Clathrin-coated vesicles (CCVs) facilitate the transport of cargo between the trans-Golgi network, endosomes, and the plasma membrane. This study presents the first comparative proteomics investigation of CCVs. A CCV-enriched fraction was isolated from HeLa cells and a “mock CCV” fraction from clathrin-depleted cells. We used a combination of 2D difference gel electrophoresis and isobaric tags for relative and absolute quantification (iTRAQ) in conjunction with mass spectrometry to analyze and compare the two fractions. In total, 63 bona fide CCV proteins were identified, including 28 proteins whose association with CCVs had not previously been established. These include numerous post-Golgi SNAREs; subunits of the AP-3, retromer, and BLOC-1 complexes; lysosomal enzymes; CHC22; and five novel proteins of unknown function. The strategy outlined in this paper should be widely applicable as a means of distinguishing genuine organelle components from contaminants.
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20 November 2006
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November 20 2006
Comparative proteomics of clathrin-coated vesicles
Georg H.H. Borner,
Georg H.H. Borner
1Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, England, UK
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Michael Harbour,
Michael Harbour
1Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, England, UK
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Svenja Hester,
Svenja Hester
2Cambridge Centre for Proteomics, University of Cambridge, Cambridge CB2 1QW, England, UK
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Kathryn S. Lilley,
Kathryn S. Lilley
2Cambridge Centre for Proteomics, University of Cambridge, Cambridge CB2 1QW, England, UK
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Margaret S. Robinson
Margaret S. Robinson
1Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, England, UK
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Georg H.H. Borner
1Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, England, UK
Michael Harbour
1Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, England, UK
Svenja Hester
2Cambridge Centre for Proteomics, University of Cambridge, Cambridge CB2 1QW, England, UK
Kathryn S. Lilley
2Cambridge Centre for Proteomics, University of Cambridge, Cambridge CB2 1QW, England, UK
Margaret S. Robinson
1Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, England, UK
Correspondence to Margaret S. Robinson: [email protected]
Abbreviations used in this paper: AP, adaptor protein; CCV, clathrin-coated vesicle; CHC, clathrin heavy chain; DIGE, difference gel electrophoresis; iTRAQ, isobaric tags for relative and absolute quantification; LC, liquid chromatography; MS/MS, tandem mass spectrometry; NSF, N-ethylmaleimide–sensitive fusion protein.
Received:
July 31 2006
Accepted:
October 13 2006
Online ISSN: 1540-8140
Print ISSN: 0021-9525
The Rockefeller University Press
2006
J Cell Biol (2006) 175 (4): 571–578.
Article history
Received:
July 31 2006
Accepted:
October 13 2006
Citation
Georg H.H. Borner, Michael Harbour, Svenja Hester, Kathryn S. Lilley, Margaret S. Robinson; Comparative proteomics of clathrin-coated vesicles . J Cell Biol 20 November 2006; 175 (4): 571–578. doi: https://doi.org/10.1083/jcb.200607164
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