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Figure 1. Identification and characterization of CVAK104.A) 2D-DIGE analysis of CCVs. The CCV-enriched fractions were prepared from untreated HeLa cells, and ‘mock’ CCV fractions from clathrin-depleted HeLa cells. Fractions were labelled with fluorescent dyes and analysed in single 2D gels. The figure shows a false-colour overlay of control CCV (red) and mock CCV (green) fractions. Red spots correspond to proteins that are depleted from the mock CCVs, and thus to candidate CCV components. Yellow spots correspond to contaminants, which are similarly abundant in both fractions. Arrowheads indicate proteins that were identified by MS. B) Schematic diagram of CVAK104 organization, showing the positions of the kinase-like domain and the clathrin-binding domain [mapped by Düwel and Ungewickell (11)]. Homology between the CVAK104 and the Saccharomyces cerevisiae protein Scy1p was determined by blast (http://www.ncbi.nlm.nih.gov/BLAST/). The predicted secondary structure downstream from the kinase-like domain was determined by JPred (http://www.compbio.dundee.ac.uk/). C) Western blots of cell homogenates (∼7.5 μg protein/lane) and CCV fractions (∼2 μg protein/lane) were probed with a new polyclonal antibody against CVAK104. The antibody labels an ∼104-kDa band, which disappears when CVAK104 is knocked down with siRNA. The protein is highly enriched in CCVs, and undetectable in mock CCVs. This particular enrichment/depletion profile is characteristic of proteins involved in intracellular clathrin-mediated transport (9). D) HeLa cells were extracted with 0.5% IGEPAL and immunoprecipitated with antibodies against CVAK104, AP-2 α and AP-1 γ in conjunction with protein A, or with protein A beads only (control). Proteins identified by MS are indicated with numbers. The identification of AP-1 and AP-2 subunits other than those directly recognized by the antibodies shows that the experimental conditions were appropriate to co-immunoprecipitate stable protein complexes. CHC, clathrin heavy chain; kd, knockdown; TfR, transferrin receptor; EF, ribosomal elongation factor. Image published in: Borner GH et al. (2007) Image downloaded from an Open Access article in PubMed Central. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd Larger Image Printer Friendly View

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