Deletions in the N-terminal segment of the plasma membrane Ca(2+) pump impair the expression of a correctly folded functional enzyme.
Mutant cDNAs encoding h4 plasma membrane Ca(2+) pumps with deletions in the N-terminal segment have been constructed and expressed in COS cells. As judged by immunoblotting, each construct was expressed at a high level similar to that of the wild-type enzyme. The removal of the first six amino acids had no effect on the Ca(2+) transport activity, but deletions in the segment 15-75 reduced the activity to undetectable levels. The d(43-56)h4 mutant, lacking amino acids 43-56, was also efficiently expressed in stable form in CHO cells. The Ca(2+) transport activity of d(43-56)h4 in this system was about 40% of that of the wild type. The d(43-56)h4 enzyme exhibited a similar affinity for Ca(2+), a slightly increased apparent affinity for ATP, and a slightly lower sensitivity to inhibition by vanadate than the wild-type enzyme. Analysis of the phosphoenzyme intermediate formed in the presence of lanthanum showed that the phosphorylation reaction was not affected, but the maximum amount of phosphoenzyme was reduced to the same extent as the Ca(2+) transport activity. These results suggest that the expressed d(43-56)h4 was a mixture of fully active and inactive enzyme. The d(43-56)h4 enzyme was more easily degraded by proteases and had a higher sensitivity to heat inactivation than the wild type suggesting that the loss of function was due to the improper folding and instability of the mutant protein. On the basis of these findings, it appears that the N-terminal segment of the plasma membrane Ca(2+) pump is neither essential for synthesis nor for catalytic activity but is critical for the expression of a correctly folded functional enzyme.
Adamo
H P
HP
IQUIFIB-Facultad de Farmacia y Bioquímica (UBA), Junín 956, Buenos Aires 1113, Argentina. hpadamo@mail.retina.ar
Grimaldi
M E
ME
García Arguinzonis
M I
MI
D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion
D011994:Recombinant Proteins; D000252:Calcium-Transporting ATPases; D002118:Calcium