Identification and characterization of membrane-bound calpains in clathrin-coated vesicles from bovine brain.
Calpains are intracellular cysteine proteases activated in a Ca(2+)-dependent manner. Previously, we found that the differentiation of K562 cells induced by 4 beta-phorbol 12-myristate 13-acetate treatment is accompanied by an increase in m-calpain levels and, at the same time, m-calpain becomes localized on the inside of plasma membranes, coated pits, and coated vesicles [Nakamura, M., Mori, M., Morishita, Y., Mori, S. & Kawashima, S. (1992) Exp. Cell Res. 200, 513-522]. We also reported that mu-calpain plays an essential role in morphological changes and membrane fusion of erythrocytes through the degradation of spectrin, a lining protein [Hayashi, M., Saito, Y. & Kawashima, S. (1992) Biochim. Biophys. Res. Commun. 182, 939-946]. Thus, calpains are implicated in endocytosis and/or exocytosis, processes stimulated by Ca2+ and involving intracellular membrane fusion. In this study, we report the biochemical characterization of calpains as components of purified coated vesicles from bovine brain. It was found by Western-blot analysis and chemical cross-linking of proteins that calpains are bound to the membranes of coated vesicles, and not to the coats. The binding of m-calpain to vesicles is Ca(2+)-dependent, while that of mu-calpain is less dependent on the presence of Ca2+. We also identified substrate proteins for calpains in coated vesicles. Upon activation of endogenous calpains, component proteins of coated vesicles such as the clathrin light chain, tubulins, and adaptins, but not the clathrin heavy chain, are highly sensitive to calpain digestion. In the case of exogenously added calpains, low concentrations degraded the same protein components. The degradation pattern differs slightly between added mu-calpain and m-calpain. These results strongly suggest that calpains are involved in the formation of coated vesicles and/or vesicle fusion to endosomes.
Sato
K
K
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan.
Saito
Y
Y
Kawashima
S
S
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002154:Calpain / Q000032:analysis* / Q000502:physiology; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002966:Clathrin / Q000494:pharmacology*; D004705:Endocytosis
D002966:Clathrin; D002154:Calpain