The endoplasmic reticulum (ER) is a site of protein biogenesis in eukaryotic cells. Perturbing ER homeostasis activates stress programs collectively called the unfolded protein response (UPR). The UPR enhances production of ER-resident chaperones and enzymes to reduce the burden of misfolded proteins. On resolution of ER stress, ill-defined, selective autophagic programs remove excess ER components. Here we identify Sec62, a constituent of the translocon complex regulating protein import in the mammalian ER, as an ER-resident autophagy receptor. Sec62 intervenes during recovery from ER stress to selectively deliver ER components to the autolysosomal system for clearance in a series of events that we name recovER-phagy. Sec62 contains a conserved LC3-interacting region in the C-terminal cytosolic domain that is required for its function in recovER-phagy, but is dispensable for its function in the protein translocation machinery. Our results identify Sec62 as a critical molecular component in maintenance and recovery of ER homeostasis. DOI: https://doi.org/10.1038/ncb3423Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-127515 Accepted Version Originally published at: Fumagalli, Fiorenza; Noak, Julia; Bergmann, Timothy J; Presmanes, Eduardo Cebollero; Pisoni, Giorgia Brambilla; Fasana, Elisa; Fregno, Ilaria; Galli, Carmela; Loi, Marisa; Solda, Tatiana; D'Antuono, Rocco; Raimondi, Andrea; Jung, Martin; Melnyk, Armin; Schorr, Stefan; Schreiber, Anne; Simonelli, Luca; Varani, Luca; Wilson-Zbinden, Caroline; Zerbe, Oliver; Hofmann, Kay; Peter, Matthias; Quadroni, Manfredo; Zimmermann, Richard; Molinari, Maurizio (2016 To define mechanisms that regulate the return of ER-resident chaperones and folding factors to their physiologic intracellular level after resolution of an ER stress, we established a protocol for reversible induction of UPR in cultured mammalian cells (Fig. 1a). Briefly, human embryonic kidney cells (HEK293) or mouse embryonic fibroblasts (MEF) were exposed for 12 h to non-toxic doses of cyclopiazonic acid (CPA), a reversible inhibitor of the sarco/endoplasmic reticulum calcium pump 6 . The return of ER-resident gene products at their pre-stress level was monitored during resolution of the UPR obtained upon CPA wash out ( CPA wash out initiated a recovery phase characterized by the rapid return of ER stress-induced transcripts at, or below, their pre-stress levels (Fig. 1b, recovery, T 1/2 average ≈ 1 h, blue line). The corresponding ER stress-induced proteins returned to their physiologic levels with much slower kinetics (Fig. 1c, d, T 1/2 average ≈ 10 h, blue). 3With the exception of Herp, which is rapidly turned over with intervention of proteasomes (Fig. 1c, d (Fig. 1g, 2a) and other membrane and luminal ER marker proteins such as Sec62 and Crt ( Fig. 2b and Extended data Fig. 3) in 0.5-1.5 µm diameter cytoplasmic puncta that rapidly disappeared upon BafA1 wash out (Extended data Fig. 4). Cytosolic puncta containing ER marker prot...
Maintenance of cellular proteostasis relies on efficient clearance of defective gene products. For misfolded secretory proteins, this involves dislocation from the endoplasmic reticulum (ER) into the cytosol followed by proteasomal degradation. However, polypeptide aggregation prevents cytosolic dislocation and instead activates ill-defined lysosomal catabolic pathways. Here, we describe an ER-to-lysosome-associated degradation pathway (ERLAD) for proteasome-resistant polymers of alpha1-antitrypsin Z (ATZ). ERLAD involves the ER-chaperone calnexin (CNX) and the engagement of the LC3 lipidation machinery by the ER-resident ER-phagy receptor FAM134B, echoing the initiation of starvation-induced, receptor-mediated ER-phagy. However, in striking contrast to ER-phagy, ATZ polymer delivery from the ER lumen to LAMP1/RAB7-positive endolysosomes for clearance does not require ER capture within autophagosomes. Rather, it relies on vesicular transport where single-membrane, ER-derived, ATZ-containing vesicles release their luminal content within endolysosomes upon membrane:membrane fusion events mediated by the ER-resident SNARE STX17 and the endolysosomal SNARE VAMP8. These results may help explain the lack of benefits of pharmacologic macroautophagy enhancement that has been reported for some luminal aggregopathies.
