FIGURE 5. CC3 binds membranes directly.

A, Rat CCCP1/CCDC186 is a peripheral membrane protein. In insulinoma 832/13 cell fractions, rat CCCP1/CCDC186 was found primarily in the post-nuclear P90 membrane fraction and could be extracted by 1 M NaCl or Triton X-100. RAB2A associates with membranes via a lipid anchor and could be extracted by Triton X-100 but not by 1 M NaCl. Tubulin served as a control soluble protein. S13: the supernatant obtained by a 13,000g spin of the post-nuclear supernatant, containing the cytosolic proteins and proteins associated with small membrane compartments. S90, P90: supernatant and pellet fractions obtained by a 90,000g spin of the S13 fraction, containing cytosolic and membrane-associated proteins respectively. B, Schematic of the liposome flotation assay. The CCCP-1 protein (1 µM) was incubated with Golgi-mix liposomes (1 mM lipids) containing a rhodamine-labeled lipid. The suspension was adjusted to 40% w/v sucrose and overlaid with three cushions of decreasing sucrose concentration. The tube was centrifuged for two hours at 200,000g. The efficiency of the flotation is demonstrated by observing the rhodamine-labeled lipid near the top of the tube. C, CCCP-1 binds to membranes directly. CCCP-1 membrane-binding activity was assayed by liposome flotation. Untagged recombinant CCCP-1 was assayed in the absence (top) or presence (bottom) of Golgi-mix liposomes. After centrifugation, fractions were collected from the top (lane 6) to the bottom (lane 1) and analyzed by Coomassie-stained SDS-PAGE. The protein input shown here was part of the same Coomassie gel. D, CC3 is necessary and sufficient for CCCP-1 membrane association. Representative blots from flotation assays using Golgi-mix liposomes and His₆-tagged recombinant CCCP-1 fragments. After centrifugation, fractions were collected from the top (lane 7) to the bottom (lane 2) and analyzed by Western blot against the His₆ tag.