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The quality control of intracellular proteins is achieved by degrading misfolded proteins which cannot be refolded by molecular chaperones. In eukaryotes, such degradation is handled primarily by the ubiquitin-proteasome system. However, it remained unclear whether and how protein quality control deploys various deubiquitinases. To address this question, we screened deletions or mutation of the 20 deubiquitinase genes in Saccharomyces cerevisiae and discovered that almost half of the mutations slowed the removal of misfolded proteins whereas none of the remaining mutations accelerated this process significantly. Further characterization revealed that Ubp6 maintains the level of free ubiquitin to promote the elimination of misfolded cytosolic proteins, while Ubp3 supports the degradation of misfolded cytosolic and ER luminal proteins by different mechanisms. [ABSTRACT FROM AUTHOR]

Membrane-bound and soluble proteins of the secretory pathway are commonly glycosylated in the endoplasmic reticulum. These adducts have many biological functions, including, notably, their contribution to the maturation of glycoproteins. N-linked glycans are of oligomeric structure, forming configurations that provide blueprints to precisely instruct the folding of protein substrates and the quality control systems that scrutinize it. O-linked mannoses are simpler in structure and were recently found to have distinct functions in protein quality control that do not require the complex structure of N-linked glycans. Together, recent studies reveal the breadth and sophistication of the roles of these glycan-directed modifications in protein biogenesis. [ABSTRACT FROM AUTHOR]

Background: Endoplasmic reticulum (ER) quality control mechanisms are part of a comprehensive system to manage cell stress. The flux of molecules is monitored to retain folding intermediates and target misfolded molecules to ER-associated degradation (ERAD) pathways. The mechanisms of sorting remain unclear. While some proteins are retained statically, the classical model substrate CPY* is found in COPII transport vesicles, suggesting a retrieval mechanism for retention. However, its management can be even more dynamic. If ERAD is saturated under stress, excess CPY* traffics to the vacuole for degradation. These observations suggest that misfolded proteins might display different signals for their management. Methodology/Principal Findings: Here, we report the existence of a functional ER exit signal in the pro-domain of CPY*. Compromising its integrity causes ER retention through exclusion from COPII vesicles. The signal co-exists with other signals used for retention and degradation. Physiologically, the export signal is important for stress tolerance. Disabling it converts a benign protein into one that is intrinsically cytotoxic. Conclusions/Significance: These data reveal the remarkable interplay between opposing signals embedded within ERAD substrate molecules and the mechanisms that decipher them. Our findings demonstrate the diversity of mechanisms deployed for protein quality control and maintenance of protein homeostasis. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) maintains an environment essential for secretory protein folding. Consequently, the premature transport of polypeptides would be harmful to the cell. To avert this scenario, mechanisms collectively termed "ER quality control" prevent the transport of nascent polypeptides until they properly fold. Irreversibly misfolded molecules are sorted for disposal by the ER-associated degradation (ERAD) pathway. To better understand the relationship between quality control and ERAD, we studied a new misfolded variant of carboxypeptidase Y (CPY). The molecule was recognized and retained by ER quality control but failed to enter the ERAD pathway. Systematic analysis revealed that a single, specific N-linked glycan of CPY was required for sorting into the pathway. The determinant is dependent on the putative lectin-like receptor Htm1/Mnl1p. The discovery of a similar signal in misfolded proteinase A supported the generality of the mechanism. These studies show that specific signals embedded in glycoproteins can direct their degradation if they fail to fold. [ABSTRACT FROM AUTHOR]

Proteins synthesized in the endoplasmic reticulum (ER) encounter quality control checkpoints that verify their fitness to proceed in the secretory pathway. Molecules undergoing folding and assembly are kept out of the exocytic pathway until maturation is complete. Misfolded side products that inevitably form are removed from the mixture of conformers and returned to the cytosol for degradation. How unfolded proteins are recognized and how irreversibly misfolded proteins are sorted to ER-associated degradation pathways was poorly understood. Recent developments from a combination of genetic and biochemical analyses has revealed new insights into these mechanisms. The emerging view shows distinct pathways working in collaboration to filter the diverse range of unfolded proteins from the transport flow and to divert misfolded molecules for destruction. [ABSTRACT FROM AUTHOR]

