Mechanism of protein quality control at the endoplasmic reticulum

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

The endoplasmic reticulum (ER) is the major site of protein biosynthesis in eukaryotes. Polypeptides entering the ER may frequently adopt aberrant conformations, resulting in aggregation-prone, misfolded proteins. Accumulation of misfolded proteins induces ER stress, which has been implicated in the pathogenesis of many human diseases. To preserve ER protein homeostasis, eukaryotes have evolved a conserved quality control pathway termed retro-translocation/dislocation or ER-associated degradation (ERAD), which eliminates misfolded proteins from the ER by exporting them into the cytosol. Polypeptides undergoing retro-translocation are disposed of by the cytosolic proteasome. The retro-translocation pathway is hijacked by certain viruses to destroy folded cellular proteins required for immune response, allowing the virus to evade host immune surveillance. For example, the Human Immunodeficiency Virus uses a protein named Vpu to target newly synthesized CD4 co-receptor for degradation, which promote viral infection. We previously identified a cytosolic enzyme called p97, which acts with two co-factors Ufd1 and Npl4 to move retrotranslocating substrates into the cytosol for degradation. We also used an affinity purification approach to identify two novel ER membrane proteins, Derlin-1 and VIMP, which associate with p97. VIMP functions as a receptor to recruit p97 to the ER membrane. The conserved multi-spanning membrane protein Derlin-1 plays a central role in retro-translocation. It appears to receive substrates from the ER lumen to promote their translocation via a yet-to-be defined membrane pore. We further identified an ubiquitin ligase-associated multiprotein complex comprising Bag6, Ubl4A, and Trc35, which chaperones retrotranslocated polypeptides en route to the proteasome to improve ERAD efficiency. Our results reveal an ubiquitin ligase-associated holdase that maintains polypeptide solubility to enhance protein quality control in mammalian cells. Our research also addressed a surprising paradox emerging from recent studies that ubiquitin ligases (E3s) and deubiquitinases (DUBs), enzymes with opposing activities, can both promote ERAD. We demonstrate that the ERAD E3 gp78 can ubiquitinate not only ERAD substrates, but also the machinery protein Ubl4A, a key component of the Bag6 chaperone complex. Remarkably, instead of targeting Ubl4A for degradation, polyubiquitination is associated with irreversible proteolytic processing and inactivation of Bag6. Importantly, we identify USP13 as a gp78-associated DUB that eliminates ubiquitin conjugates from Ubl4A to maintain the functionality of Bag6. Our study reveals an unexpected paradigm in which a DUB prevents undesired ubiquitination to sharpen substrate specificity for an associated ubiquitin ligase partner and to promote ER quality control. We recently show that ribosome stalling during protein translocation induces the attachment of UFM1, a ubiquitin-like modifier, to two conserved lysine residues near the COOH-terminus of the 60S ribosomal subunit RPL26 (uL24) at the ER. Strikingly, RPL26 UFMylation enables the degradation of stalled nascent chains, but unlike ERAD or previously established cytosolic ribosome-associated quality control (RQC), which uses proteasome to degrade their client proteins, ribosome UFMylation promotes the targeting of a translocation-arrested ER protein to lysosomes for degradation. RPL26 UFMylation is upregulated during erythroid differentiation to cope with increased secretory flow, and compromising UFMylation impairs protein secretion, and ultimately hemoglobin production. We propose that in metazoan, co-translational protein translocation (TAQC) into the ER is safeguarded by a UFMylation-dependent, translocation-associated protein quality control mechanism, which when impaired causes anemia in mice and abnormal neuronal development in humans. We also used a genome-wide CRISPR/Cas9 screen to identify an uncharacterized membrane protein named SAYSD1 that facilitates TAQC. SAYSD1 associates with the Sec61 translocon and engages a stalled nascent chain-ribosome complex by directly recognizing both ribosome and UFM1, ensuring the export of stalled substrates via the TRAPP complex for lysosomal degradation. Like UFM1 deficiency, SAYSD1 depletion causes the build-up of translocation-stalled proteins at the ER, which triggers ER stress. Importantly, disrupting UFM1- and SAYSD1-dependent quality control in Drosophila leads to the accumulation of translocation-stalled collagen, defective collagen deposition, abnormal basement membranes, and reduced stress tolerance. Together, our data support a model that SAYSD1 acts as an ER-associated UFM1 sensor to collaborate with ribosome UFMylation at the site of translocon-jamming, safeguarding ER homeostasis during animal development. More recently, we combined genome-wide CRISPR screen with live cell fluorescence confocal microscopy to dissect the molecular linchpins of TAQC. We show that substrates translated from mRNAs bearing a ribosome stalling poly-(A) sequence can be degraded by lysosomes and proteasome. By contrast, the degradation of a TAQC substrate encoded by non-stop mRNA is mediated by an unconventional ER-associated degradation (ERAD) mechanism that involves ER-to-Golgi trafficking and KDEL-dependent ER retrieval of non-stop substrate. The diversity in triaging option appear to result from the heterogeneity of NEMF-mediated CATylation, as an AT-rich tail can serve as a degron for ERAD, while an AG-rich tail allows an otherwise secretory protein to be sorted to lysosomes or retrieved back to the ER from the Golgi. Thus, our study reveals an unexpected protein triaging function of CAT-tailing that safeguards protein biogenesis at the ER.