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Comparative Study
. 2006 Nov 20;175(4):571-8.
doi: 10.1083/jcb.200607164.

Comparative proteomics of clathrin-coated vesicles

Georg H H Borner  1 , Michael Harbour, Svenja Hester, Kathryn S Lilley, Margaret S Robinson
Affiliations
Comparative Study

Comparative proteomics of clathrin-coated vesicles

Georg H H Borner et al. J Cell Biol. .

Abstract

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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Figures

Figure 1.
Figure 1.
Unbiased comparative proteomics of CCVs. (a) Schematic representation of the experimental approach. (b) Control and mock CCV fractions were prepared and analyzed by Western blotting (∼7.5 μg protein/lane for homogenates and ∼2 μg for CCV fractions). (c) Comparison of control and mock CCV fractions by 1D SDS-PAGE. Proteins were identified by mass spectrometry. EF, elongation factor; GDE, glycogen debranching enzyme; MVP, major vault protein; TfR, transferrin receptor.
Figure 2.
Figure 2.
2D DIGE analysis of CCVs. A CCV-enriched fraction (control) and a mock CCV fraction (CHC knockdown) were labeled with Cy3 and Cy5, respectively, pooled, and analyzed in a single 2D gel. (a) Control CCV fraction. (b) CHC-knockdown mock CCV fraction. (c) Overlay of a and b with control CCVs in red and mock CCVs in green. Yellow spots correspond to proteins with similar abundance in both fractions. White arrowheads indicate proteins that were identified by mass spectrometry. EF, elongation factor; MVP, major vault protein; TfR, transferrin receptor.
Figure 3.
Figure 3.
iTRAQ quantification of control and mock CCV fractions. Fractions were tagged with iTRAQ reagents, and the relative abundance of identified proteins in control and mock CCVs was ascertained by quantifying the iTRAQ tags. The 522 proteins were sorted by decreasing ratios of control/mock CCVs. The figure shows a normalized plot of ratio over rank. Error bars indicate standard deviations from means of two technical replicates.
Figure 4.
Figure 4.
Western blots of selected proteins. Blots of control and mock CCV fractions were probed with antibodies against AP-3, AP-4, retromer, and BLOC-1 subunits (a) or with various SNARE antibodies (b). Loading was as in Fig. 1.
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References

    1. Andersen, J.S., and M. Mann. 2006. Organellar proteomics: turning inventories into insights. EMBO Rep. 7:874–879. - PMC - PubMed
    1. Blondeau, F., B. Ritter, P.D. Allaire, S. Wasiak, M. Girard, N.K. Hussain, A. Angers, V. Legendre-Guillemin, L. Roy, D. Boismenu, et al. 2004. Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling. Proc. Natl. Acad. Sci. USA. 101:3833–3838. - PMC - PubMed
    1. Bonifacino, J.S., and P. Rojas. 2006. Retrograde transport from endosomes to the trans-Golgi network. Nat. Rev. Mol. Cell Biol. 7:568–579. - PubMed
    1. Borner, G.H.H., K.S. Lilley, T.J. Stevens, and P. Dupree. 2003. Identification of glycosylphosphatidylinositol-anchored proteins in Arabidopsis. A proteomic and genomic analysis. Plant Physiol. 132:568–577. - PMC - PubMed
    1. Boutros, R., S. Fanayan, M. Shehata, and J.A. Byrne. 2004. The tumor protein D52 family: many pieces, many puzzles. Biochem. Biophys. Res. Commun. 325:1115–1121. - PubMed

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