Expression and functional characterization of isoforms 4 of the plasma membrane calcium pump.
PMCA isoforms 4CII (generated by splicing at the C-terminus) and 4BICI (a pump version lacking the 10th transmembrane domain) were expressed in Sf9 cells using the baculovirus system. The purified PMCA4CII had a 20-fold lower affinity for calmodulin than the PMCA4CI, the PMCA4 isoform of the erythrocytes' membranes, but had a higher activity in the absence of calmodulin. The amount of phosphoenzyme intermediate formed by PMCA4CII in the presence of Ca2+ alone was almost 3 times higher than in PMCA4CI and was increased by La3+ less than in the PMCA4CI. The isoform lacking the 10th transmembrane domain (PMCA4BICI) had no Ca2+-dependent ATPase activity, but was still able to form the phosphoenzyme intermediate starting from phosphate. When expressed in COS cells, this isoform was retained in the endoplasmic reticulum; changes in membrane architecture apparently occurred during its expression; the C-terminal portion of the isoform was located in the cytosol, indicating that the deletion of the 10th transmembrane domain resulted in the loss of at least another transmembrane domain.
Preianò
B S
BS
Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zürich, Switzerland.
Guerini
D
D
Carafoli
E
E
D017398:Alternative Splicing; D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004910:Erythrocyte Membrane / Q000201:enzymology; D015870:Gene Expression; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008961:Models, Structural; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection; D014357:Trypsin
D002147:Calmodulin; D007527:Isoenzymes; D011994:Recombinant Proteins; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D002122:Calcium Chloride