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.
Murphy
J E
JE
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Pleasure
I T
IT
Puszkin
S
S
Prasad
K
K
Keen
J H
JH
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D008970:Molecular Weight; D034041:Monomeric Clathrin Assembly Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D000911:Antibodies, Monoclonal; D002966:Clathrin; D034041:Monomeric Clathrin Assembly Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C439023:clathrin assembly protein AP180; D014357:Trypsin