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Mechanism of mtDNA deletion in short lived mutant of Neurospora.

脉孢菌短命突变体线粒体DNA缺失的机制。

基本信息

批准号：
20570001
负责人：
INOUE Hirokazu
金额：
$ 3.08万
依托单位：
Saitama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2010
项目状态：
已结题

项目摘要

The mus-10 mutant was isolated which showed highly sensitivity to alkylating agent methylmethane sulfonate (MMS). It had been forecasted that mus-10 gene belonged to the some DNA repair pathway, because of it sensitivity to mutagen. This mutant have other unique characteristics; unable to grow after several times sequential inoculation, or stop growing after 2 to 3 weeks culture. Furthermore, these phenotypes are accompanied the deletion of mitochondrial DNA and fragmented mitochondrial feature comparing to the normal (tubular) shape in wild type strain. The responsible gene of mus-10 was cloned by complementation of its MMS sensitivity. This gene encodes the F-box domain containing polypeptide, and deletion of F-box domain showed identical phenotype with mus-10 mutant. Since F-box protein is known as a counterpart of SCF (Skp-Cullin-F-box) comlex, which have a role for the degradation of some target protein via ubiquitination following degrading in The mus-10 mutant was isolated which … More showed highly sensitivity to alkylating agent methylmethane sulfonate (MMS). It had been forecasted that mus-10 gene belonged to the some DNA repair pathway, because of it sensitivity to mutagen. This mutant have other unique characteristics ; unable to grow after several times sequential inoculation, or stop growing after 2 to 3 weeks culture. Furthermore, these phenotypes are accompanied the deletion of mitochondrial DNA and fragmented mitochondrial feature comparing to the normal (tubular) shape in wild type strain. The responsible gene of mus-10 was cloned by complementation of its MMS sensitivity. This gene encodes the F-box domain containing polypeptide, and deletion of F-box domain showed identical phenotype with mus-10 mutant. Since F-box protein is known as a counterpart of SCF (Skp-Cullin-F-box) comlex, which have a role for the degradation of some target protein via ubiquitination following degrading in proteasome.To uncover the mus-10 gene function, we focused the feature of mitochondria. We examined whether 1) mitochondrial fusion is inhibited, or 2) mitochondrial fission is stimulated in mus-10 mutant. Double mutation of mus-10 and fis-1, which was essential for mitochondrial fission, showed quite resemble feature of mitochondria with wild type strain. And also this double mutant suppressed sensitivity to mutagen and short life span. These results suggested that MUS-10 protein prevent from the mutagen sensitivity and short life span according to maintain a mitochondrial feature. MUS-10 protein was considered to have a function of degradation of some target protein. So MUS-10 protein should be bound to that protein. Considering MUS-10 protein was correlated to maintenance of mitochondria feature, one candidate FZO-1 arose which had functions in the mitochondrial fusion. Using immunoprecipitation mthod, we could show that MUS-10 protein bound to FZO-1. Next, we tried to make fzo-1 knock out strain, but couldn't. The fzo-1 gene thought to be essential gene and fusion of mitochondria was important for maintenance of life span in Neurospora. Further, we forecasted that constitutive expression of FZO-1 protein in mus-10 mutant might show eviler phenotype than the mus-10 single mutant, because FZO-1, target of MUS-10, might be accumulated in that strain by escaping degradation. However, any evil phenotypes were not observed. Above these results, it was suggested that there were complex mechanism to maintain the mitochondrial feature. Less

mus-10突变株对烷基化剂甲基甲烷磺酸盐（MMS）高度敏感。由于mus-10基因对诱变剂的敏感性，推测其属于DNA修复途径。该突变体还具有其他独特的特性，即连续接种几次后不能生长，或在培养2 - 3周后停止生长。此外，与野生型菌株中的正常（管状）形状相比，这些表型伴随着线粒体DNA的缺失和片段化的线粒体特征。通过互补mus-10的MMS敏感性，克隆了mus-10的相关基因。该基因编码含F-box结构域的多肽，缺失F-box结构域后表现出与mus-10突变体相同的表型。由于F-box蛋白是SCF（Skp-Cullin-F-box）复合物的对应物，其在降解后通过泛素化降解某些靶蛋白，因此分离到mus-10突变体， ...更多信息对烷基化剂甲基甲烷磺酸盐（MMS）具有很高的敏感性。由于mus-10基因对诱变剂的敏感性，推测其属于DNA修复途径。该突变体还具有其他独特的特性，即连续接种几次后不能生长，或在培养2 - 3周后停止生长。此外，与野生型菌株中的正常（管状）形状相比，这些表型伴随着线粒体DNA的缺失和片段化的线粒体特征。通过互补mus-10的MMS敏感性，克隆了mus-10的相关基因。该基因编码含F-box结构域的多肽，缺失F-box结构域后表现出与mus-10突变体相同的表型。由于F-box蛋白是SCF（Skp-Cullin-F-box）复合物的一个对应物，在蛋白酶体中降解后，通过泛素化途径降解某些靶蛋白，因此为了揭示mus-10基因在线粒体中的功能，我们将研究重点放在了mus-10基因在线粒体中的功能。我们研究了在mus-10突变体中是否1）线粒体融合被抑制，或2）线粒体分裂被刺激。mus-10和fis-1的双突变是线粒体分裂所必需的，与野生型菌株的线粒体特征十分相似。而且这种双突变体对诱变剂的敏感性降低，寿命缩短。这些结果表明，MUS-10蛋白通过维持线粒体特征来防止诱变剂敏感性和短寿命。MUS-10蛋白被认为具有降解某些靶蛋白的功能。所以MUS-10蛋白应该与该蛋白结合。考虑到MUS-10蛋白与线粒体特征的维持相关，出现了一个在线粒体融合中具有功能的候选FZO-1。免疫沉淀法显示MUS-10蛋白与FZO-1蛋白结合。接下来，我们试图使fzo-1敲除菌株，但不能。fzo-1基因被认为是链孢霉的必需基因，线粒体的融合对链孢霉寿命的维持具有重要意义。此外，我们预测，FZO-1蛋白在mus-10突变体中的组成型表达可能比mus-10单突变体表现出更差的表型，因为MUS-10的靶标FZO-1可能通过逃避降解而在该菌株中积累。然而，没有观察到任何邪恶的表型。以上结果表明，线粒体的功能维持有复杂的机制。少