Deletion of amino acid residues 18-75 inactivates the plasma membrane Ca2+ pump.
A mutant of the plasma membrane Ca2+ pump hPMCA4b(d18-75)(ct120) containing a deletion of the N-terminal amino acid residues 18-75 and lacking the C-terminal 120 amino acid residues was expressed in COS-1 cells. The deletion in the N-terminal region did not significantly affect the level of expression of the Ca2+ pump. Tryptic digestion of the hPMCA4b(d18-75)(ct120) mutant resulted in the appearance of the same fragments obtained by proteolysis of the hPMCA4b(ct120) enzyme, suggesting that deletion of residues 18-75 neither impeded the insertion in the membrane nor extensively affected the folding of the mutant protein. The functional competence of the hPMCA4b(d18-75)(ct120) enzyme was examined by measuring the Ca2+ transport and the Ca2+ ATPase activity of COS-1 cell microsomes expressing the mutant protein. Both tests proved the mutant to be inactive. Under conditions in which hPMCA4b(ct120) becomes phosphorylated, hPMCA4b(d18-75)(ct120) was incapable of reacting with ATP and Ca2+ to form the phosphoenzyme. Taken together these results suggest that the segment of amino acids 18-75 is essential for the activity of the plasma membrane Ca2+ pump.
Instituto de Química y Fisicoquímica Biológicas-Facultad de Farmacia y Bioquímica (Universidad de Buenos Aires), Junin 956, 1113 Capital Federal, Buenos Aires, Argentina.
M E
ME
Grimaldi
H P
HP
Adamo
A F
AF
Rega
J T
JT
Penniston
D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology; D018076:DNA, Complementary; D006868:Hydrolysis; D016296:Mutagenesis; D010766:Phosphorylation; D017384:Sequence Deletion
D000596:Amino Acids; D018076:DNA, Complementary; D000252:Calcium-Transporting ATPases