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
收稿日期: 2013-04-06
修回日期: 2013-05-16
网络出版日期: 2013-09-25
基金资助
国家重点基础研究发展计划(973计划)项目(编号：2009CB825601)和国家自然科学基金项目(编号：31200961)资助
SAGA complex subunit Spt20 involves in the calcineurin-mediated Cl- homeostasis in Schizosaccharomyces pombe
Received date: 2013-04-06
Revised date: 2013-05-16
Online published: 2013-09-25
SAGA(Spt-Ada-Gcn5 acetyltransferase)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(Schizosaccharomyces 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。
周楠 雷秉坤 周幸 余垚 吕红 . 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[J]. 遗传, 2013 , 35(9) : 1135 -1142 . DOI: 10.3724/SP.J.1005.2013.01135
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. Ppb1, catalytic subunit of calcineruin was identified in the test. Calcineurin is a key regulator of signal transduction. The interaction between Spt20 and Ppb1 was confirmed by yeast two-hybrid assay and co-immunoprecipitation. In S. pombe, ppb1Δ was hypersensitive to high concentration of Cl?. In contrast, spt20Δ could resist high concentration of Cl?, which maintained normal growth of cells. Fluorescent colocalization analysis showed that Ppb1 was translocated from cytoplasm to nucleus and colocalized with Spt20 upon the increase of extracellular Cl?. Further genetic analysis revealed that loss of spt20+ suppressed the hypersensitive phenotype to Cl? of ppb1Δ. Thus, spt20+ and ppb1+ stayed in the same pathway of regulating Cl? homeostasis and spt20+ functioned downstream of ppb1+. Our data suggest that spt20Δ is able to resist high concentration of extracellular Cl? and Spt20 involves in the calcineurin-mediated Cl? homeostasis. The aberrant up-regulation of intracellular Cl? 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 Cl? imbalance related diseases.
Key words： SAGA complex; Spt20; calcineurin; Cl?homeostasis; S. pombe
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