Guerini D, Foletti D, Vellani F, Carafoli E
Institute of Biochemistry, Swiss Federal Institute of Technology, Zurich.
Biochemistry. 1996 Mar 12;35(10):3290-6. doi: 10.1021/bi952572f.
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.
已将跨膜结构域（TM）4、6和8中的氨基酸被替换的质膜Ca2+泵（PMCA）突变体在COS-7细胞中进行了表达。通过测量微粒体制剂中Ca2+的摄取以及追踪由ATP和磷酸盐形成的磷酸化中间体，对它们进行了功能分析。突变的残基对应于肌浆网泵（SERCA）中那些突变会导致泵蛋白丧失Ca2+转运能力的氨基酸：然而，在PMCA泵中，六个SERCA残基中只有四个是保守的。Glu423（TM4）、Asn879或Asp883（TM6）或Gln971（TM8）的突变抑制了泵的Ca2+转运及其从ATP形成磷酸化中间体的能力。相比之下，这些突变体从磷酸盐形成中间体的能力并未受损。在两个突变体（Glu423和Asp883）中，这种能力甚至增强了。TM4的两个保守Pro残基也发生了突变，导致泵形成Ca2+和ATP依赖性磷酸化中间体的能力丧失。出乎意料的是，其中两个突变（Asn879和Gln971）导致突变蛋白的靶向错误，即它们滞留在内质网中。
