Regulation of chromosome segregation in the budding yeast, S.cerevisiae

NPS1/STH1编码RSC的一个催化亚基，在有丝分裂生长中起重要作用。我们之前对NPS1温度敏感突变等位基因NPS1 -105的分析表明，NPS1 /RSC在着丝粒周围染色质结构的组装和/或维持中起重要作用。为了了解RSC在体内的功能，我们进行了这项研究，得到了以下结果。1)我们分离了15个抑制突变体（rtn突变体），它们可以在TBZ存在的情况下恢复nps1-105的生长。这些突变体被划分为9个互补组。利用温度敏感性，我们克隆了RTN2、RTN4和RTN5，分别鉴定为NUP82、ABD1和CET1。2)筛选温度-噻苯达唑（TBZ） NPS1 -105突变体的高拷贝抑制基因，鉴定出包括NPS1在内的9个基因。其中，PKC1和BIM1分别编码哺乳动物蛋白激酶C的同源物和与人类EB1同源的保守微管结合蛋白，在抑制中发挥重要作用。对这些基因之间功能关系的遗传分析表明，PKC1通过BIM1的功能抑制nps1-105的缺陷，而不是通过激活MPK1/MAPK途径。stt1突变在半允许温度下表现出TBZ敏感性和延迟g2相进展。这两种stt1表型都被BIM1的过表达所抑制。此外，在允许温度下，stt1和nps1-105比野生型有更高的错误分离小染色体的频率。在nps1-105 stt1双突变体中，错分离现象增强。这些结果表明Pkc1p在微管功能中起着相关的作用，并且这种作用是由一个未知的PKC效应分支和Bim1p介导的。

NPS1/STH1 encodes a catalytic subunit of RSC and plays essential role for mitotic growth. Our previous analysis on temperature-sensitive mutant allele of NPS1, nps1-105, revealed that Nps1/RSC plays important role on the assembly and/or maintenance of the chromatin structure around centromeres. We performed this study on an attempt to understand the in vivo function of RSC and obtained the following results.1) We isolated fifteen suppressor mutants (rtn mutants) that can restore the growth of nps1-105 in the presence of TBZ.These mutants were classified in nine complementation groups. Taking an advantage of temperature sensitivity, we cloned RTN2, RTN4 and RTN5 and identified them as NUP82, ABD1 and CET1, respectively.2) We screened high-copy suppressor genes for the temperature- and thiabendazole (TBZ) nps1-105 mutant and identified nine genes including NPS1. Among those, PKC1 and BIM1 that respectively encoded a homolog of mammalian protein kinase C and a conserved microtubule binding protein homologous to human EB1 were found to play important role on suppression. Genetic analysis of the functional relationships between these genes revealed that PKC1 suppressed the defect of nps1-105 via the function of BIM1 but not through activation of the MPK1/MAPK pathway. The stt1 mutation showed TBZ sensitivity and delayed G2-phase progression at semi-permissive temperatures. Both of these stt1 phenotypes were suppressed by the overexpression of BIM1. In addition, stt1 as well as nps1-105, mis-segregated a mini-chromosome at higher frequency than the wild type at permissive temperature. The mis-segregation was enhanced in the nps1-105 stt1 double mutant. These results suggested that Pkc1p plays a role relevant to microtubule function and that this role is mediated by an unknown PKC effector branch and by Bim1p.