Substitution of the N-terminal segment of the plasma membrane Ca pump isoform 4 by that of isoform 1 results in a fully functional chimeric enzyme.
The N-terminal segment of the plasma membrane Ca2+ pump (PMCA) is one of the most variable regions among the four isoforms of the enzyme and its functional importance is unknown. In the present work, the N-terminal segment of the highly active C-terminally truncated h4 mutant, h4(ct120) was modified either by substituting residues 18-43 by residues 43-75 or by replacing residues 1-75 by the homologous region from isoform h1 (residues 1-79). Immunoblot analysis of microsomal membranes from transfected COS-1 cells showed that the two N-terminally mutated proteins were correctly expressed at a level similar to that of h4(ct120). Measurements of the Ca2+ uptake by microsomal vesicles from transfected COS-1 cells indicated that mutant (18-43-->43-75)h4(ct120) had only negligible Ca2+ transport activity while the chimeric (n1-79)h1h4(ct120) enzyme was fully capable of functioning as a calcium pump. Like h4(ct120), the chimeric mutant was not stimulated further by calmodulin, and was inhibited to a similar degree by the C28R2 peptide corresponding to the calmodulin binding autoinhibitory region of the pump. Moreover, the apparent affinity for Ca2+ and the ATP dependence of the chimeric enzyme were similar to those of the h4(ct120) pump suggesting that the variability of sequence between the N-terminal segment of PMCA isoforms h1 and h4 involves amino acid substitutions that do not substantially change the behavior of the h4 enzyme. Altogether, these results demonstrate that for activity the h4 Ca pump requires a specific amino acid sequence at its N-terminus, and the essential elements for a fully active enzyme can be provided by the N-terminal segment of isoform h1 despite the variability.
Talgham
S A
SA
Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113, Buenos Aires, Argentina.
Adamo
H P
HP
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D015870:Gene Expression; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D014162:Transfection
D007527:Isoenzymes; D000252:Calcium-Transporting ATPases; D002118:Calcium