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Identification of minor components of coated vesicles by use of permeation chromatography

Coated vesicles are thought to be vehicles for the intracellular transport of membranes. Clathrin is the major protein component of coated vesicles. Minor components of these organelles can be identified in highly purified preparations if they can be shown to copurify with clathrin. To show copurifi...

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Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1981
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2111959/
https://www.ncbi.nlm.nih.gov/pubmed/7309787
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description Coated vesicles are thought to be vehicles for the intracellular transport of membranes. Clathrin is the major protein component of coated vesicles. Minor components of these organelles can be identified in highly purified preparations if they can be shown to copurify with clathrin. To show copurification we have made use of the relatively uniform diameter of coated vesicles (50-150 nm) to fractionate conventionally purified coated vesicles according to size in glass bead columns of 200-nm pore size. We have found that bovine brain coated vesicles prepared by the standard procedure of Pearse can be contaminated with large membrane fragments that are removed by permeation chromatography on such glass bead columns. Gel electrophoretic analysis of column fractions shows that only three major polypeptide chains, and a family of polypeptides with molecular weights close to 100,000 are always in constant ratio to clathrin, and are unique to fractions containing coated vesicles. Two other major polypeptides that appear to be components of coated vesicles are also present in other membrane fractions. We have also used permeation chromatography to monitor artifactual membrane trapping during vesicle isolation. Pure radiolabeled synaptic vesicle membranes were added to bovine brain tissue before homogenization. Considerable amounts of the added radioactivity could be recovered in the fractions conventionally pooled in the preparation of coated vesicles. After permeation chromatography, the radioactivity in the coated vesicle peak was reduced essentially to background.
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spelling pubmed-21119592008-05-01 Identification of minor components of coated vesicles by use of permeation chromatography J Cell Biol Articles Coated vesicles are thought to be vehicles for the intracellular transport of membranes. Clathrin is the major protein component of coated vesicles. Minor components of these organelles can be identified in highly purified preparations if they can be shown to copurify with clathrin. To show copurification we have made use of the relatively uniform diameter of coated vesicles (50-150 nm) to fractionate conventionally purified coated vesicles according to size in glass bead columns of 200-nm pore size. We have found that bovine brain coated vesicles prepared by the standard procedure of Pearse can be contaminated with large membrane fragments that are removed by permeation chromatography on such glass bead columns. Gel electrophoretic analysis of column fractions shows that only three major polypeptide chains, and a family of polypeptides with molecular weights close to 100,000 are always in constant ratio to clathrin, and are unique to fractions containing coated vesicles. Two other major polypeptides that appear to be components of coated vesicles are also present in other membrane fractions. We have also used permeation chromatography to monitor artifactual membrane trapping during vesicle isolation. Pure radiolabeled synaptic vesicle membranes were added to bovine brain tissue before homogenization. Considerable amounts of the added radioactivity could be recovered in the fractions conventionally pooled in the preparation of coated vesicles. After permeation chromatography, the radioactivity in the coated vesicle peak was reduced essentially to background. The Rockefeller University Press 1981-11-01 /pmc/articles/PMC2111959/ /pubmed/7309787 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
Identification of minor components of coated vesicles by use of permeation chromatography
title Identification of minor components of coated vesicles by use of permeation chromatography
title_full Identification of minor components of coated vesicles by use of permeation chromatography
title_fullStr Identification of minor components of coated vesicles by use of permeation chromatography
title_full_unstemmed Identification of minor components of coated vesicles by use of permeation chromatography
title_short Identification of minor components of coated vesicles by use of permeation chromatography
title_sort identification of minor components of coated vesicles by use of permeation chromatography
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2111959/
https://www.ncbi.nlm.nih.gov/pubmed/7309787