ANNEXIN-VI IS REQUIRED FOR BUDDING OF CLATHRIN-COATED PITS

Isolated plasma membranes attached to a solid substratum at 4-degrees-C have numerous clathrin-coated pits. These pits initially are flat but become deeply invaginated after warming to 37-degrees-C. The pits remain tethered to the membrane in this rounded condition unless supplied with ATP, Ca2+, and cytosol. We now show that when cytosol is treated to remove the Ca2+-dependent, phospholipid-binding protein annexin VI, coated pit budding no longer takes place. Addition of purified annexin VI back to the annexin VI-depleted cytosol restores budding activity to normal. Purified annexin VI alone shows only a modest budding activity, suggesting that the cytosol contains a factor(s) in addition to annexin VI that is required for full activity. Cytosol-dependent activation of annexin VI requires both ATP and Ca2+. Annexin VI appears to be not only an active component in the detachment of coated pits from the membrane but also a site for regulating the formation of coated vesicles.