MUTATION OF CONSERVED RESIDUES IN TRANSMEMBRANE DOMAIN-4, DOMAIN-6, AND DOMAIN-8 CAUSES LOSS OF CA2-MEMBRANE CA2+ PUMP( TRANSPORT BY THE PLASMA)

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 measurin g the uptake of Ca2+ in microsomal preparations and by following the f ormation of the phosphorylated intermediate from ATP and from phosphat e. 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 Asp88 3 (TM6), or Gln971 (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 start ing from phosphate was not impaired. In two mutants (Glu423 and Asp883 ) it was even enhanced. Two conserved Pro residues of TM4 were also mu tated, 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 mutat ed proteins, i.e., to their retention in the endoplasmic reticulum.