Clathrin assembly protein AP-3
| Autor: | Saul Puszkin, J.E. Murphy, K Prasad, I T Pleasure, James H. Keen |
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| Rok vydání: | 1991 |
| Předmět: | |
| Zdroj: | Journal of Biological Chemistry. 266:4401-4408 |
| ISSN: | 0021-9258 1325-1333 |
| DOI: | 10.1016/s0021-9258(20)64336-8 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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