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Functional Dissection of COP-I Subunits in the Biogenesis of Multivesicular Endosomes
In the present paper, we show that transport from early to late endosomes is inhibited at the restrictive temperature in a mutant CHO cell line (ldlF) with a ts-defect in ε coatomer protein (εCOP), although internalization and recycling continue. Early endosomes then appear like clusters of thin tub...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
The Rockefeller University Press
1997
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2140201/ https://www.ncbi.nlm.nih.gov/pubmed/9382865 |
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author | Gu, Feng Aniento, Fernando Parton, Robert G. Gruenberg, Jean |
author_facet | Gu, Feng Aniento, Fernando Parton, Robert G. Gruenberg, Jean |
author_sort | Gu, Feng |
collection | PubMed |
description | In the present paper, we show that transport from early to late endosomes is inhibited at the restrictive temperature in a mutant CHO cell line (ldlF) with a ts-defect in ε coatomer protein (εCOP), although internalization and recycling continue. Early endosomes then appear like clusters of thin tubules devoid of the typical multivesicular regions, which are normally destined to become vesicular intermediates during transport to late endosomes. We also find that the in vitro formation of these vesicles from BHK donor endosomes is inhibited in cytosol prepared from ldlF cells incubated at the restrictive temperature. Although εCOP is rapidly degraded in ldlF cells at the restrictive temperature, cellular amounts of the other COP-I subunits are not affected. Despite the absence of εCOP, we find that a subcomplex of β, β′, and ζCOP is still recruited onto BHK endosomes in vitro, and this binding exhibits the characteristic properties of endosomal COPs with respect to stimulation by GTPγS and sensitivity to the endosomal pH. Previous studies showed that γ and δCOP are not found on endosomes. However, αCOP, which is normally present on endosomes, is no longer recruited when εCOP is missing. In contrast, all COP subunits, except obviously εCOP itself, still bind BHK biosynthetic membranes in a pH-independent manner in vitro. Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins. Our data also show that membrane association and function of endosomal COPs can be dissected: whereas β, β′, and ζCOP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of α and/or εCOP. |
format | Text |
id | pubmed-2140201 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 1997 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-21402012008-05-01 Functional Dissection of COP-I Subunits in the Biogenesis of Multivesicular Endosomes Gu, Feng Aniento, Fernando Parton, Robert G. Gruenberg, Jean J Cell Biol Article In the present paper, we show that transport from early to late endosomes is inhibited at the restrictive temperature in a mutant CHO cell line (ldlF) with a ts-defect in ε coatomer protein (εCOP), although internalization and recycling continue. Early endosomes then appear like clusters of thin tubules devoid of the typical multivesicular regions, which are normally destined to become vesicular intermediates during transport to late endosomes. We also find that the in vitro formation of these vesicles from BHK donor endosomes is inhibited in cytosol prepared from ldlF cells incubated at the restrictive temperature. Although εCOP is rapidly degraded in ldlF cells at the restrictive temperature, cellular amounts of the other COP-I subunits are not affected. Despite the absence of εCOP, we find that a subcomplex of β, β′, and ζCOP is still recruited onto BHK endosomes in vitro, and this binding exhibits the characteristic properties of endosomal COPs with respect to stimulation by GTPγS and sensitivity to the endosomal pH. Previous studies showed that γ and δCOP are not found on endosomes. However, αCOP, which is normally present on endosomes, is no longer recruited when εCOP is missing. In contrast, all COP subunits, except obviously εCOP itself, still bind BHK biosynthetic membranes in a pH-independent manner in vitro. Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins. Our data also show that membrane association and function of endosomal COPs can be dissected: whereas β, β′, and ζCOP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of α and/or εCOP. The Rockefeller University Press 1997-12-01 /pmc/articles/PMC2140201/ /pubmed/9382865 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 | Article Gu, Feng Aniento, Fernando Parton, Robert G. Gruenberg, Jean Functional Dissection of COP-I Subunits in the Biogenesis of Multivesicular Endosomes |
title | Functional Dissection of COP-I Subunits in the Biogenesis of Multivesicular Endosomes |
title_full | Functional Dissection of COP-I Subunits in the Biogenesis of Multivesicular Endosomes |
title_fullStr | Functional Dissection of COP-I Subunits in the Biogenesis of Multivesicular Endosomes |
title_full_unstemmed | Functional Dissection of COP-I Subunits in the Biogenesis of Multivesicular Endosomes |
title_short | Functional Dissection of COP-I Subunits in the Biogenesis of Multivesicular Endosomes |
title_sort | functional dissection of cop-i subunits in the biogenesis of multivesicular endosomes |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2140201/ https://www.ncbi.nlm.nih.gov/pubmed/9382865 |
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