Posttranslational protein transport in yeast reconstituted with a purified complex of Sec proteins and Kar2p

Panzner, Steffen; Dreier, Lars; Hartmann, Enno; Kostka, Susanne; Rapoport, Tom A.

doi:10.1016/0092-8674(95)90077-2

Cited by 366 publications

(400 citation statements)

References 36 publications

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“…An alternative explanation may be that import of p⌬gp␣f into the ER is extremely rapid and efficient, whereas export proceeds with much slower kinetics and may therefore result in prolonged exposure of O-mannosyl acceptor sites in the appropriate vicinity of Pmt2p. A third possibility is that the Sec63 complex associated with the Sec61 channel during posttranslational p⌬gp␣f import into the ER interferes with access of Pmt2p to proteins in the translocon (Panzner et al, 1995). The Sec63 complex is not required for misfolded protein export from the ER and thus most likely absent from Sec61 channels engaged in export, which may allow mannosylation of export substrates by Pmt2p (Pilon et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

O-Mannosylation Protects Mutant Alpha-Factor Precursor from Endoplasmic Reticulum-associated Degradation

Harty

Strahl

Römisch

2001

MBoC

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Secretory proteins that fail to fold in the endoplasmic reticulum (ER) are transported back to the cytosol and degraded by proteasomes. It remains unclear how the cell distinguishes between folding intermediates and misfolded proteins. We asked whether misfolded secretory proteins are covalently modified in the ER before export. We found that a fraction of mutant alpha-factor precursor, but not the wild type, was progressively O-mannosylated in microsomes and in intact yeast cells by protein O-mannosyl transferase 2 (Pmt2p). O-Mannosylation increased significantly in vitro under ER export conditions, i.e., in the presence of ATP and cytosol, and this required export-proficient Sec61p in the ER membrane. Deletion of PMT2, however, did not abrogate mutant alpha-factor precursor degradation but, rather, enhanced its turnover in intact yeast cells. In vitro, O-mannosylated mutant alpha-factor precursor was stable and protease protected, and a fraction was associated with Sec61p in the ER lumen. Thus, prolonged ER residence allows modification of exposed O-mannosyl acceptor sites in misfolded proteins, which abrogates misfolded protein export from the ER at a posttargeting stage. We conclude that there is a limited window of time during which misfolded proteins can be removed from the ER before they acquire inappropriate modifications that can interfere with disposal through the Sec61 channel.

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Section: Discussionmentioning

confidence: 99%

O-Mannosylation Protects Mutant Alpha-Factor Precursor from Endoplasmic Reticulum-associated Degradation

Harty

Strahl

Römisch

2001

MBoC

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“…Secretory proteins are conducted through the membrane via interactions with translocon protein components, which may vary according to the type of secretory protein involved [5,6]. In yeast, Sec61p (Sec l secretory) and its two paralogues, Sbh1p (Sbh l Sec61 beta homologue) and Sss1p (Sss l Sec61 suppressor), as well as Sec62p, Sec63p, Sec71p and binding protein (BiP), are required to reconstitute post-translational translocation in protoliposomes [7]. In mammals, a heterotrimer of Sec61p homologues with the translocation-associated membrane protein (' TRAM ') are similarly required for translocation of most proteins.…”

Section: Introductionmentioning

confidence: 99%

Proteins of the endoplasmic-reticulum-associated degradation pathway: domain detection and function prediction

Ponting

2000

Biochemical Journal

113

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Sequence database searches, using iterative-profile and Hidden-Markov-model approaches, were used to detect hitherto-undetected homologues of proteins that regulate the endoplasmic reticulum (ER)-associated degradation pathway. The translocon-associated subunit Sec63p (Sec = secretory) was shown to contain a domain of unknown function found twice in several Brr2p-like RNA helicases (Brr2 = bad response to refrigeration 2). Additionally, Cue1p (Cue=coupling of ubiquitin conjugation to ER degradation), a yeast protein that recruits the ubiquitin-conjugating (UBC) enzyme Ubc7p to an ER-associated complex, was found to be one of a large family of putative scaffolding-domain-containing proteins that include the autocrine motility factor receptor and fungal Vps9p (Vps=vacuolar protein sorting). Two other yeast translocon-associated molecules, Sec72p and Hrd3p (Hrd = 3-hydroxy-3-methylglutaryl-CoA reductase degradation), were shown to contain multiple tetratricopeptide-repeat-like sequences. From this observation it is suggested that Sec72p associates with a heat-shock protein, Hsp70, in a manner analogous to that known for Hop (Hsp70/Hsp90 organizing protein). Finally, the luminal portion of Ire1p (Ire=high inositol-requiring), thought to convey the sensing function of this transmembrane kinase and endoribonuclease, was shown to contain repeats similar to those in β-propeller proteins. This finding hints at the mechanism by which Ire1p may sense extended unfolded proteins at the expense of compact folded molecules.

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“…These include SEC61 that is an essential gene encoding the 53-kDa integral membrane protein, Sec61p (13). Sec61p appears to exist as part of a stable complex together with Sss1p and Sbh1p (14,15). Like Sec61p, Sss1p is essential for viability and for protein translocation in vivo (9).…”

mentioning

confidence: 99%

Determination of the Transmembrane Topology of Yeast Sec61p, an Essential Component of the Endoplasmic Reticulum Translocation Complex

Wilkinson

Critchley

Stirling

1996

Journal of Biological Chemistry

142

147

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Sec61p is a highly conserved integral membrane protein that plays a role in the formation of a proteinconducting channel required for the translocation of polypeptides into, and across, the membrane of the endoplasmic reticulum. As a major step toward elucidating the structure of the endoplasmic reticulum translocation apparatus, we have determined the transmembrane topology of Sec61p using a combination of C-terminal reporter-domain fusions and the in situ digestion of specifically inserted factor Xa protease cleavage sites. Our data indicate the presence of 10 transmembrane domains, including several with surprisingly limited hydrophobicity. Furthermore, we provide evidence for complex intramolecular interactions in which these weakly hydrophobic domains require C-terminal sequences for their correct topogenesis. The incorporation of sequences with limited hydrophobicity into the bilayer may play a vital role in the formation of an aqueous membrane channel required for the translocation of hydrophilic polypeptide chains.

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Posttranslational protein transport in yeast reconstituted with a purified complex of Sec proteins and Kar2p

Cited by 366 publications

References 36 publications

O-Mannosylation Protects Mutant Alpha-Factor Precursor from Endoplasmic Reticulum-associated Degradation

O-Mannosylation Protects Mutant Alpha-Factor Precursor from Endoplasmic Reticulum-associated Degradation

Proteins of the endoplasmic-reticulum-associated degradation pathway: domain detection and function prediction

Determination of the Transmembrane Topology of Yeast Sec61p, an Essential Component of the Endoplasmic Reticulum Translocation Complex

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