Rincón Sergio A, Santos Beatriz, Pérez Pilar
Instituto Microbiología-Bioquímica, CSIC/Universidad de Salamanca, Campus Unamuno, 37007 Salamanca, Spain.
Eukaryot Cell. 2006 Mar;5(3):435-46. doi: 10.1128/EC.5.3.435-446.2006.
The Rho GTPase family and their effectors are key regulators involved in many eukaryotic cell functions related to actin organization and polarity establishment. Schizosaccharomyces pombe Rho1p is essential, directly activates the (1,3)-beta-d-glucan synthase, and participates in regulation of cell wall growth and morphogenesis. Here we describe the characterization of the fission yeast Rho5p GTPase, highly homologous to Rho1p, sharing 86% identity and 95% similarity. Overexpression of the hyperactive allele rho5-G15V causes a morphological effect similar to that of rho1-G15V, but the penetrance is significantly lower, and overexpression of the dominant-negative allele rho5-T20N causes lysis like that of rho1-T20N. Importantly, overexpression of rho5(+) but no other rho genes is able to rescue the lethality of rho1Delta cells. Shutoff experiments indicated that Rho5p can replace Rho1p, but it is not as effective in maintaining cell wall integrity or actin organization. rho5(+) expression is hardly detected during log-phase growth but is induced under nutritional starvation conditions. rho5Delta cells are viable and do not display any defects during logarithmic growth. However, when rho1(+) expression is repressed during stationary phase, rho5Delta cells display reduced viability. Ascospores lacking Rho5p are less resistant to heat or lytic enzymes than wild-type spores. Moreover, h(90) mutant strains carrying the hyperactive rho5-G15V or the dominant-negative rho5-T20N alleles display severe ascospore formation defects. These results suggest that Rho5p functions in a way similar to, but less efficient than, Rho1p, plays a nonessential role during stationary phase, and participates in the spore wall formation.
Rho GTP酶家族及其效应分子是参与许多与肌动蛋白组织和极性建立相关的真核细胞功能的关键调节因子。粟酒裂殖酵母Rho1p是必需的,它直接激活(1,3)-β-D-葡聚糖合酶,并参与细胞壁生长和形态发生的调节。在此,我们描述了裂殖酵母Rho5p GTP酶的特性,它与Rho1p高度同源,同一性为86%,相似性为95%。超活性等位基因rho5-G15V的过表达会产生与rho1-G15V相似的形态学效应,但外显率显著较低,而显性负等位基因rho5-T20N的过表达会导致细胞裂解,类似于rho1-T20N。重要的是,rho5(+)的过表达而非其他rho基因能够挽救rho1Delta细胞的致死性。关闭实验表明Rho5p可以替代Rho1p,但在维持细胞壁完整性或肌动蛋白组织方面效果不佳。在对数生长期几乎检测不到rho5(+)的表达,但在营养饥饿条件下会被诱导。rho5Delta细胞是有活力的,在对数生长期不表现出任何缺陷。然而,当在稳定期抑制rho1(+)的表达时,rho5Delta细胞的活力会降低。缺乏Rho5p的子囊孢子比野生型孢子对热或裂解酶的抵抗力更弱。此外,携带超活性rho5-G15V或显性负rho5-T20N等位基因的h(90)突变株表现出严重的子囊孢子形成缺陷。这些结果表明,Rho5p的功能与Rho1p相似,但效率较低,在稳定期发挥非必需作用,并参与孢子壁的形成。