线粒体信号肽引导葡萄糖调节蛋白75-增强型绿色荧光蛋白(GRP75-EGFP)融合蛋白定位于线粒体  

Mitochondrial signal peptide guides EGFP-GRP75 fusion proteins into mitochondria

作　　者：陈航[1] 牛秀然[1] 高嫒嫒[1] 张思河[1] CHEN Hang NIU Xiuran GAO Aiai ZHANG Sihe(Department of Biochemistry, School of Medicine, Nankai University, Tianjin 300071, China)

机构地区：[1]南开大学医学院生物化学教研室,天津300071

出　　处：《细胞与分子免疫学杂志》2016年第10期1311-1316,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81373318);高等学校博士点专项科研基金(20130031120037);天津市自然科学基金(16JCYBJC23900);药物化学国家重点实验室开放基金(20130575)

摘　　要：目的构建葡萄糖调节蛋白75(GRP75)不同结构域、不同形式突变体与线粒体信号肽重组蛋白并鉴定其亚细胞定位。方法通过重叠延伸PCR,将线粒体靶向信号肽序列分别与GRP75不同结构域基因拼接。利用定点突变PCR分别扩增实现62和65位氨基酸突变的磷酸化失活型、磷酸化激活型突变体、482位氨基酸突变的底物结合缺陷型突变体及三位点同时突变重组体。将上述重组基因克隆入p EGFPC1真核表达质粒,酶切和测序验证后分别用脂质体转染He La细胞,Western blot法检测重组蛋白表达水平,激光共聚焦显微镜观察比较重组蛋白亚细胞定位情况。结果成功构建了线粒体信号肽融合或缺失的GRP75结构域、磷酸化失活和激活突变体、底物结合缺陷突变体真核表达质粒。各重组质粒的片段插入、位点突变、信号肽融合情况均符合设计目的,在He La细胞表达后产物的相对分子质量大小均符合预期。EGFP蛋白C端插入信号肽能引导下游融合的GRP75全长蛋白、结构域片段、突变体蛋白表达定位于He La细胞线粒体中,而缺失信号肽的对应重组蛋白则主要分布于胞质和局部核周。结论构建了一系列GRP75重组真核表达质粒,EGFP融合信号肽能引导重组蛋白在线粒体中表达定位。Objective To investigate the role of mitoch-ondrial signal peptide in guiding enhanced green fluorescent protein-glucose-regulated protein 75 fusion proteins（ EGFP-GRP75） into mitochondria. Methods At first,the signal peptide gene and GRP75 domain genes were spliced by overlap extension PCR. Unphosphorylatable mutant T62 A / S65 A,phospho-mimic mutant T62 D / S65 D and substrate binding-defective mutant V482 F were further created through site-directed mutagenesis PCR. The fusion gene fragments were ligated into p EGFPC1 expression plasmid, respectively. The expressions of EGFP-GRP75 fusion constructs in He La cells were examined directly with Western blotting and laser scanning confocal microscopy. Results All the fusion proteins were highly expressed. Signal peptide remarkably reduced the expression of EGFP-GRP75 fusions compared with recombinant plasmids without signal peptide. Fluorescence was seen exclusively located in mitochondria of the cells transfected by signal peptide-contained plasmids,whereas signal peptide-absent EGFP-GRP75 fusion proteins were homogeneously distributed in the whole cell body. In addition,no changes were observed in the subcellular localization of EGFP-GRP75 fusion proteins that contained double or triple mutations at Thr 62 residues,Ser65 residues and Val 482 residues. Conclusion Signal peptide is essential for targeting EGFP-GRP75 fusion proteins into mitochondria, and 62 threonine /65 serine /482 valine residues contribute to phosphorylation or substrate-binding characteristics are dispensable in the mitochondrial targeting function.

关 键 词：葡萄糖调节蛋白75(GRP75) 线粒体靶向信号肽 重叠延伸PCR 亚细胞定位 

分 类 号：R394.2[医药卫生—医学遗传学] R392.33[医药卫生—基础医学]