Stirling D A, Welch K A, Stark M J
Department of Biochemistry, The University, Dundee, UK.
EMBO J. 1994 Sep 15;13(18):4329-42. doi: 10.1002/j.1460-2075.1994.tb06753.x.
NUF1/SPC110, encoding a nuclear filament-related protein which is a component of the yeast spindle pole body (SPB), has been identified in a screen designed to isolate genes encoding targets of yeast calmodulin. Spc110p interacts with calmodulin by two different criteria and the calmodulin interacting region has been localized within the C-terminus of the protein. Point mutations between residues 898 and 917 further define the calmodulin binding site within this region. Mutations in this domain which abolish calmodulin binding in vitro prevent Spc110p function in vivo, demonstrating that calmodulin binding by Spc110p has important functional consequences. In keeping with a role for calmodulin in Spc110p function, we show that calmodulin localizes to the yeast SPB when cells are prepared under appropriate conditions. Non-functional mutant Spc110 proteins which cannot bind calmodulin are present at lowered steady-state levels in the cell; when their level is increased by elevated gene dosage, partial recovery of Spc110p function is seen. Overexpression of calmodulin suppresses the defect(s) associated with the mutant Spc110 proteins, supporting the notion that Spc110p stability is a consequence of its ability to bind calmodulin and pointing to a direct role for calmodulin in Spc110p function.
NUF1/SPC110编码一种与核丝相关的蛋白质,它是酵母纺锤体极体(SPB)的一个组成部分,该基因是在一个旨在分离编码酵母钙调蛋白靶标的基因的筛选中被鉴定出来的。Spc110p通过两种不同的标准与钙调蛋白相互作用,并且钙调蛋白相互作用区域已定位在该蛋白质的C末端内。898至917位残基之间的点突变进一步确定了该区域内的钙调蛋白结合位点。该结构域中的突变在体外消除了钙调蛋白结合,在体内也阻止了Spc110p的功能,这表明Spc110p与钙调蛋白的结合具有重要的功能后果。与钙调蛋白在Spc110p功能中的作用一致,我们表明,在适当条件下制备细胞时,钙调蛋白定位于酵母SPB。不能结合钙调蛋白的无功能突变Spc110蛋白在细胞中的稳态水平较低;当通过提高基因剂量增加其水平时,可以看到Spc110p功能部分恢复。钙调蛋白的过表达抑制了与突变Spc110蛋白相关的缺陷,支持了Spc110p稳定性是其结合钙调蛋白能力的结果这一观点,并表明钙调蛋白在Spc110p功能中起直接作用。