Expression of avian Ca2+-ATPase in cultured mouse myogenic cells.

Abstract

cDNA encoding Ca2+-ATPase was cloned from a chicken skeletal muscle library. The cDNA (termed FCa) comprised 3,239 base pairs, including an open reading frame encoding 994 amino acids which showed the highest degree of homology with the adult rabbit fast-twitch Ca2+-ATPase isoform (C. J. Brandl, S. de Leon, D. R. Martin, and D. H. MacLennan, J. Biol. Chem. 262:3768-3774, 1987). Radiolabeled FCa hybridized to a 3.2-kilobase transcript in chicken skeletal muscle RNA but not to cardiac muscle RNA, which confirmed its identity as encoding the fast Ca2+-ATPase isoenzyme. FCa was transfected into the mouse myogenic line C2C12, from which a protein of 100 kilodaltons was immunopurified by using a monoclonal antibody specific for the avian fast Ca2+-ATPase. Immunofluorescence microscopy of a line (designated C2FCa2) stably expressing the avian Ca2+-ATPase localized the protein to the nuclear envelope and a population of cytoplasmic vesicles. A similar pattern was observed when C2FCa2 cells were stained with DiOC6(3), a cyanine dye that labels endoplasmic reticulum and mitochondria (M. Terasaki, J. Song, J. R. Wong, M. J. Weiss, and L. B. Chen, Cell 38:101-108, 1984). We conclude that the avian Ca2+-ATPase fast isoform is expressed and correctly targeted to the endoplasmic reticulum in mouse C2C12 cells.