Nicolson T A, Weisman L S, Payne G S, Wickner W T
Molecular Biology Institute, University of California, Los Angeles 90024-1570, USA.
J Cell Biol. 1995 Aug;130(4):835-45. doi: 10.1083/jcb.130.4.835.
Partitioning of the vacuole during cell division in Saccharomyces cerevisiae begins during early S phase and ends in late G2 phase before the yeast nucleus migrates into the bud neck. We have isolated and characterized a new mutant, vac5-1, which is defective in vacuole segregation. Cells with the vac5-1 mutation can form large buds without vacuoles. The VAC5 gene was cloned and is identical to PHO80. PHO80 encodes a cyclin which acts in a complex with a cdc-like kinase, PHO85, as a negative regulator of two transcription factors (PHO2 and PHO4) that govern the expression of metabolic phosphatases. The vacuole inheritance defect in vac5-1 cells is dependent on the presence of the Pho85 kinase and its targets Pho4p and Pho2p. As with other alleles of PHO80, phosphatase levels are elevated in vac5-1 mutants. A suppressor, the COOH-terminal half of the Gal11 transcription factor, rescues the vac5-1 phenotype of defective vacuole inheritance without altering the vac5-1 phenotype of elevated phosphatase levels. In addition, neither maximal nor minimal levels of expression of the inducible "PHO" system phosphatases causes a vacuole inheritance defect. Though vac5-1 is recessive, pho80 delta or pho85 delta strains do not show a defect in vacuole inheritance, suggesting that vac5-1 is not a complete loss-of-function allele. Sequence analysis shows that the vac5-1 allele encodes a truncated form of the Pho80 cyclin and overexpression of vac5-1 in pho80 delta cells causes a vacuole inheritance defect. We conclude that the vac5-1 allele directs the Pho85 kinase to regulate, via transcription factors Pho4 and Pho2, genes that affect vacuole inheritance but which are not known to be under normal PHO pathway control.
酿酒酵母细胞分裂过程中液泡的分配在S期早期开始，在酵母细胞核迁移到芽颈之前的G2期晚期结束。我们分离并鉴定了一个新的突变体vac5-1，它在液泡分离方面存在缺陷。具有vac5-1突变的细胞可以形成没有液泡的大芽。VAC5基因被克隆出来，它与PHO80相同。PHO80编码一种细胞周期蛋白，它与一种类cdc激酶PHO85形成复合物，作为两个调控代谢磷酸酶表达的转录因子（PHO2和PHO4）的负调节因子。vac5-1细胞中的液泡遗传缺陷依赖于Pho85激酶及其靶标Pho4p和Pho2p的存在。与PHO80的其他等位基因一样，vac5-1突变体中的磷酸酶水平升高。一个抑制子，即Gal11转录因子的COOH末端一半，挽救了vac5-1有缺陷的液泡遗传表型，而没有改变磷酸酶水平升高的vac5-1表型。此外，诱导型“PHO”系统磷酸酶的最大或最小表达水平都不会导致液泡遗传缺陷。虽然vac5-1是隐性的，但pho80δ或pho85δ菌株在液泡遗传方面没有缺陷，这表明vac5-1不是一个完全功能丧失的等位基因。序列分析表明，vac5-1等位基因编码一种截短形式的Pho80细胞周期蛋白，在pho80δ细胞中过表达vac5-1会导致液泡遗传缺陷。我们得出结论，vac5-1等位基因指导Pho85激酶通过转录因子Pho4和Pho2调节影响液泡遗传的基因，但这些基因在正常PHO途径控制下并不为人所知。
