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ER translocation intermediates are adjacent to a nonglycosylated 34-kD integral membrane protein

We have used the homobifunctional cross-linking reagent disuccinimidyl suberate (DSS) to identify proteins that are adjacent to nascent polypeptides undergoing translocations across mammalian rough ER. Translocation intermediates were assembled by supplementing cell free translations of truncated mR...

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Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1991
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2289059/
https://www.ncbi.nlm.nih.gov/pubmed/1646822
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description We have used the homobifunctional cross-linking reagent disuccinimidyl suberate (DSS) to identify proteins that are adjacent to nascent polypeptides undergoing translocations across mammalian rough ER. Translocation intermediates were assembled by supplementing cell free translations of truncated mRNAs with the signal recognition particle (SRP) and microsomal membrane vesicles. Two prominent cross-linked products of 45 and 64 kD were detected. The 64-kD product was obtained when the cell free translation contained SRP, while formation of the 45- kD product required both SRP and translocation competent microsomal membrane vesicles. In agreement with previous investigators, we suggest that the 64-kD product arises by cross-linking of the nascent polypeptide to the 54-kD subunit of SRP. The 45-kD product resists alkaline extraction from the membrane, so we conclude that the 11-kD nascent polypeptide has been crosslinked to an integral membrane protein of approximately 34 kD (imp34). The cross-linked product does not bind to ConA Sepharose, nor is it sensitive to endoglycosidase H digestion; hence imp34 is not identical to the alpha or beta subunits of the signal sequence receptor (SSR). We propose that imp34 functions in concert with SSR to form a translocation site through which nascent polypeptides pass in traversing the membrane bilayer of the rough endoplasmic reticulum.
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spelling pubmed-22890592008-05-01 ER translocation intermediates are adjacent to a nonglycosylated 34-kD integral membrane protein J Cell Biol Articles We have used the homobifunctional cross-linking reagent disuccinimidyl suberate (DSS) to identify proteins that are adjacent to nascent polypeptides undergoing translocations across mammalian rough ER. Translocation intermediates were assembled by supplementing cell free translations of truncated mRNAs with the signal recognition particle (SRP) and microsomal membrane vesicles. Two prominent cross-linked products of 45 and 64 kD were detected. The 64-kD product was obtained when the cell free translation contained SRP, while formation of the 45- kD product required both SRP and translocation competent microsomal membrane vesicles. In agreement with previous investigators, we suggest that the 64-kD product arises by cross-linking of the nascent polypeptide to the 54-kD subunit of SRP. The 45-kD product resists alkaline extraction from the membrane, so we conclude that the 11-kD nascent polypeptide has been crosslinked to an integral membrane protein of approximately 34 kD (imp34). The cross-linked product does not bind to ConA Sepharose, nor is it sensitive to endoglycosidase H digestion; hence imp34 is not identical to the alpha or beta subunits of the signal sequence receptor (SSR). We propose that imp34 functions in concert with SSR to form a translocation site through which nascent polypeptides pass in traversing the membrane bilayer of the rough endoplasmic reticulum. The Rockefeller University Press 1991-07-01 /pmc/articles/PMC2289059/ /pubmed/1646822 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Articles
ER translocation intermediates are adjacent to a nonglycosylated 34-kD integral membrane protein
title ER translocation intermediates are adjacent to a nonglycosylated 34-kD integral membrane protein
title_full ER translocation intermediates are adjacent to a nonglycosylated 34-kD integral membrane protein
title_fullStr ER translocation intermediates are adjacent to a nonglycosylated 34-kD integral membrane protein
title_full_unstemmed ER translocation intermediates are adjacent to a nonglycosylated 34-kD integral membrane protein
title_short ER translocation intermediates are adjacent to a nonglycosylated 34-kD integral membrane protein
title_sort er translocation intermediates are adjacent to a nonglycosylated 34-kd integral membrane protein
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2289059/
https://www.ncbi.nlm.nih.gov/pubmed/1646822