Kusano Ayumi, Yoshioka Tomoko, Nishijima Hitoshi, Nishitani Hideo, Nishimoto Takeharu
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Mol Biol Cell. 2004 Nov;15(11):4960-70. doi: 10.1091/mbc.e04-01-0067. Epub 2004 Aug 18.
We isolated 11 independent temperature-sensitive (ts) mutants of Schizosaccharomyces pombe RanGAP, SpRna1 that have several amino acid changes in the conserved domains of RanGAP. Resulting Sprna1ts showed a strong defect in mitotic chromosome segregation, but did not in nucleocytoplasmic transport and microtubule formation. In addition to Sprna1+ and Spksp1+, the clr4+ (histone H3-K9 methyltransferase), the S. pombe gene, SPAC25A8.01c, designated snf2SR+ (a member of the chromatin remodeling factors, Snf2 family with DNA-dependent ATPase activity), but not the spi1+ (S. pombe Ran homolog), rescued a lethality of Sprna1ts. Both Clr4 and Snf2 were reported to be involved in heterochromatin formation essential for building the centromeres. Consistently, Sprna1ts was defective in gene-silencing at the centromeres. But a silencing at the telomere, another heterochromatic region, was normal in all of Sprna1ts strains, indicating SpRna1 in general did not function for a heterochromatin formation. snf2SR+ rescued a centromeric silencing defect and Deltaclr4+ was synthetic lethal with Sprna1ts. Taken together, SpRna1 was suggested to function for constructing the centromeres, by cooperating with Clr4 and Snf2SR. Loss of SpRna1 activity, therefore, caused chromosome missegregation.
我们分离出了粟酒裂殖酵母RanGAP（SpRna1）的11个独立的温度敏感（ts）突变体，这些突变体在RanGAP的保守结构域中有几个氨基酸变化。产生的Sprna1ts在有丝分裂染色体分离中表现出强烈缺陷，但在核质运输和微管形成中没有。除了Sprna1 +和Spksp1 +外，粟酒裂殖酵母基因clr4 +（组蛋白H3-K9甲基转移酶）、名为snf2SR +（具有DNA依赖性ATP酶活性的染色质重塑因子Snf2家族成员），而不是spi1 +（粟酒裂殖酵母Ran同源物），挽救了Sprna1ts的致死性。据报道，Clr4和Snf2都参与了构建着丝粒所必需的异染色质形成。一致地，Sprna1ts在着丝粒处的基因沉默有缺陷。但是在端粒（另一个异染色质区域）的沉默在所有Sprna1ts菌株中都是正常的，这表明SpRna1一般不参与异染色质形成。snf2SR +挽救了着丝粒沉默缺陷，并且Deltaclr4 +与Sprna1ts是合成致死的。综上所述，SpRna1被认为通过与Clr4和Snf2SR合作来构建着丝粒。因此，SpRna1活性的丧失导致染色体错分离。
