Cytosolic Quality Control of Mislocalized Proteins Requires RNF126 Recruitment to Bag6

摘要

class="head no_bottom_margin" id="sec1title">IntroductionProtein quality control is essential for cellular homeostasis (). Failure to promptly recognize and degrade defective or superfluous proteins leads to their accumulation. This accumulation of aberrant proteins can have several consequences, including protein aggregation, inappropriate interactions, damage to cellular membranes, induction of stress responses, and many others. Each of these consequences can be detrimental at both the cellular and organismal level, and is causative of numerous human diseases (). Thus, deciphering the molecular basis of protein misfolding diseases requires an understanding of the cell’s various protein quality control pathways.Proteins in most, if not all, cellular compartments are subjected to quality control. The cytosol is the largest cellular compartment and houses the most diverse proteome. The range of proteins it handles, and the myriad ways in which they can be defective, requires a highly flexible quality control system. This challenging task is not handled by a single pathway, but by multiple parallel pathways dedicated to different types of aberrant proteins (). The pathways range widely, including ribosome-associated systems for partially synthesized proteins (), chaperone-assisted pathways of degradation (), and autophagy-based pathways for large multiprotein aggregates (). A current goal in the quality control field is to define the full complement of pathways, the components that comprise each pathway, and their respective client specificities.Earlier work has shown that one cellular process requiring quality control is protein translocation into organelles. For example, import into the endoplasmic reticulum is not perfectly efficient, resulting inevitably in at least some polypeptides mislocalized to the cytosol (). This mislocalization can be enhanced during ER stress (), by rare mutations in signal peptides (), and possibly by mutant translocation machinery (). Importantly, protein mislocalization can lead to disease in both animal models and humans (). Thus, cells are likely to have evolved mechanisms to deal with mislocalized proteins (MLPs) to avoid their accumulation.Previous studies have shown that ∼10%–20% of mammalian prion protein (PrP) is mislocalized to the cytosol, and this population of PrP is rapidly degraded via the ubiquitin-proteasome system (). Using mislocalized PrP as a model substrate, an in vitro system was used to search for protein factors involved in its ubiquitination (). A combination of crosslinking and functional analyses led to the identification of the heterotrimeric Bag6 complex as a factor that interacts specifically with the unprocessed hydrophobic domains of PrP and other MLPs (). Bag6 complex was necessary for maximal MLP ubiquitination in vitro, and for efficient degradation of mislocalized PrP in cultured cells. Thus, the Bag6 complex was proposed to be a component of the quality control pathway for MLPs.Parallel studies widened the scope of Bag6 in protein quality control. In one set of studies, Bag6 was found to interact with proteins dislocated from the endoplasmic reticulum (href="#bib7" rid="bib7 bib47" class=" bibr popnode">Claessen and Ploegh, 2011; Wang et al., 2011). In this case, Bag6 appears to interact after substrate ubiquitination, and is needed to maintain client solubility until delivery to the proteasome. Other studies found Bag6 associated with newly synthesized polyubiquitinated proteins that were proposed to generate peptides for MHC class I presentation (href="#bib51" rid="bib51" class=" bibr popnode">Minami et al., 2010). Collectively, these findings implicate Bag6 in multiple quality control pathways (href="#bib26" rid="bib26" class=" bibr popnode">Kawahara et al., 2013), although its exact role in any of them remains poorly understood.The heterotrimeric Bag6 complex is composed of Bag6, TRC35, and Ubl4A (href="#bib33" rid="bib33" class=" bibr popnode">Mariappan et al., 2010). In addition to mediating ubiquitination, the Bag6 complex was shown to be involved in the capture and loading of tail-anchored (TA) membrane proteins onto the targeting factor TRC40 (href="#bib33" rid="bib33" class=" bibr popnode">Mariappan et al., 2010). The TRC35 and Ubl4A subunits appear to be conserved in all eukaryotes and participate in the TA targeting pathway (href="#bib46" rid="bib46" class=" bibr popnode">Wang et al., 2010). By contrast, the Bag6 subunit appears to be a later evolutionary acquisition that has embellished a targeting factor complex with protein quality control capability. It was therefore proposed that the Bag6 complex is at the center of a triage reaction that routes hydrophobic proteins toward either targeting (in the case of TA proteins) or degradation (for other hydrophobic proteins) (href="#bib21" rid="bib21" class=" bibr popnode">Hessa et al., 2011).Photocrosslinking and mutagenesis experiments demonstrated that Bag6 interacts with hydrophobic domains (href="#bib21" rid="bib21 bib32 bib33" class=" bibr popnode">Hessa et al., 2011; Leznicki et al., 2010; Mariappan et al., 2010). The client then becomes ubiquitinated in a reaction that does not seem to require TRC35 or Ubl4A, but does need the ubiquitin-like (Ubl) domain at the N terminus of Bag6 (href="#bib21" rid="bib21" class=" bibr popnode">Hessa et al., 2011). These observations led to a model in which the Ubl domain recruits a ubiquitin ligase to target Bag6-associated proteins for degradation. However, alternative explanations, such as Bag6 itself acting as an atypical ligase, could not be excluded. Thus, the mechanism of ubiquitination in the MLP degradation pathway was unknown. Here, we have continued our investigation of MLP degradation and identify RNF126 as a Bag6-dependent ubiquitin ligase in this pathway.