account for over half of all the reported mutations in the Human Gene Mutation Database. Here we establish a model cytosolic protein substrate whose stability is temperature dependent. We then perform a flow cytometry based screen to identify factors that promote the degradation of our model substrate. We identified the E3 ubiquitin ligase Ubr1 and the prefoldin chaperone complex subunit Gim3. Prefoldin forms a “jellyfish-like” structure and aids in nascent protein folding and prevents protein aggregation. We show that prefoldin promotes protein degradation by maintaining substrate solubility. Our work adds to that of others highlighting the importance of the prefoldin complex in preventing potentially toxic protein aggregation.