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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.

The plasma membrane Ca(2+)-ATPase isoform 4b (PMCA4CI) with truncations in the cytoplasmically exposed COOH-terminal tail was expressed in COS and HeLa cells and in Sf9 cells using the baculovirus system. The truncated protein terminating with the acidic sequence Glu1067-Arg1087 was retained within the endoplasmic reticulum (ER), whereas mutants lacking this sequence or having it at a distance from the COOH terminus were delivered to the plasma membrane. Although the truncated protein retained in the endoplasmic reticulum was still able to form a Ca(2+)-dependent phosphoenzyme, it underwent partial degradation. Substitution of glutamic and aspartic residue(s) in the acidic region promoted rescue of the protein to the plasma membrane. The results suggest that the sequence Glu1067-Arg1087 encodes a masked signal for ER retention and for the degradation of the protein. However, its presence at the COOH terminus was not sufficient to induce ER-retention and degradation; when the sequence was attached to the full-length PMCA protein, normal plasma delivery was observed. Evidently, ER retention and degradation required the presence of the sequence in its specific location within the PMCA structure. The degradation of the protein retained in the endoplasmic reticulum occurred through the proteolytic attack at cytoplasmically exposed residues (amino acid sequence 720-750) by a cytoplasmic PEST sequence-related protease different from calpain.