The alternative carboxyl termini of avian cardiac and brain sarcoplasmic reticulum/endoplasmic reticulum Ca(2+)-ATPases are on opposite sides of the membrane.
The sarcoplasmic/endoplasmic reticulum slow-twitch or cardiac Ca(2+)-ATPase (SERCA2) is expressed as two forms (SERCA2a and SERCA2b) which vary at their extreme carboxyl termini. SERCA2a and SERCA2b are derived from alternatively spliced primary transcripts of the same gene. These two alternative carboxyl termini are highly conserved in mammals (Eggermont, J. A., Wuytack, F., De Jaegere, S., Nelles, L., and Casteels, R. (1989) Biochem. J. 260, 757-761; Lytton, J., and MacLennan, D. H. (1988) J. Biol. Chem. 263, 15024-15031) and birds (Campbell, A. M., Kessler, P. D., Sagara, Y., Inesi, G., and Fambrough, D. M. (1991) J. Biol. Chem. 266, 16050-16055). The topology of SERCA2a is believed to be identical to the fast-twitch Ca(2+)-ATPase (SERCA1) with 10 membrane-spanning domains. Based on hydropathy analysis, the extended carboxyl terminus of SERCA2b is predicted to span the endoplasmic reticulum (ER) membrane an additional (i.e. 11th) time. We have added the human c-myc epitope, a 10-amino acid sequence recognized by monoclonal antibody 9E10, onto the carboxyl termini of SERCA2a and SERCA2b to test whether or not their carboxyl termini are on the same side of the ER membrane. The added epitopes do not appear to disrupt topology as judged from unaltered Ca2+ transport. Immunocytochemical studies demonstrate that SERCA2a and SERCA2b have their carboxyl termini on opposite sides of the ER membrane; SERCA2a's is in the cytosol and SERCA2b's is in the ER lumen.
Campbell
A M
AM
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Kessler
P D
PD
Fambrough
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001717:Birds; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D004247:DNA / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D016259:Genes, myc; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D012326:RNA Splicing; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D004247:DNA; D000252:Calcium-Transporting ATPases