DELETION OF AMINO-ACID-RESIDUES-18-75 INACTIVATES THE PLASMA-MEMBRANECA2+ PUMP

A mutant of the plasma membrane Ca2+ pump hPMCA4b(d18-75)(ct120) conta ining a deletion of the N-terminal amino acid residues 18-75 and lacki ng the C-terminal 120 amino acid residues was expressed in COS-1 cells , The deletion in the N-terminal region did not significantly affect t he level of expression of the Ca2+ pump. Tryptic digestion of the hPMC A4b(d18-75)(ct120) mutant resulted in the appearance of the same fragm ents obtained by proteolysis of the hPMCA4b(ct120) enzyme, suggesting that deletion of residues 18-75 neither impeded the insertion in the m embrane nor extensively affected the folding of the mutant protein. Th e functional competence of the hPMCA4b(d18-75)(ct120) enzyme was exami ned by measuring the Ca2+ transport and the Ca2+ ATPase activity of CO S-1 cell microsomes expressing the mutant protein. Both tests proved t he mutant to be inactive, Under conditions in which hPMCA4b(ct120) bec omes phosphorylated, hPMCA4b(d18-75)(ct120) was incapable of reacting with ATP and Ca2+ to form the phosphoenzyme. Taken together these resu lts suggest that the segment of amino acids 18-75 is essential for the activity of the plasma membrane Ca2+ pump.