Valosin-containing protein, VCP, is a ubiquitous clathrin-binding protein.

Clathrin is the structural protein of coated membranes involved in receptor-mediated endocytosis and aspects of Golgi sorting in eukaryotic cells. We have now detected a stoichiometric complex of clathrin with a novel protein of M(r) approximately 100,000 (100K) in lysates of different mammalian cells. Formation of the complex, which also includes the 70K heat-shock protein Hsc70, occurs within 15 min of synthesis. The 100K protein has been identified as valosin-containing protein (VCP; ref. 1), an early substrate for tyrosine phosphorylation on T-cell receptor activation. Further, VCP is the mammalian homologue of yeast Cdc48p (ref. 3) and is a member of a larger gene family that includes putative ATP-binding proteins involved in vesicle transport and fusion, 26S proteasome function, regulation of the expression of human immunodeficiency virus, and assembly of peroxisomes. The association with clathrin and the morphological and catalytic similarity to the chaperonin proteins indicate that VCP may modulate protein-protein interactions in membrane transport processes.

Stable clathrin: uncoating protein (hsc70) complexes in intact neurons and their axonal transport.

We have studied the organization of clathrin during its transport in axons. Using immunoprecipitation techniques we have confirmed earlier findings that clathrin is transported as part of slow component b, but we also detect small amounts of clathrin in fast component. As fast component is known to correspond to the transport of membraneous material, including coated vesicle membrane components, our findings suggest that some clathrin in axons undergoes transport in the form of coated membranes and that a portion of the clathrin delivered to axons and axon terminals arrives by way of fast component. The organizational form of clathrin in slow component b (SCb) was examined in more detail, as it is thought to represent a non-membrane-associated species, is relatively long-lived, and at any instant represents the major transport species in axons. We used nondenaturing immunoprecipitation methods with stringent wash procedures to identify other SCb proteins that interact with clathrin. The immunoprecipitates contained major labeled bands that corresponded to clathrin heavy and light chains, along with a prominent 70-kDa band and several minor bands that ranged in apparent Mr from 70,000 to 150,000; the 70-kDa band was shown to be the ATP-dependent uncoating protein by two-dimensional gel electrophoresis. A very similar profile of polypeptides was also immunoprecipitated from extracts of cultured neurons. The results from a variety of control immunoprecipitations, including the use of antisera preadsorbed with purified clathrin trimers or clathrin light chains, indicate that coprecipitation of clathrin and uncoating protein with the other 70,000-150,000-Da polypeptides from SCb reflects specific interactions. Including exogenous uncoating protein in the lysis buffer had no detectable effect on the levels of endogenous uncoating protein recovered in the immunoprecipitates, indicating that complexes of clathrin, uncoating protein, and the other coimmunoprecipitating SCb protein existed in the intact neurons prior to lysis. Finally, a specific and functional association is further supported by the release of uncoating protein, but not the other 70,000-150,000-Da polypeptides, from the immunoprecipitated complexes on the addition of ATP. Collectively, these observations provide the first direct evidence of interaction between clathrin and uncoating protein in intact cells, lend strong support to the concept that uncoating protein plays an intimate role in clathrin dynamics within cells, and reveal a family of 70,000-150,000-Da polypeptides that form a stable nonmembranous association with clathrin in intact cells.

Clathrin assembly protein AP-3. The identity of the 155K protein, AP 180, and NP185 and demonstration of a clathrin binding domain.

Three independently isolated clathrin-associated proteins have been reported that have molecular weights of approximately 155,000-185,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: the 155K protein (Keen, J. H., and Black, M. M. (1986) J. Cell Biol. 102, 1325-1333), AP 180 (Ahle, S., and Ungewickell, E. (1986) EMBO J. 5, 3143-3149), and NP185 (Kohtz, D. S., and Puszkin, S. (1988) J. Biol. Chem. 263, 7418-7425). Using two-dimensional isoelectric focusing polyacrylamide gel electrophoresis and one- and two-dimensional immunoblots with two different monoclonal antibodies, we show that these three proteins are identical. The term AP-3 is used to denote this protein. A preliminary analysis of the domain structure of AP-3 was done by controlled proteolysis. Trypsin treatment of AP-3 yields two distinct classes of products. The larger fragments obtained (100,000-135,000 apparent Mr) are acidic and behave anomalously on gel electrophoresis, yielding aberrantly high Mr and exhibiting poor dye binding; these characteristics are shared with intact AP-3. Trypsin also generates a smaller neutral species of approximately 30,000 Da which migrates appropriately on sodium dodecyl sulfate-gel electrophoresis, binds dye comparatively strongly, and behaves as a monomeric globular species in solution. In addition, this species, which is also released by a variety of other proteases, binds specifically and reversibly to clathrin-Sepharose, identifying it as a clathrin recognition domain.