The DNA-binding domain of the yeast Spt10p activator includes a zinc finger that is homologous to foamy virus integrase

The yeast SPT10 gene encodes a putative histone acetyltransferase that binds specifically to pairs of upstream activating sequence (UAS) elements found only in the histone gene promoters. Here, we demonstrate that the DNA-binding domain of Spt10p is located between residues 283 and 396 and includes a His(2)-Cys(2) zinc finger. The binding of Spt10p to the histone UAS is zinc-dependent and is disabled by a zinc finger mutation (C388S). The isolated DNA-binding domain binds to single histone UAS elements with high affinity. In contrast, full-length Spt10p binds with high affinity only to pairs of UAS elements with very strong positive cooperativity and is unable to bind to a single UAS element. This implies the presence of a "blocking" domain in full-length Spt10p, which forces it to search for a pair of UAS elements. Chromatin immunoprecipitation experiments indicate that, unlike wild-type Spt10p, the C388S protein does not bind to the promoter of the gene encoding histone H2A (HTA1) in vivo. The C388S mutant has a phenotype similar to that of the spt10Delta mutant: poor growth and global aberrations in gene expression. Thus, the C388S mutation disables the DNA-binding function of Spt10p in vitro and in vivo. The zinc finger of Spt10p is homologous to that of foamy virus integrase, perhaps suggesting that this integrase is also a sequence-specific DNA-binding protein.     

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(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。