Misfolded proteins retained in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation pathway. The mechanisms used to sort them from correctly folded proteins remain unclear. Analysis of substrates with defined folded and misfolded domains has revealed a system of sequential checkpoints that recognize topologically distinct domains of polypeptides. The first checkpoint examines the cytoplasmic domains of membrane proteins. If a lesion is detected, it is retained statically in the ER and rapidly degraded without regard to the state of its other domains. Proteins passing this test face a second checkpoint that monitors domains localized in the ER lumen. Proteins detected by this pathway are sorted from folded proteins and degraded by a quality control mechanism that requires ER-to-Golgi transport. Although the first checkpoint is obligatorily directed at membrane proteins, the second monitors both soluble and membrane proteins. Our data support a model whereby "properly folded" proteins are defined biologically as survivors that endure a series of distinct checkpoints. [ABSTRACT FROM AUTHOR]

How cells decide that a protein is misfolded is a mystery. The endoplasmic reticulum integrates N-linked glycosylation into the decision as to whether a protein is misfolded. The basic strategy of glycan-based recognition, previously identified in yeast, is conserved in mammals but is expanded, possibly to accommodate a more complex client portfolio. [ABSTRACT FROM AUTHOR]

Misfolded cytosolic proteins are degraded by the ubiquitin proteasome system through quality control (QC) pathways defined by E3 ubiquitin ligases and associated chaperones. Although they work together as a comprehensive system to monitor cytosolic protein folding, their respective contributions remain unclear. To bridge existing gaps, the pathways mediated by the San1 and Ubr1 E3 ligases were studied coordinately. We show that pathways share the same complement of chaperones needed for substrate trafficking, ubiquitination, and degradation. The significance became clear when Ubr1, like San1, was localized primarily to the nucleus. Appending nuclear localization signals to cytosolic substrates revealed that Ydj1 and Sse1 are needed for substrate nuclear import, whereas Ssa1/Ssa2 is needed both outside and inside the nucleus. Sis1 is required to process all substrates inside the nucleus, but its role in trafficking is substrate specific. Together, these data show that using chaperones to traffic misfolded cytosolic proteins into the nucleus extends the nuclear protein QC pathway to include cytosolic clients. [ABSTRACT FROM AUTHOR]

The secretory pathway maintains multiple quality control checkpoints. Initially, endoplasmic reticulum-associated degradation pathways monitor protein folding to retain and eliminate aberrant products. Despite its broad client range, some molecules escape detection and traffic to Golgi membranes. There, a poorly understood mechanism termed Golgi quality control routes aberrant proteins for lysosomal/vacuolar degradation. To better understand Golgi quality control, we examined the processing of the obligate substrate Wsc1p. Misfolded Wsc1p does not use routes of typical vacuolar membrane proteins. Instead, it partitions into intralumenal vesicles of the multivesicular body (MVB) pathway, mediated by the E3 ubiquitin ligase Rsp5p. Its subsequent transport to the vacuolar lumen is essential for complete molecule breakdown. Surprisingly, the transport mode plays a second crucial function in neutralizing potential substrate toxicity. Eliminating the MVB sorting signal diverted molecules to the vacuolar limiting membrane, resulting in the generation of toxic by-products. These data demonstrate a new role of the MVB pathway in protein quality control. [ABSTRACT FROM AUTHOR]

Secretory protein folding is monitored by endoplasmic reticulum (ER) quality control mechanisms. Misfolded proteins are retained and targeted to ER-associated degradation (ERAD) pathways. At their core are E3 ubiquitin ligases, which organize factors that recognize, ubiquitinate, and translocate substrates. Of these, we report that the Hrd1 complex manages three distinct substrate classes. A core complex is required for all classes and is sufficient for some membrane proteins. The accessory factors Usa1p and Der1p adapt the complex to process luminal substrates. Their integration is sufficient to process molecules bearing glycan-independent degradation signals. The presence of Yos9p extends the substrate range by mediating the recognition of glycan-based degradation signals. This modular organization enables the Hrd1 complex to recognize topologically diverse substrates. The Hrd1 system does not directly evaluate the folding state of polypeptides. Instead, it does so indirectly, by recognizing specific embedded signals displayed upon misfolding. [ABSTRACT FROM AUTHOR]