Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors.

Abstract

Abstract. Using stage-specific assays for receptormediated endocytosis of transferrin (-Tfn) into perforated A431 cells we show that purified adaptors stimulate coated pit assembly and ligand sequestration into deeply invaginated coated pits. Late events in endocytosis involving membrane fission and coated vesicle budding which lead to the internalization of Tfn are unaffected. AP2, plasma membrane adaptors, are active at physiological concentrations, whereas AP1, Golgi adaptors, are inactive. Adaptor-dependent stimulation of Tfn sequestration requires cytosolic clathrin, but is unaffected by clathrin purified from coated vesicles suggesting that soluble and assembled clathrin pools are functionally distinct. In addition to adaptors and cytosolic clathrin other, as yet unidentified, cytosolic factors are also required for efficient coated pit invagination. These results provide new insight into the mechanisms and regulation of coated pit assembly and invagination.

Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis.

“…To directly measure de novo coated pit assembly, assays are performed in the presence of limiting concentrations of cytosol supplemented with purified adaptor proteins. When perforated A431 cells are incubated under these conditions adaptors specifically stimulate coated pit assembly which increases the extent of sequestration of BSST into deeply invaginated pits (Smythe et al, 1992b). Thus, adaptor-stimulated sequestration of BSST provides a measurement of the de novo assembly of functionally active coated pits (Smythe et al, 1992b).…”

“…Extensive biochemical and morphological characterization of this assay system (Smythe et al, 1989; Schrnid and Smythe, 1991;Smythe et al, 1992b) has shown that coated vesicles which form during the in vitro reaction are largely derived from pre-existing coated pits. As a result, MesNaresistance selectively measures late events which correspond to coated vesicle budding and lead to the internalization of BSST.…”

“…Clathrin and adaptors exist as distinct soluble pools which appear to assemble sequentially to form clathrin-coated pits (Mahaffey et al, 1990;Smythe et al, 1992b). In contrast, COPs are present in the cytosol as a large multimeric precursor termed a "coatomer" which presumably self-assembles onto membranes to form COPcoated pits (Waters et al, 1991).…”

“…The data are expressed as the percent of total cell-associated BSST which became inaccessible to either probe in an ATP and cytosoldependent manner. Untreated cell lysates obtained following an incubation of cells in the presence of 2/tg/ml BSST but in the absence of ATP and cytosol were plated onto microtitre wells in serial dilutions to determine total ceU-associated BSST and to ensure that the binding capacity of the wells for BSST was not exceeded.backgrounds due to incomplete reduction of surface-bound BSST are reduced.Extensive biochemical and morphological characterization of this assay system (Smythe et al, 1989; Schrnid and Smythe, 1991;Smythe et al, 1992b) has shown that coated vesicles which form during the in vitro reaction are largely derived from pre-existing coated pits. As a result, MesNaresistance selectively measures late events which correspond to coated vesicle budding and lead to the internalization of BSST.…”

“…The cytosol was prepared from bovine brain as previously described (Schmid and Smythe, 1992) except that it was immediately gel filtered by chromatography using a Sephadex (325 column equilibrated with KSHM. Gel-filtered cytosol preparations were rapidly frozen in liquid nitrogen and stored in aliquots at -70~ Bovine brain adaptors were prepared exactly as described by Smythe et al (1992b). Internalization and sequestration assays were performed exactly as previously described (Schmid and Smythe, 1991;Smythe et al, 1992a).…”