ANNEXIN VI-MEDIATED LOSS OF SPECTRIN DURING COATED PIT BUDDING IS COUPLED TO DELIVERY OF LDL TO LYSOSOMES

Previously we reported that annexin VI is required for the budding of clathrin-coated pits from human fibroblast plasma membranes in vitro. Here we show that annexin VI bound to the NH2-terminal 28-kD portion o f membrane spectrin is as effective as cytosolic annexin VI in support ing coated pit budding. Annexin VI-dependent budding is accompanied by the loss of similar to 50% of the spectrin from the membrane and is b locked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norle ucinal (ALLN), Incubation of fibroblasts in the presence of ALLN initi ally blocks the uptake of low density lipoprotein (LDL), but the cells recover after 1 h and internalize LDL with normal kinetics. The LDL i nternalized under these conditions, however, fails to migrate to the c enter of the cell and is not degraded. ALLN-treated cells have twice a s many coated pits and twofold more membrane clathrin, suggesting that new coated pits have assembled. Annexin VI is not required for the bu dding of these new coated pits and ALLN does not inhibit, Finally, mic roinjection of a truncated annexin VI that inhibits budding in vitro h as the same effect on LDL internalization as ALLN. These findings sugg est that fibroblasts are able to make at least two types of coated pit s, one of which requires the annexin VI-dependent activation of a cyst eine protease to disconnect the clathrin lattice from the spectrin mem brane cytoskeleton during the final stages of budding.