Chen G C, Zheng L, Chan C S
Department of Microbiology, The University of Texas, Austin, 78712, USA.
Mol Cell Biol. 1996 Apr;16(4):1376-90. doi: 10.1128/MCB.16.4.1376.
Normal cell growth in the yeast Saccharomyces cerevisiae involves the selection of genetically determined bud sites where most growth is localized. Previous studies have shown that BEM2, which encodes a GTPase-activating protein (GAP) that is specific for the Rho-type GTPase Rho1p in vitro, is required for proper bud site selection and bud emergence. We show here that DBM1, which encodes another putative Rho-type GAP with two tandemly arranged cysteine-rich LIM domains, also is needed for proper bud site selection, as haploid cells lacking Dbm1p bud predominantly in a bipolar, rather than the normal axial, manner. Furthermore, yeast cells lacking both Bem2p and Dbm1p are inviable. The nonaxial budding defect of dbm1 mutants can be rescued partially by overproduction of Bem3p and is exacerbated by its absence. Since Bem3p has previously been shown to function as a GAP for Cdc42p, and also less efficiently for Rho1p, our results suggest that Dbm1p, like Bem2p and Bem3p, may function in vivo as a GAP for Cdc42p and/or Rho1p. Both LIM domains of Dbm1p are essential for its normal function. Point mutations that alter single conserved cysteine residues within either LIM domain result in mutant forms of Dbm1p that can no longer function in bud site selection but instead are capable of rescuing the inviability of bem2 mutants at 35 degrees C.
酿酒酵母中的正常细胞生长涉及到对基因决定的芽殖位点的选择,大多数生长都集中在这些位点。先前的研究表明,BEM2编码一种体外对Rho型GTP酶Rho1p具有特异性的GTP酶激活蛋白(GAP),它是正确的芽殖位点选择和芽体出现所必需的。我们在此表明,DBM1编码另一种推定的Rho型GAP,具有两个串联排列的富含半胱氨酸的LIM结构域,它也是正确的芽殖位点选择所必需的,因为缺乏Dbm1p的单倍体细胞主要以双极而非正常的轴向方式出芽。此外,同时缺乏Bem2p和Dbm1p的酵母细胞无法存活。dbm1突变体的非轴向出芽缺陷可以通过过量表达Bem3p部分挽救,而缺失Bem3p则会使其加剧。由于先前已表明Bem3p作为Cdc42p的GAP起作用,对Rho1p的作用效率较低,我们的结果表明,Dbm1p与Bem2p和Bem3p一样,可能在体内作为Cdc42p和/或Rho1p的GAP起作用。Dbm1p的两个LIM结构域对其正常功能至关重要。改变任一LIM结构域内单个保守半胱氨酸残基的点突变会导致Dbm1p的突变形式,这些突变形式不再能在芽殖位点选择中发挥作用,但能够挽救bem2突变体在35摄氏度时的致死性。