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Published 20 November 2006. doi:10.1083/jcb.200607164
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 175, Number 4, 571-578
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 Comparative proteomics of clathrin-coated vesicles

Georg H.H. Borner1, Michael Harbour1, Svenja Hester2, Kathryn S. Lilley2, and Margaret S. Robinson1

1 Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, England, UK
2 Cambridge Centre for Proteomics, University of Cambridge, Cambridge CB2 1QW, England, UK

Correspondence to Margaret S. Robinson: msr12{at}mole.bio.cam.ac.uk

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.

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.


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