ATP- and Cytosol-dependent Release of Adaptor Proteins from Clathrin-coated Vesicles: A Dual Role for Hsc70

Abstract

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP-and cytosol-dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release. INTRODUCTIONMost, if not all, intracellular transport vesicles are encased in a proteinaceous coat, one class of which is clathrin-coated vesicles (CCV). 1 CCV mediate the transport of lysosomal hydrolases from the transGolgi network, as well as the efficient internalization of extracellular solutes such as nutrients, hormones, growth factors, and immunoglobulins at the plasma membrane. Their coat contains two components: clathrin (heavy and light chains) and adaptor protein complexes (AP). There are two distinct classes of CCVassociated APs. The AP1 complex is found on transGolgi network-derived CCV, and AP2 is found on plasma membrane-derived CCV (Robinson, 1987;Ahle et al., 1988). More recently, a new AP complex AP3 has been identified that is partially associated with clathrin-coated structures in vivo (Simpson et al., 1997;Dell' Angelica et al., 1998). Both clathrin-associated AP complexes are heterotetramers consisting of two different subunits of ϳ100 kDa (␥ and ␤1 in AP1, ␣ and ␤2 in AP2), one medium chain subunit (1 or 2) of ϳ50 kDa, and a small chain subunit (1 or 2) of ϳ17 kDa (reviewed by Robinson, 1994;Kirchhausen et al., 1997;Schmid, 1997).A fully assembled clathrin coat, viewed by electron microscopy, appears as a polygonal lattice of clathrin on the outer surface of the vesicle, with APs located at each vertex (Vigers et al., 1986a(Vigers et al., , 1986bHeuser and Keen, 1988). The APs form the innermost shell of the coat and associate with the plasma membrane via saturable, high-affinity, and protease-sensitive receptors (Mahaffey et al., 1990). In low-ionic strength, low-pH buffers, clathrin can spontaneously self-assemble into a closed polygonal lattice (Kartenbeck, 1978;Woodward and Roth, 1978;Crowther and Pearse, 1981). APs coassemble with clathrin and promote coat assembly under physiological conditions that do not otherwise support clathrin self-assembly (Keen et al., 1979;Pearse and Robinson, 1984;Ahle and Ungewickell, 1989). Given the ability of APs to stimu...

Exchange of clathrin, AP2 and epsin on clathrin-coated pits in permeabilized tissue culture cells

“…Similarly, neither AP1 nor GGA dissociated with the clathrin from the TGN. These results are consistent with the observation of Hannan et al, who suggested that, Hsc70 is in some way necessary for dissociation of AP2 from CVs, but it is not sufficient (Hannan et al, 1998). A cofactor in cytosol, which has recently been identified by Ghosh et al to be a phosphatase, has been shown to be required for dissociation of AP1 from CVs (Ghosh et al, 2003).…”

Hsp70 dynamics in vivo: effect of heat shock and protein aggregation

“…However, this effect may occur because GFP-Hsp70(K71E) becomes completely immobilized in the nucleus and partially immobilized in the cytosol during heat shock. By contrast, in untreated cells, GFP-Hsp70(K71E) is only slightly less mobile than GFP-Hsp70 and, therefore, its slow rate of transport into and out of the nucleus is probably either because of its inability to bind ATP tightly and hydrolyze it (O'Brien et al, 1996) or because of its related tendency to oligomerize with itself in vitro (Rajapandi et al, 1998) and with Hsp70 in vivo (Hannan et al, 1998). This tendency to oligomerize could also explain why expression of Hsp70(K71E) had a trans-dominant effect on GFP-Hsp70 transport into and out of the nucleus before heat shock and why it prevented these rates from changing during heat shock.…”

Association of l-Glutamic Acid Decarboxylase to the 70-kDa Heat Shock Protein as a Potential Anchoring Mechanism to Synaptic Vesicles

“…The ubiquitous binding partners of HSP70 include a wide range of unfolded peptides as well as native proteins. Two native proteins found in the nerve terminal that bind with the constitutively expressed member of the heat shock protein 70 family, HSC70, are the clathrin triskelions of coated vesicles (33,34) and the intrinsic synaptic vesicle protein, CSP (35)(36)(37).…”