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Volume 271, Number 43, Issue of October 25, 1996 pp. 26995-26997
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Deletion of Amino Acid Residues 18-75 Inactivates the Plasma Membrane Ca²⁺ Pump

(Received for publication, July 9, 1996, and in revised form, July 29, 1996)

Mirta E. Grimaldi , Hugo P. Adamo , Alcides F. Rega and John T. Penniston ^¶

From the  Instituto de Química y Fisicoquímica Biológicas-Facultad de Farmacia y Bioquímica (Universidad de Buenos Aires), Junin 956, 1113 Capital Federal, Buenos Aires, Argentina and the ^¶ Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, Minnesota 55905

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.

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