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...
Background:The molecular chaperone immunoglobulin heavy-chain-binding protein (BiP) modulates gating of the polypeptide-conducting and calcium-permeable channel (Sec61 complex) in the membrane of the endoplasmic reticulum (ER). Results: Two co-chaperones, ERj3 and ERj6, support BiP in preventing ER calcium leakage via Sec61 complex. Conclusion: ERj3 and ERj6 facilitate Sec61 channel closing. Significance: Different co-chaperones assist BiP in Sec61 channel gating.
In mammalian cells, the rough endoplasmic reticulum or ER plays a central role in the biogenesis of most extracellular plus many organellar proteins and in cellular calcium homeostasis. Therefore, this organelle comprises molecular chaperones that are involved in import, folding/assembly, export, and degradation of polypeptides in millimolar concentrations. In addition, there are calcium channels/pumps and signal transduction components present in the ER membrane that affect and are affected by these processes. The ER lumenal Hsp70, termed immunoglobulin-heavy chain binding protein or BiP, is the central player in all these activities and involves up to seven different co-chaperones, i.e. ER-membrane integrated as well as ERlumenal Hsp40s, which are termed ERj or ERdj, and two nucleotide exchange factors.
Protein transport into the human endoplasmic reticulum (ER) is relevant to the biogenesis of most soluble and membrane proteins of organelles, which are involved in endo- or exo-cytsosis. It involves amino-terminal signal peptides in the precursor polypeptides and various transport components in the cytosol plus the ER, and can occur co- or post-translationally. The two mechanisms merge at the level of the ER membrane, specifically at the level of the heterotrimeric Sec61 complex, which forms a dynamic polypeptide-conducting channel in the ER membrane. Since the mammalian ER is also the main intracellular calcium storage organelle, and the Sec61 complex is calcium permeable, the Sec61 complex is tightly regulated in its equilibrium between the closed and open conformations, or "gated", by ligands, such as signal peptides of the transport substrates and the ER lumenal Hsp70-type molecular chaperone BiP. Furthermore, BiP binding to the incoming polypeptide contributes to the efficiency and unidirectionality of transport. Recent insights into the structure and dynamic equilibrium of the Sec61 complex have various mechanistic as well as medical implications.
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