CA2+ BINDING TO CALMODULIN AND ITS ROLE IN SCHIZOSACCHAROMYCES-POMBE AS REVEALED BY MUTAGENESIS AND NMR-SPECTROSCOPY
CA2+ BINDING TO CALMODULIN AND ITS ROLE IN SCHIZOSACCHAROMYCES-POMBE AS REVEALED BY MUTAGENESIS AND NMR-SPECTROSCOPY
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DOI：
10.1074/jbc.270.35.20643
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发表时间：
1995-09-01
影响因子：
4.8
通讯作者：
DAVIS, TN
中科院分区：
文献类型：
--
作者：
MOSER, MJ;LEE, SY;DAVIS, TN
作为鉴定pombe Schizosaccharomyces calmodulin的重要结构元件的第一步，我们检测了异源calmodulin和Ca2+结合位点突变S. pombe calmodulin取代cam1（+）基因的能力，一个编码脊椎动物钙调蛋白的cDNA允许S. pombe生长。然而，来自酿酒酵母的钙调素并不支持生长，即使这种蛋白的产量很高。除了一个例外，所有突变的S. pombe钙调素具有一个或多个完整的Ca2+结合位点，允许在21℃下生长。只有一个完整的Ca2+结合位点3的突变体不能支持生长，所有四个Ca2+结合位点突变的S. pombe钙调素也是如此。一些突变蛋白赋予温度敏感表型。对温度敏感性程度的分析允许Ca2+结合位点根据它们支持裂变酵母增殖的能力进行排名。2号点比1号点重要，1号点比4号点重要，4号点比3号点重要。基于裂变酵母ade1（+）基因的视觉菌落彩色屏幕被开发来执行这些遗传分析。为了比较各个位点的Ca2+结合特性及其对生存能力的功能重要性，利用H-1核磁共振光谱研究了pombe中Ca2+与钙调蛋白的结合。核磁共振分析表明Ca2+结合谱不同于先前确定的脊椎动物和酿酒酵母钙调素。Ca2+结合位点3对Ca2+的相对亲和力最高，而位点1、2和4的亲和力难以区分。体内功能测定和体外物理测定的结合表明，Ca2+位点的相对亲和力不能预测其功能重要性。
As a first step toward identifying the important structural elements of calmodulin from Schizosaccharomyces pombe, we examined the ability of heterologous calmodulins and Ca2+-binding site mutant S. pombe calmodulins to replace the essential cam1(+) gene, A cDNA encoding vertebrate calmodulin allows growth of S. pombe. However, calmodulin from Saccharomyces cerevisiae does not support growth even though the protein is produced at high levels. With one exception, all mutant S. pombe calmodulins with one or more intact Ca2+ binding sites allow growth at 21 degrees C. A mutant containing only an intact Ca2+-binding site 3 fails to support growth, as does S. pombe calmodulin with all four Ca2+ binding sites mutated. Several of the mutant proteins confer a temperature-sensitive phenotype. Analysis of the degree of temperature sensitivity allows the Ca2+ binding sites to be ranked by their ability to support fission yeast proliferation. Site 2 is more important than site 1, which is more important than site 4, which is more important than site 3. A visual colony color screen based on the fission yeast ade1(+) gene was developed to perform these genetic analyses. To compare the Ca2+ binding properties of individual sites to their functional importance for viability, Ca2+ binding to calmodulin from S. pombe was studied by H-1 NMR spectroscopy. NMR analysis indicates a Ca2+-binding profile that differs from those previously determined for vertebrate and S. cerevisiae calmodulins. Ca2+-binding site 3 has the highest relative affinity for Ca2+, while the affinities of sites 1, 2, and 4 are indistinguishable. A combination of an in vivo functional assay and an in vitro physical assay reveals that the relative affinity of a site for Ca2+ does not predict its functional importance.