Foletti D, Guerini D, Carafoli E
Institute of Biochemistry, Swiss Federal Institute of Technology, Zurich.
FASEB J. 1995 May;9(8):670-80. doi: 10.1096/fasebj.9.8.7768360.
ATP-powered Ca2+ pumps are located in the plasma membrane (PMCA) and the sarco(endo)plasmic reticulum (SERCA). The two pump types share numerous structural and functional features; nevertheless, they are strictly targeted to different cell compartments. Chimeric SERCA/PMCA pumps were constructed to investigate the structural determinants responsible for their specific cellular location. The level of expression of the chimeric constructs and of the wild-type pumps in COS-7 and Sf9 cells was the same and so was their stability. One exception was chimera D, which showed special propensity to degradation. The chimeric constructs had no Ca(2+)-dependent ATPase activity, although in one trace amounts of the Ca(2+)-dependent phosphoenzyme intermediate were detected. Thus, the exchange of the regions encompassing the (NH2-terminal) transmembrane domains apparently is incompatible with the activity of the pump. The immunofluorescence experiments showed that the 85 NH2-terminal residues of the SERCA pump, encompassing the first transmembrane domain, contain a signal-promoting retention of chimeric constructs otherwise consisting of the PMCA pump structure in the endoplasmic reticulum. Additional structural determinants most likely also contribute to the retention of the SERCA pump in the endoplasmic reticulum: one chimeric construct (E) composed of the first two transmembrane domains of the PMCA pump, followed by the remainder of the SERCA pump structure, was still retained, even if not completely, in the endoplasmic reticulum.
由ATP供能的Ca2+泵位于质膜（质膜钙泵，PMCA）和肌浆（内质）网（肌浆网钙泵，SERCA）中。这两种泵类型具有许多结构和功能特征；然而，它们严格定位于不同的细胞区室。构建了嵌合型SERCA/PMCA泵，以研究决定其特定细胞定位的结构决定因素。嵌合构建体和野生型泵在COS-7细胞和Sf9细胞中的表达水平相同，其稳定性也相同。一个例外是嵌合体D，它表现出特别的降解倾向。嵌合构建体没有Ca(2+)依赖性ATP酶活性，尽管在一个构建体中检测到微量的Ca(2+)依赖性磷酸酶中间体。因此，包含（氨基末端）跨膜结构域的区域交换显然与泵的活性不相容。免疫荧光实验表明，SERCA泵的85个氨基末端残基，包括第一个跨膜结构域，包含一个促进信号，可将原本由PMCA泵结构组成的嵌合构建体保留在内质网中。其他结构决定因素很可能也有助于SERCA泵保留在内质网中：一种嵌合构建体（E）由PMCA泵的前两个跨膜结构域组成，后面跟着SERCA泵结构的其余部分，即使没有完全保留，也仍保留在内质网中。
