Caviston Juliane P, Longtine Mark, Pringle John R, Bi Erfei
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6058, USA.
Mol Biol Cell. 2003 Oct;14(10):4051-66. doi: 10.1091/mbc.e03-04-0247. Epub 2003 Jul 25.
The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42V36G allele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42V36G or GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth.
Septin蛋白家族是一类保守的GTP结合且能形成丝状结构的蛋白质。在酿酒酵母中，Septin蛋白在母细胞与芽体的颈部形成一个环，其主要功能似乎是作为一个支架，用于招募其他蛋白质至颈部，这些蛋白质在颈部参与胞质分裂及多种其他过程。Septin环的形成依赖于Rho型GTP酶Cdc42p，但似乎与肌动蛋白细胞骨架无关。在本研究中，我们进一步探究了Septin环形成的机制。光漂白后荧光恢复（FRAP）实验表明，在假定的芽体部位最初的Septin结构是不稳定的（亚基可自由交换），但随着芽体出现，它会转变为一个稳定的环。携带cdc42V36G等位基因或缺失两个或全部三个已知的Cdc42p GTP酶激活蛋白（GAPs：Bem3p、Rga1p和Rga2p）的突变体能够将Septin蛋白招募至细胞皮层，但在形成正常的Septin环时受阻或延迟，并且伴有形态发生缺陷。在Cla4p或Gin4p也存在缺陷的突变体中，这些表型显著增强，Cla4p和Gin4p这两种蛋白激酶先前已被证明对正常的Septin环形成很重要。Cdc42p GAPs在细胞周期的早期和晚期均与Septin蛋白共定位，并且GAPs的过表达能够抑制温度敏感型Septin突变体的Septin组织和形态发生缺陷。综合来看，这些数据表明成熟Septin环的形成是一个至少由两个可区分步骤组成的过程，即Septin蛋白招募至假定的芽体部位以及它们组装成稳定的Septin环。这两个步骤似乎都依赖于Cdc42p，而Cdc42p GAPs以及其他已知促进正常Septin环形成的蛋白质似乎在组装步骤中以部分冗余的方式发挥作用。此外，由于在cdc42V36G或GAP突变体中最终形成正常的Septin环总是伴随着从异常伸长的芽形态转变为环远端更正常的芽形态，因此似乎Septin环在决定芽生长模式中起直接作用。
