Mammalian Sec61 Is Associated with Sec62 and Sec63

Meyer, Hellmuth-Alexander; Grau, H.; Kraft, Regine; Kostka, Susanne; Prehn, Siegfried; Kalies, Kai‐Uwe; Hartmann, Enno

doi:10.1074/jbc.275.19.14550

Cited by 171 publications

(158 citation statements)

References 46 publications

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“…This novel role of Sec62 in ER physiology is enhanced after successful resolution of ER stress, contribute to re-establish pre-stress ER homeostasis and opens several lines of research aiming at better understanding of how mammalian cells maintain or re-establish proteostasis. The activity of Sec62 in translocation of nascent proteins across the ER membrane is carried out in complex with Sec63 and requires formation of higher order complexes with other components of the Sec61 translocon 9,10,[12][13][14][15] . However, ectopic expression of Sec62 or of reporter proteins displaying its LIR containing cytosolic domain is sufficient to enhance delivery of ER protein markers to the autolysosomal system for clearance, and Sec63 is not required for Sec62 function in ER stress recovery.…”

Section: Fig 8h)mentioning

confidence: 99%

Translocon component Sec62 acts in endoplasmic reticulum turnover during stress recovery

et al. 2016

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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...

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Section: Fig 8h)mentioning

confidence: 99%

Translocon component Sec62 acts in endoplasmic reticulum turnover during stress recovery

et al. 2016

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“…Extraction of Microsomal Membranes-Dog pancreas (19) and cow pancreas microsomes (20) were prepared as described previously. For definition of microsome equivalents see Ref.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Posttranslational Formylglycine Formation by Luminal Components of the Endoplasmic Reticulum

Fey¹,

Balleininger²,

Borissenko³

et al. 2001

Journal of Biological Chemistry

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C ␣ -formylglycine is the key catalytic residue in the active site of sulfatases. In eukaryotes formylglycine is generated during or immediately after sulfatase translocation into the endoplasmic reticulum by oxidation of a specific cysteine residue. We established an in vitro assay that allowed us to measure formylglycine modification independent of protein translocation. The modifying enzyme was recovered in a microsomal detergent extract. As a substrate we used ribosome-associated nascent chain complexes comprising in vitro synthesized sulfatase fragments that were released from the ribosomes by puromycin. Formylglycine modification was highly efficient and did not require a signal sequence in the substrate polypeptide. Ribosome association helped to maintain the modification competence of nascent chains but only after their release efficient modification occurred. The modifying machinery consists of soluble components of the endoplasmic reticulum lumen, as shown by differential extraction of microsomes. The in vitro assay can be performed under kinetically controlled conditions. The activation energy for formylglycine formation is 61 kJ/mol, and the pH optimum is Ϸ10. The activity is sensitive to the SH/SS equilibrium and is stimulated by Ca 2؉ . Formylglycine formation is efficiently inhibited by a synthetic sulfatase peptide representing the sequence directing formylglycine modification. The established assay system should make possible the biochemical identification of the modifying enzyme.

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“…ER components distribute differently between SER patches and polygonal meshwork ER In cells in which RER and SER are spatially separated, ER proteins involved in the translocation and processing of nascent peptides are generally confined to the rough domains (Marcantonio et al, 1984;Meyer et al, 2000;Rolls et al, 2002;Vogel et al, 1990). Also reported to be more concentrated in the rough than in the smooth ER is CLIMP-63, a membrane protein that mediates the interaction between the ER and microtubules (Klopfenstein et al, 1998;Schweizer et al, 1995).…”

Section: Pdmp-induced Ser Patches Are Dynamic and Rapidly Disperse Upmentioning

confidence: 99%

Dynamic and reversible restructuring of the ER induced by PDMP in cultured cells

Sprocati

Ronchi

Raimondi

et al. 2006

Journal of Cell Science

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In many cells, the endoplasmic reticulum (ER) contains segregated smooth and rough domains, but the mechanism of this segregation is unclear. Here, we used a HeLa cell line, inducibly expressing a GFP fusion protein [GFP-b(5)tail] anchored to the ER membrane, as a tool to investigate factors influencing ER organisation. Induction of GFP-b(5)tail expression caused proliferation of the ER, but its normal branching polygonal meshwork architecture was maintained. Experiments designed to test the effects of drugs that alter ceramide levels revealed that treatment of these cells with Phenyl-2-decanoyl-amino-3-morpholino-1-propanol-hydrocholride (PDMP) generated patches of segregated smooth ER, organised as a random tubular network, which rapidly dispersed after removal of the drug. The effect of PDMP was independent of its activity as sphingolipid synthesis inhibitor, but could be partially reversed by a membrane-permeant Ca2+ chelator. Although the smooth ER patches maintained connectivity with the remaining ER, they appeared to represent distinct domains differing in protein and lipid composition from the remaining ER. PDMP did not cause detachment of membrane-bound ribosomes, indicating that smooth ER patch generation was due to a reorganisation of pre-existing ribosome-free areas. Our results demonstrate a dynamic relationship between smooth and rough ER and have implications for the mechanisms regulating ER architecture.

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Mammalian Sec61 Is Associated with Sec62 and Sec63

Cited by 171 publications

References 46 publications

Translocon component Sec62 acts in endoplasmic reticulum turnover during stress recovery

Translocon component Sec62 acts in endoplasmic reticulum turnover during stress recovery

Characterization of Posttranslational Formylglycine Formation by Luminal Components of the Endoplasmic Reticulum

Dynamic and reversible restructuring of the ER induced by PDMP in cultured cells

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