Faculty Opinions recommendation of The Get1/2 transmembrane complex is an endoplasmic-reticulum membrane protein insertase.

Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (sec61, sec62, sec63) are not impaired in the assembly and glycosylation of the type II membrane protein dipeptidylaminopeptidase B (DPAPB) or of a chimeric membrane protein consisting of the multiple membrane-spanning domain of yeast hydroxymethylglutaryl CoA reductase (HMG1) fused to yeast histidinol dehydrogenase (HIS4C). This chimera is assembled in wild-type or mutant cells such that the His4c protein is oriented to the ER lumen and thus is not available for conversion of cytosolic histidinol to histidine. Cells harboring the chimera have been used to select new translocation defective sec mutants. Temperature-sensitive lethal mutations defining two complementation groups have been isolated: a new allele of sec61 and a single isolate of a new gene sec65. The new isolates are defective in the assembly of DPAPB, as well as the secretory protein alpha-factor precursor. Thus, the chimeric membrane protein allows the selection of more restrictive sec mutations rather than defining genes that are required only for membrane protein assembly. The SEC61 gene was cloned, sequenced, and used to raise polyclonal antiserum that detected the Sec61 protein. The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec61p antiserum detects an integral protein localized to the endoplasmic reticulum membrane. Sec61p appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum.

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KEG1/YFR042w Encodes a Novel Kre6-binding Endoplasmic Reticulum Membrane Protein Responsible for β-1,6-Glucan Synthesis in Saccharomyces cerevisiae

Journal of Biological Chemistry ◽

10.1074/jbc.m706486200 ◽

2007 ◽

Vol 282 (47) ◽

pp. 34315-34324 ◽

Cited By ~ 17

Author(s):

Kosuke Nakamata ◽

Tomokazu Kurita ◽

M. Shah Alam Bhuiyan ◽

Keisuke Sato ◽

Yoichi Noda ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Membrane Protein ◽

Endoplasmic Reticulum Membrane ◽

Glucan Synthesis

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Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3288 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3288-3296 ◽

Cited By ~ 161

Author(s):

D Feldheim ◽

J Rothblatt ◽

R Schekman

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Protein ◽

Protein Translocation ◽

Protein A ◽

Integral Membrane Protein ◽

Secretory Protein ◽

Carboxyl Terminus ◽

Endoplasmic Reticulum Membrane ◽

Cell Fractionation ◽

Network Cell

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.

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An ATP-binding membrane protein is required for protein translocation across the endoplasmic reticulum membrane.

Cell Regulation ◽

10.1091/mbc.2.10.851 ◽

1991 ◽

Vol 2 (10) ◽

pp. 851-859 ◽

Cited By ~ 12

Author(s):

D L Zimmerman ◽

P Walter

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Membrane Protein ◽

Protein Translocation ◽

Signal Sequence ◽

Microsomal Membrane ◽

Atp Binding ◽

Endoplasmic Reticulum Membrane ◽

Receptor Complex

The role of nucleotides in providing energy for polypeptide transfer across the endoplasmic reticulum (ER) membrane is still unknown. To address this question, we treated ER-derived mammalian microsomal vesicles with a photoactivatable analogue of ATP, 8-N3ATP. This treatment resulted in a progressive inhibition of translocation activity. Approximately 20 microsomal membrane proteins were labeled by [alpha 32P]8-N3ATP. Two of these were identified as proteins with putative roles in translocation, alpha signal sequence receptor (SSR), the 35-kDa subunit of the signal sequence receptor complex, and ER-p180, a putative ribosome receptor. We found that there was a positive correlation between inactivation of translocation activity and photolabeling of alpha SSR. In contrast, our data demonstrate that the ATP-binding domain of ER-p180 is dispensable for translocation activity and does not contribute to the observed 8-N3ATP sensitivity of the microsomal vesicles.

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