THE CYTOPLASMIC LOOP BETWEEN PUTATIVE TRANSMEMBRANE SEGMENT-6 AND SEGMENT-7 IN SARCOPLASMIC-RETICULUM CA2-ATPASE BINDS CA2+ AND IS FUNCTIONALLY IMPORTANT()

Limited proteolysis Toy proteinase K of rabbit SERCA1 Ca2+-ATPase gene rates 21 number of fragments which have been identified recently, Here , we have focused on two proteolytic C-terminal fragments, p20C and p1 9C, starting at Gly-808 and Asp-818, respectively. The longer peptide p20C binds Ca2+, as deduced from changes in migration rate by SDS-poly acrylamide gel electrophoresis performed in the presence of Ca2+ as we ll as from labeling with Ca-45(2+) in overlay experiments. In contrast , the shorter peptide p19C, a proteolysis fragment identical to p20C b ut for 10 amino acids missing at the N-terminal side, did not bind Ca2 + when submitted to the same experiments, Two cluster mutants of Ca2+- ATPase, D813A/D818A and D813A/D815A/D818A, expressed in the yeast Sacc haromyces cerevisiae, were found to have a very low Ca2+-ATPase activi ty. Region 808-818 is thus essential for both Ca2+ binding and enzyme activity, in agreement with similar results recently reported for the homologous gastric H+, K+-ATPase (Swarts, H, G. P., Klaassen, C, H. W. , de Boer, M., Fransen, J. A, M., and De Pont, J, J, H, Pi, M., (1996) J. Biol. Chem. 271, 29764-29772). However, the accessibility of prote inase K to the peptidyl huh between Leu-807 and Oly-808 clearly shows that the transmembrane segment M6 ends before region 808-818, It is re markable that critical residues]far enzyme activity are located in a c ytoplasmic loop starting at Gly-808.