Ling K Y, Maley M E, Preston R R, Saimi Y, Kung C
Laboratory of Molecular Biology, University of Wisconsin-Madison.
Eur J Biochem. 1994 Jun 1;222(2):433-9. doi: 10.1111/j.1432-1033.1994.tb18882.x.
The mechanisms by which calmodulin coordinates its numerous molecular targets in living cells remain largely unknown. To further understand how this pivotal Ca(2+)-binding protein functions in vivo, we isolated and studied nine new Paramecium behavioral mutants defective in calmodulin. Nucleotide sequences of mutant calmodulin genes indicated single amino-acid substitutions in mutants cam4(E104K), cam5-1 (D95G), cam6 (A102V), cam7 (H135R), cam14-1 (G59S) and cam15 (D50G). In addition, we encountered a second occurrence of three identified substitutions; they are cam1-2 (S101F), cam5-2 (D95G) and cam14-2 (G59S). Most of these mutational changes occurred in sites that have been highly conserved throughout evolution. Furthermore, most of these changes were not among the amino acids known to interact with the basic amphiphilic peptides of calmodulin targets. Consistent with our previous finding [Kink, J. A., Maley, M. E., Preston R. R., Ling, K.-Y., Wallen-Friedman, M. A., Saimi, Y. & Kung, C. (1990) Cell 62, 165-174], mutants that under-reacted to certain stimuli (allele number above 10) had substitutions in the N-terminal lobe of calmodulin, and those that over-reacted (below 10) had substitutions in the C-terminal lobe. No mutations were found in the central helix that connects the lobes. Thus, through undirected in vivo mutation analyses of Paramecium, we discovered that each of the two lobes of calmodulin has a distinct role in regulating the function of a specific ion channel and eventually the behavior of Paramecium. We, therefore, propose a hypothesis of functional bipartition of calmodulin that reflects its structural bipartition.
钙调蛋白在活细胞中协调其众多分子靶点的机制在很大程度上仍然未知。为了进一步了解这种关键的钙结合蛋白在体内的功能，我们分离并研究了九个新的钙调蛋白缺陷型草履虫行为突变体。突变型钙调蛋白基因的核苷酸序列表明，突变体cam4（E104K）、cam5-1（D95G）、cam6（A102V）、cam7（H135R）、cam14-1（G59S）和cam15（D50G）存在单氨基酸替换。此外，我们发现了已鉴定的三个替换的第二次出现；它们是cam1-2（S101F）、cam5-2（D95G）和cam14-2（G59S）。这些突变变化大多发生在整个进化过程中高度保守的位点。此外，这些变化大多不在已知与钙调蛋白靶点的碱性两亲肽相互作用的氨基酸之中。与我们之前的发现[Kink, J. A., Maley, M. E., Preston R. R., Ling, K.-Y., Wallen-Friedman, M. A., Saimi, Y. & Kung, C. (1990) Cell 62, 165 - 174]一致，对某些刺激反应不足的突变体（等位基因编号大于10）在钙调蛋白的N端叶中有替换，而反应过度的突变体（小于10）在C端叶中有替换。在连接叶的中央螺旋中未发现突变。因此，通过对草履虫进行无定向的体内突变分析，我们发现钙调蛋白的两个叶在调节特定离子通道的功能以及最终调节草履虫的行为方面各有不同作用。因此，我们提出了一个反映其结构二分性的钙调蛋白功能二分假说。
