Fig. 1.

Vps8a associates with five other subunits in a hexameric CORVET complex. (A) Co-immunoprecipitation of Vps8a–FLAG with Myc-tagged Vps16b, Vps18d and Vps33b subunits. Cells were transformed to endogenously express Vps8a–FLAG in pairwise combination with Myc-tagged Vps16a or Vps16b, Vps33a or Vps33b, and Vps18a, Vps18b, Vps18c or Vps18d. Cell lysates were split and incubated with anti-c-Myc or anti-FLAG beads. SDS-PAGE samples were immunoblotted with anti-c-Myc and anti-FLAG antibodies. (B,C) Sedimentation analysis of 8A-CC. (B) Vps8a–FLAG and associated proteins were immuno-isolated using anti-FLAG beads. Eluted proteins were sedimented in glycerol gradients, and 250 µl fractions were harvested from top to bottom, of which 25 µl aliquots were subjected to SDS-PAGE (4–20% gel). Fractions (F)17–29 (top–bottom) are shown. Vps8a was detected by western blotting with anti-FLAG antibodies. ‘INPUT’ corresponds to 1% of total eluate. F4–F16 (top–bottom) were similarly analyzed (see Fig. S1C). (C) 4% of the total eluate (INPUT) and 35 µl aliquots of gradient fractions (F21 to F28) were subjected to SDS-PAGE (8% gel) and visualized by silver staining. The Vps8a subunit, as well as bands of the expected sizes for five additional CORVET subunits, are identified on the right. Thyroglobulin, sedimented in parallel as a size standard, appeared in F24–F29 (top-bottom), with a peak in F26 (arrow at the top). 8A-CC sediments more slowly than expected for a 727 kDa complex. (D) Mass spectrometric identification of proteins co-isolated with Vps8a. Cryopowders (150 g) from wild-type and Vps8a–FLAG-expressing cells were solubilized and treated as in B, except bound proteins were eluted with LDS sample buffer. The total eluates were prepared for mass spectrometric analysis. On volcano plots, such as the one shown here, proteins falling above the threshold line are considered significant. To generate the plot, the −log10 t-test P-value was plotted versus the t-test difference (difference between means). The cut-off curve is based on the false discovery rate (FDR=0.01) and the artificial factor s0 (s0=1), controlling the relative importance of the t-test P-value and difference between means. The open circle marks the Vps8a subunit used as bait, while the black circle marks the unique Vps11 subunit. The light gray circles indicate specific paralogs of the other four CORVET subunits. Each sample was prepared in duplicate.