Mutation of conserved residues in transmembrane domains 4,6 and 8 causes loss of Ca2+ transport by the plasma membrane Ca2+ pump.
Mutants of the plasma membrane Ca2+ pump (PMCA), in which amino acids in transmembrane domains (TM) 4, 6, and 8 had been replaced, have been expressed in COS-7 cells. They were analyzed functionally by measuring the uptake of Ca2+ in microsomal preparations and by following the formation of the phosphorylated intermediate from ATP and from phosphate. The mutated residues corresponded to amino acids whose mutation in the sarcoplasmic reticulum pump (SERCA) caused loss of Ca2+ transport by the pump protein: however, only four of the six SERCA residues were conserved in the PMCA pump. Mutation of Glu423 (TM4), Asn879 or Asp883 (TM6), or Gln97l (TM8) suppressed Ca2+ transport by the pump and its ability to form the phosphorylated intermediate starting from ATP. By contrast, the ability of these mutants to form the intermediate starting from phosphate was not impaired. In two mutants (Glu423 and Asp883) it was even enhanced. Two conserved Pro residues of TM4 were also mutated, leading to the loss of the ability of the pump to form the Ca2+- and ATP-dependent phosphorylated intermediate. Unexpectedly, two of the mutations (Asn879 and Gln971) led to the mistargeting of the mutated proteins, i.e., to their retention in the endoplasmic reticulum.
Guerini
D
D
Institute of Biochemistry, Swiss Federal Institute of Technology, Zurich.
Foletti
D
D
Vellani
F
F
Carafoli
E
E
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D027682:Cation Transport Proteins; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D017124:Conserved Sequence; D007150:Immunohistochemistry; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D053499:Plasma Membrane Calcium-Transporting ATPases; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D027682:Cation Transport Proteins; D010710:Phosphates; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium