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Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles.

Authors: Borner, GH  Antrobus, R  Hirst, J  Bhumbra, GS  Kozik, P  Jackson, LP  Sahlender, DA  Robinson, MS 
Citation: Borner GH, etal., J Cell Biol. 2012 Apr 2;197(1):141-60. doi: 10.1083/jcb.201111049.
Pubmed: (View Article at PubMed) PMID:22472443
DOI: Full-text: DOI:10.1083/jcb.201111049

Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a "profiling" cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions.


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CRRD Object Information
CRRD ID: 11250428
Created: 2016-06-15
Species: All species
Last Modified: 2016-06-15
Status: ACTIVE


RGD is funded by grant HL64541 from the National Heart, Lung, and Blood Institute on behalf of the NIH.