Members of the protein-disulfide isomerase superfamily catalyze the formation of intra-and intermolecular disulfide bonds, a ratelimiting step of protein folding in the endoplasmic reticulum (ER).Here we compared maturation of one obligate and two facultative calnexin substrates in cells with and without ERp57, the calnexinassociated, glycoprotein-specific oxidoreductase. ERp57 deletion did not prevent the formation of disulfide bonds during co-translational translocation of nascent glycopolypeptides in the ER. It affected, however, the post-translational phases of oxidative influenza virus hemagglutinin (HA) folding, resulting in significant loss of folding efficiency for this obligate calnexin substrate. Without ERp57, HA also showed reduced capacity to recover from an artificially induced aberrant conformation, thus revealing a crucial role of ERp57 during post-translational reshuffling to the native set of HA disulfides. ERp57 deletion did not affect maturation of the model facultative calnexin substrates E1 and p62 (and of most cellular proteins, as shown by lack of induction of ER stress). ERp72 was identified as one of the ER-resident oxidoreductases associating with the orphan ERp57 substrates to maintain their folding competence.Polypeptide asparagines emerging in the ER 4 lumen as part of an Asn-X-Ser/Thr motif are covalently modified with preassembled glycans containing 2 N-acetylglucosamine, 9 mannose, and 3 glucose residues (1). N-Glycosylation prepares nascent chains for association with the two lectin-like molecular chaperones calnexin and calreticulin and the glycoprotein-dedicated oxidoreductase ERp57 (2-5).Deletion of individual members of the calnexin chaperone system, namely calreticulin (6), UGT1 (7), and ERp57 (8), is embryonic lethal in mice. Deletion of calnexin does not result in embryonic lethality but causes a severe progressive pathology leading to premature death (9). The suboptimal glycoprotein folding efficiency upon chaperone deletion might be detrimental for organism viability, but all of these deletions are well tolerated at the cellular level. In fact, only a restricted number of endogenous, recombinant, or virus-encoded glycoproteins (e.g. the major histocompatibility complex class I peptide loading complex (8, 10), the influenza virus HA (11), and the ADAM1/ADAM2 fertilin complex (12)) have so far been shown to strongly depend on the calnexin chaperone system for maturation. Consistently, and as previously shown for calnexin, calreticulin, and UGT1 deletions, cells lacking ERp57 show normal viability and proliferation rates and unperturbed maturation and transport of several surface glycoproteins containing disulfide bonds (8). Conditional deletion of ERp57 in B lymphocytes/ plasma cells revealed no defect in the production of immunoglobulin chains, the most abundant N-glycosylated/disulfide-bonded products of these cells (8). The thorough analysis by Garbi et al. showed that ERp57 deletion specifically affected assembly and stability of the major histocompatibility class I p...
Newly synthesized glycoproteins displaying monoglucosylated N-glycans bind to the endoplasmic reticulum (ER) chaperone calnexin, and their maturation is catalyzed by the calnexin-associated oxidoreductase ERp57. Folding substrates are eventually released from calnexin, and terminal glucoses are removed from N-glycans. The UDP-glucose:glycoprotein glucosyltransferase (UGT1, UGGT, GT) monitors the folding state of polypeptides released from calnexin and adds back a glucose residue on N-glycans of nonnative polypeptides, thereby prolonging retention in the calnexin chaperone system for additional folding attempts. Here we show that for certain newly synthesized glycoproteins UGT1 deletion has no effect on binding to calnexin. These proteins must normally complete their folding program in one binding event. Other proteins normally undergo multiple binding events, and UGT1 deletion results in their premature release from calnexin. For other proteins, UGT1 deletion substantially delays release from calnexin, unexpectedly showing that UGT1 activity might be required for a structural maturation needed for substrate dissociation from calnexin and export from the ER.
The endoplasmic reticulum-associated degradation (ERAD) machinery selects native and misfolded polypeptides for dislocation across the ER membrane and proteasomal degradation. Regulated degradation of native proteins is an important aspect of cell physiology. For example, it contributes to the control of lipid biosynthesis, calcium homeostasis and ERAD capacity by setting the turnover rate of crucial regulators of these pathways. In contrast, degradation of native proteins has pathologic relevance when caused by viral or bacterial infections, or when it occurs as a consequence of dysregulated ERAD activity. The efficient disposal of misfolded proteins prevents toxic depositions and persistent sequestration of molecular chaperones that could induce cellular stress and perturb maintenance of cellular proteostasis. In the first section of this review, we survey the available literature on mechanisms of selection of native and non-native proteins for degradation from the ER and on how pathogens hijack them. In the second section, we highlight the mechanisms of ERAD activity adaptation to changes in the ER environment with a particular emphasis on the post-translational regulatory mechanisms collectively defined as ERAD tuning. The cellular proteome is mostly synthesized by cytosolic ribosomes to operate in the cytosol and, upon appropriate targeting, in various intracellular organelles or in the extracellular space. Cellular compartments where protein folding occurs [e.g. the cytosol, mitochondria, the endoplasmic reticulum (ER)] contain two classes of non-native polypeptides: (i) newly synthesized polypeptide chains that must be assisted by folding chaperones and enzymes to attain the native mono-or oligomeric structure; (ii) terminally misfolded conformers that must efficiently be degraded to prevent the formation of toxic deposits and the persistent sequestration of chaperones that could eventually inhibit the cellular protein folding capacity and elicit stress (1-3) ( Figure 1A). The distinction between the two classes of non-native chains, one to be preserved, the second to be cleared from the folding compartment, is not an easy task for the cellular quality control machineries. Selection for disposal might be a stochastic process: the longer the persistency of structural defects in the ER, the greater is the probability to be selected for destruction. Therefore, mutations that delay folding may channel the polypeptide into destructive pathways, even if they do not compromise the function of the mutated protein.In the ER, non-native, but also native proteins might be selected for degradation by components of the endoplasmic reticulum-associated degradation (ERAD) machinery that deliver them at dislocation sites embedded in the ER membrane. Dislocation sites consist of a multitude of luminal and membrane-bound specialized ER-resident proteins as well as a number of cytosolic factors insuring dislocation across the ER membrane, poly-ubiquitylation and disposal of ERAD substrates by 26S proteasomes (Specificity of...
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