Verboomen H, Wuytack F, Van den Bosch L, Mertens L, Casteels R
Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Belgium.
Biochem J. 1994 Nov 1;303 ( Pt 3)(Pt 3):979-84. doi: 10.1042/bj3030979.
Ca(2+)-uptake experiments in microsomal fractions from transfected COS-1 cells have revealed a functional difference between the non-muscle SERCA2b Ca2+ pump and its muscle-specific SERCA2a splice variant. Structurally, the two pumps differ only in their C-terminal tail. The last four amino acids of SERCA2a are replaced in SERCA2b by a 49-residue-long peptide chain containing a very hydrophobic stretch which could be an additional transmembrane segment. The functionally important subdomains in the SERCA2b tail were analysed by constructing three SERCA2b deletion mutants lacking 12, 31 or 49 amino acids. The mutants and the parental SERCA2 pumps were expressed in COS-1 cells and analysed for functional difference. SERCA2b had a twofold higher Ca2+ affinity, a twofold lower turnover rate and a 10-fold lower vanadate-sensitivity than SERCA2a and the mutants. Since each of the three truncated versions of SERCA2b acquire the characteristic properties of SERCA2a, it is concluded that the stretch of the last 12 residues of SERCA2b is of critical importance.
对转染的COS-1细胞微粒体部分进行的钙离子摄取实验揭示了非肌肉型SERCA2b钙离子泵与其肌肉特异性剪接变体SERCA2a之间的功能差异。从结构上看，这两种泵仅在其C末端尾巴上有所不同。SERCA2a的最后四个氨基酸在SERCA2b中被一条49个残基长的肽链取代，该肽链包含一个非常疏水的片段，可能是一个额外的跨膜片段。通过构建三个分别缺失12、31或49个氨基酸的SERCA2b缺失突变体，分析了SERCA2b尾巴中功能重要的亚结构域。这些突变体和亲本SERCA2泵在COS-1细胞中表达，并分析其功能差异。SERCA2b的钙离子亲和力比SERCA2a和突变体高两倍，周转率低两倍，钒酸盐敏感性低10倍。由于SERCA2b的三个截短版本中的每一个都获得了SERCA2a的特征性质，因此得出结论，SERCA2b最后12个残基的片段至关重要。
