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© The Rockefeller University Press, 0021-9525/1998//937 $5.00
The Journal of Cell Biology, Volume 142, Number 4, , 1998 937-947

Articles

Annexin VI-mediated Loss of Spectrin during Coated Pit Budding Is Coupled to Delivery of LDL to Lysosomes



Adeela Kamal, Yun-shu Ying, and Richard G.W. Anderson

Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235

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 of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding. Annexin VI–dependent budding is accompanied by the loss of ~50% of the spectrin from the membrane and is blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Incubation of fibroblasts in the presence of ALLN initially blocks the uptake of low density lipoprotein (LDL), but the cells recover after 1 h and internalize LDL with normal kinetics. The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded. ALLN-treated cells have twice as many coated pits and twofold more membrane clathrin, suggesting that new coated pits have assembled. Annexin VI is not required for the budding of these new coated pits and ALLN does not inhibit. Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN. These findings suggest that fibroblasts are able to make at least two types of coated pits, one of which requires the annexin VI–dependent activation of a cysteine protease to disconnect the clathrin lattice from the spectrin membrane cytoskeleton during the final stages of budding.

Key Words: annexin VI • coated pit • spectrin • endocytosis

Abbreviations used in this paper: ALLN, N-acetyl-leucyl-leucyl-norleucinal; GST, glutathione-S-transferase; LDL, low density lipoprotein.

Address all correspondence to Richard G.W. Anderson, Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75235-9039. Tel.: (214) 648-2346. Fax: (214) 648-7577. E-mail: anders06{at}utsw.swmed.edu


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