Smith Jean A, Rose Mark D
Department of Molecular Biology, Princeton University, New Jersey 08544.
Department of Molecular Biology, Princeton University, New Jersey 08544
Genetics. 2016 Apr;202(4):1421-35. doi: 10.1534/genetics.115.185207. Epub 2016 Feb 10.
Cell fusion is ubiquitous among eukaryotes. Although little is known about the molecular mechanism, several proteins required for cell fusion in the yeast Saccharomyces cerevisiae have been identified. Fus2p, a key regulator of cell fusion, localizes to the shmoo tip in a highly regulated manner. C-terminal truncations of Fus2p cause mislocalization and fusion defects, which are suppressed by overexpression of Kel1p, a kelch-domain protein of unknown function previously implicated in cell fusion. We hypothesize that Fus2p mislocalization is caused by auto-inhibition, which is alleviated by Kel1p overexpression. Previous work showed that Fus2p localization is mediated by both Fus1p- and actin-dependent pathways. We show that the C-terminal mutations mainly affect the actin-dependent pathway. Suppression of the Fus2p localization defect by Kel1p is dependent upon Fus1p, showing that suppression does not bypass the normal pathway. Kel1p and a homolog, Kel2p, are required for efficient Fus2p localization, acting through the actin-dependent pathway. Although Kel1p overexpression can weakly suppress the mating defect of a FUS2 deletion, the magnitude of suppression is allele specific. Therefore, Kel1p augments, but does not bypass, Fus2p function. Fus2p mediates cell fusion by binding activated Cdc42p Although Kel1p overexpression suppresses a Cdc42p mutant that is defective for Fus2p binding, cell fusion remains dependent upon Fus2p These data suggest that Fus2p, Cdc42p, and Kel1p form a ternary complex, which is stabilized by Kel1p Supporting this hypothesis, Kel1p interacts with two domains of Fus2p, partially dependent on Cdc42p We conclude that Kel1p enhances the activity of Fus2p/Cdc42p in cell fusion.
细胞融合在真核生物中普遍存在。尽管对其分子机制了解甚少，但酿酒酵母细胞融合所需的几种蛋白质已被鉴定出来。Fus2p是细胞融合的关键调节因子，以高度调控的方式定位于shmoo尖端。Fus2p的C末端截短会导致定位错误和融合缺陷，而Kel1p（一种功能未知的kelch结构域蛋白，先前被认为与细胞融合有关）的过表达可抑制这些缺陷。我们假设Fus2p定位错误是由自身抑制引起的，而Kel1p过表达可缓解这种抑制。先前的研究表明，Fus2p的定位由Fus1p和肌动蛋白依赖性途径介导。我们发现C末端突变主要影响肌动蛋白依赖性途径。Kel1p对Fus2p定位缺陷的抑制依赖于Fus1p，这表明抑制作用不会绕过正常途径。Kel1p及其同源物Kel2p是Fus2p有效定位所必需的，它们通过肌动蛋白依赖性途径发挥作用。尽管Kel1p过表达可微弱抑制FUS2缺失的交配缺陷，但抑制程度具有等位基因特异性。因此，Kel1p增强但不绕过Fus2p的功能。Fus2p通过结合活化的Cdc42p介导细胞融合。尽管Kel1p过表达可抑制对Fus2p结合有缺陷的Cdc42p突变体，但细胞融合仍依赖于Fus2p。这些数据表明Fus2p、Cdc42p和Kel1p形成三元复合物，该复合物由Kel1p稳定。支持这一假设的是，Kel1p与Fus2p的两个结构域相互作用，部分依赖于Cdc42p。我们得出结论，Kel1p增强了Fus2p/Cdc42p在细胞融合中的活性。
