裂殖酵母SAGA复合物亚基Spt20参与钙调蛋白磷酸酶调节的Cl^-胞内平衡  

SAGA complex subunit Spt20 involves in the calcineurin-mediated Cl^homeostasis in Schizosaccharomyces pombe

作　　者：周楠[1] 雷秉坤[1] 周幸[1] 余垚[1] 吕红[1] 

机构地区：[1]复旦大学生命科学学院,遗传工程国家重点实验室,上海200433

出　　处：《遗传》2013年第9期1135-1142,共8页Hereditas（Beijing)

基　　金：国家重点基础研究发展计划(973计划)项目(编号:2009CB825601);国家自然科学基金项目(编号:31200961)资助

摘　　要：SAGA(Spt-Ada-Gcn5acetyltransferase)复合物是真核生物中高度保守的蛋白复合体,参与转录激活、mRNA转运等诸多生物学过程。为了探究SAGA复合物亚基的潜在生物学功能,文章以裂殖酵母(Schizosac-charomyces pombe)SAGA复合物核心结构亚基Spt20为诱饵蛋白进行酵母双杂交筛选,获得了Ppb1蛋白。Ppb1是真核生物重要信号分子-钙调蛋白磷酸酶的催化亚基。酵母双杂交验证及免疫共沉淀实验均表明Spt20与Ppb1可以在体内发生蛋白相互作用。裂殖酵母ppb1+缺失突变体对高浓度Cl敏感,而spt20+缺失突变体则能抵抗高浓度的外源Cl,维持细胞的正常生长。荧光共定位分析表明,当外源Cl浓度升高时,Ppb1蛋白能够从细胞质迁移入核,与Spt20蛋白在细胞核内发生共定位。遗传分析显示,spt20+缺失可以抑制ppb1+缺失突变体对Cl高度敏感的表型,spt20+与ppb1+处于Cl平衡调节的同一通路,且spt20+位于ppb1+的下游。上述结果表明,spt20+缺失突变体耐受外源高浓度Cl,Spt20参与了钙调蛋白磷酸酶调节的Cl胞内平衡。在高等生物中胞内Cl浓度异常升高与心肌缺血/再灌注损伤等疾病的发生密切相关。鉴于Spt20在真核生物中高度保守,Spt20可能成为潜在的药物靶点应用于Cl失衡相关疾病的防治中。SAGA （Spt-Ada-Gcn5 acetyltransferase） is a highly conserved protein complex in eukaryotes, which plays a role in many important cellular processes, including transcriptional activation and mRNA exportation. In order to investigate the potential biological function of SAGA subunit, we performed a yeast two-hybrid screen using a core structural subunit of SAGA in fission yeast, Spt20, as the bait. Ppbl, catalytic subunit of calcineruin was identified in the test. Calcineurin is a key regulator of signal transduction. The interaction between Spt20 and Ppbl was confirmed by yeast two-hybrid assay and co-immunoprecipitation. In S. pombe, ppblA was hypersensitive to high concentration of C1. In contrast, spt20A could resist high concentration of C1-, which maintained normal growth of cells. Fluorescent colocalization analysis showed that Ppbl was translocated from cytoplasm to nucleus and colocalized with Spt20 upon the increase of extracellular C1-. Further genetic analysis revealed that loss of spt20＋ suppressed the hypersensitive phenotype to C1 of ppblA. Thus, spt20＋ and ppbl stayed in the same pathway of regulating C1- homeostasis and spt20＋ functioned down- stream ofppbl＋. Our data suggest that spt20A is able to resist high concentration of extracellular C1- and Spt20 involves in the calcineurin-mediated CF homeostasis. The aberrant up-regulation of intracellular C1- is correlated with the diseases like myocardial ischemia reperfusion injury in higher organism. As Spt20 is highly conserved in eukaryotes, it might serve as a potential drug target in C1- imbalance related diseases.

关 键 词：裂殖酵母 SAGA复合物 Spt20 钙调蛋白磷酸酶 Cl胞内平衡 

分 类 号：Q933[生物学—微生物学]