Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity.

D T Mahaffey,J S Peeler,F M Brodsky,R G Anderson

doi:10.1016/s0021-9258(17)46253-3

D T Mahaffey, J S Peeler + Show 2 more

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https://doi.org/10.1016/s0021-9258(17)46253-3

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Abstract

Coated pits will assemble onto purified plasma membranes that are attached to a poly-L-lysine coated substratum (Moore, M. S., Mahaffey, D. T., Brodsky, F. M., and Anderson, R. G. W. (1987) Science 236, 558-563; Mahaffey, D. T., Moore, M. S., Brodsky, F. M., and Anderson, R. G. W. (1989) J. Cell Biol. 108, 1615-1624). To better understand the assembly reaction, we have purified both clathrin triskelion and AP-2 subunits from bovine brain and assayed for their ability to bind to the cytoplasmic surface of attached membranes. Two types of membranes were analyzed: those washed with a high pH buffer that selectively removes triskelions and those washed with a high salt buffer that removes both the AP-2 and the triskelion subunits. We found that purified AP-2 subunits bind with high affinity (Kd approximately 3 x 10(-8) M) to salt stripped membranes. Binding is saturable and abolished by treating membranes with less than 20 micrograms/ml of elastase. When membranes were treated with elastase before the salt wash and then salt washed and assayed for AP-2 binding, normal binding was seen, which indicates that the presence of clathrin-coated pits protects the binding site from the protease. Membranes that had rebound AP-2 did not bind purified triskelions, even though high pH buffer-washed membranes that bear endogenous AP-2 bound triskelions with high affinity (Kd approximately 3 x 10(-9) M) and supported lattice assembly. We conclude that coated pit assembly is initiated by the binding of AP-2 to an integral membrane protein but that the AP-2 complex must be activated by an unknown process before the coated pit lattice will assemble.

Highlights

From the *Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235 and the YDepartment of Pharmacy, University of California at San Francisco, San Francisco, California 74143
Membranes that had been treated with high pH buffer had lost clathrin but not AP-2, whereas the Tris salt-treated membranes had diminished amounts of both clathrin and AP-2 (Fig. 2)
The ability to control the molecular composition of the coated pits allowed us to determine how purified clathrin and AP-2 interacted with membranes that contained either clathrin and AP-2, only AP-2, or neither of these subunits

Summary

Present address

There is no information available, about whether these same interactions occur during coated pit formation, which is vital knowledge for understanding the assembly of this organelle For this reason, we have employed the in vitro coated pit assembly assay we developed [17,18] to measure the interaction of AP2 with the inner membrane surface, as well as the interaction of triskelions with AP-2 that is bound to these membranes. We have employed the in vitro coated pit assembly assay we developed [17,18] to measure the interaction of AP2 with the inner membrane surface, as well as the interaction of triskelions with AP-2 that is bound to these membranes These studies have revealed the presence of an integral membrane protein in the coated pit that binds AP-2 with high affinity

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