粟酒裂殖酵母中Shy1定位及功能的研究

Study on the Localization and Function of Shy1 in Schizosaccharomyces pombe

旨在揭示芽殖酵母线粒体蛋白SHY1(YGR112W)在粟酒裂殖酵母中同源蛋白Shy1的功能。借助基因敲除方法获得shy1基因缺失菌株获Δshy1,并观察其在以甘油为唯一碳源的非发酵培养基上的生长表型;生物信息学分析显示粟酒裂殖酵母Shy1在N端含有大概30个氨基酸的线粒体定位序列(MTS),为进一步确定Shy1蛋白的定位,借助nmt1启动子调控下的Shy1蛋白C端GFP荧光标记,观察GFP绿色荧光位置。最后利用Western blotting检测shy1的缺失对线粒体蛋白的影响。研究结果表明,shy1基因缺失菌株在以甘油为唯一碳源的非发酵培养基上表现出生长缺陷,是线粒体呼吸缺陷型菌株;当GFP标记于nmt1启动子调控下的Shy1蛋白C端时,绿色荧光观察确定Shy1蛋白定位在线粒体;Western blotting检测结果显示shy1缺失导致线粒体相关蛋白的表达量明显下降;结果表明,粟酒裂殖酵母Shy1定位于线粒体且是线粒体呼吸链正常发挥功能所必须的。

Mitochondria are the main place for cell respiration,and mitochondrial dysfunction causes various human diseases. Herewe aimed to explore the function of the Schizosaccharomyces cerevisiae mitochondrial protein SHY1（YGR112W）in homologous protein Shy1 of Schizosaccharomyces pombe. Firstly,we generated the strain Δshy1 of shy1 gene deletion by gene knockout,and examined its phenotypein the non-fermentation medium with glycerol as the sole carbon source. Then,bioinformatics analyses revealed that Shy1 of S. pombecontained the conserved MTS（mitochondrial targeting sequence）of about 30 amino acids at the N-terminus. To further study the localizationof Shy1,the location of green fluorescent protein（GFP）was observed by labeling GFP at the C-terminus of Shy1 protein regulated by thenmt1 promoter. Lastly,we detected the influence of the deletion of shy1 on the expression of mitochondrial protein by Western blotting. Theresults demonstrated that the strain of shy1 gene deletion presented its growing deletion in the non-fermentation medium with glycerol as thesole carbon source,and was one with mitochondrial respiratory defect. When GFP was labeled at the C terminus of Shy1 protein regulatedby the nmt1 promoter,the location of Shy1 protein was in the mitochondria as the green fluorescence was observed. Moreover,the results ofWestern blotting revealed that the expression of protein related to mitochondria resulted from the shy1 gene deletion was dramatically declined.Therefore,these findings indicate that Shy1 is localized in mitochondria and is obligatory for mitochondrial respiratory chain to play ordinaryfunction in S. pombe.