粟酒裂殖酵母蛋白Atp4定位和功能的研究

Research on the Localization and Function of Atp4 in Schizosaccharomyces pombe

研究粟酒裂殖酵母(Schizosaccharomyces pombe)中Atp4的定位和参与线粒体功能的机制。借助于基因敲除、荧光显微镜观察、线粒体提取、生化试剂处理和Western blotting等技术开展研究。在甘油培养基上,Δatp4突变菌株表现出生长缺陷,是线粒体呼吸缺陷菌;Fluorescence microscope观察到用GFP标记的Atp4和线粒体是共定位的;采用生化试剂TritonX-100和蛋白酶K处理以及碳酸钠处理Atp4-Flag和yHL6381线粒体,结果表明Atp4定位在线粒体内膜;Western blotting实验结果表明Atp4缺失导致Cob1、Cox1、Cox2和Atp6表达量剧烈下降,Cox3和Cox4蛋白表达量也有略微下降。综上所述,Atp4在线粒体内膜发挥功能,对于维持线粒体编码蛋白的正常表达至关重要,是线粒体呼吸链功能的发挥所必需的。

In this study,we studied the localization of Atp4 in Saccharomyces pombe and the mechanism of its involving in the mitochondrial function. The technologies utilized in the article included gene knock,fluorescence microscope,mitochondrial extraction, biochemical treatment,Western blotting,and so on. The Δatp4 deletion mutant glycerin medium grew defectively,and it was mitochondrial respiratory-deficient strain. The Atp4 with GFP label in C terminal showed co-localization with mitochondrion by fluorescence microscope observation. Treating Atp4-Flag and vyHL6381 mitochondria by biochemical reagents TritonX-100 and protease K and sodium carbonate revealed that Atp4 located in mitochondrial endometrium. Western blotting assays demonstrated that Atp4 deletion led to the sharp drop of Cob1、Cox1、Cox2 and Atp6 protein and the light drop of Cox and Cox4. Sum of above results,Atp4 plays its function in mitochondrion,and it is essential in maintaining the normal expressions of mitochondrial proteins and function of mitochondrial respiratory chain.