The cytoplasmic loop between putative transmembrane segments 6 and 7 in sarcoplasmic reticulum Ca2+-ATPase binds Ca2+ and is functionally important.

Limited proteolysis by proteinase K of rabbit SERCA1 Ca2+-ATPase generates a number of fragments which have been identified recently. Here, we have focused on two proteolytic C-terminal fragments, p20C and p19C, starting at Gly-808 and Asp-818, respectively. The longer peptide p20C binds Ca2+, as deduced from changes in migration rate by SDS-polyacrylamide gel electrophoresis performed in the presence of Ca2+ as well as from labeling with 45Ca2+ in overlay experiments. In contrast, the shorter peptide p19C, a proteolysis fragment identical to p20C but 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 Saccharomyces cerevisiae, were found to have a very low Ca2+-ATPase activity. 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. H. M. (1996) J. Biol. Chem. 271, 29764-29772). However, the accessibility of proteinase K to the peptidyl link between Leu-807 and Gly-808 clearly shows that the transmembrane segment M6 ends before region 808-818. It is remarkable that critical residues for enzyme activity are located in a cytoplasmic loop starting at Gly-808.