Hoke Stephen M T, Genereaux Julie, Liang Gaoyang, Brandl Christopher J
Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Canada.
J Mol Biol. 2008 Dec 26;384(4):743-55. doi: 10.1016/j.jmb.2008.09.088. Epub 2008 Oct 11.
The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex of Saccharomyces cerevisiae contains more than 20 components that acetylate and deubiquitylate nucleosomal histones. Its acetyltransferase, Gcn5, preferentially acetylates histones H3 and H2B and is regulated through interactions with Ada2 and Ngg1/Ada3. Sequence alignments of Ada2 homologs indicate a conserved approximately 120-amino-acid-residue central region. To examine the function of this region, we constructed ada2 alleles with mutations of clustered conserved residues. One of these alleles, ada2-RLR (R211S, L212A, and R215A), resulted in an approximately threefold reduction in transcriptional activation of the PHO5 gene and growth changes that parallel deletion of ada2. Microarray analyses further revealed that ada2-RLR alters expression of a subset of those genes affected by deletion of ada2. Indicative of Ada2-RLR affecting Gcn5 function, Ada2-RLR resulted in a decrease in Gcn5-mediated histone acetylation in vitro to a level approximately 40% that with wild-type Ada2. In addition, in vivo acetylation of K16 of histone H2B was almost totally eliminated at Ada2-regulated promoters in the ada2-RLR strain, while acetylation of K9 and K18 of histone H3 was reduced to approximately 40% of wild-type levels. We also show that the central region of Ada2 interacts with phospholipids. Since phosphatidylserine binding paralleled Ada2 function, we suggest that lipid binding may play a role in the function or regulation of the SAGA complex.
酿酒酵母的SAGA（Spt-Ada-Gcn5乙酰转移酶）复合物包含20多个成分，这些成分可对核小体组蛋白进行乙酰化和去泛素化修饰。其乙酰转移酶Gcn5优先对组蛋白H3和H2B进行乙酰化修饰，并通过与Ada2和Ngg1/Ada3的相互作用进行调控。Ada2同源物的序列比对表明，其中心区域存在一个保守的约120个氨基酸残基的区域。为了研究该区域的功能，我们构建了具有成簇保守残基突变的ada2等位基因。其中一个等位基因ada2-RLR（R211S、L212A和R215A）导致PHO5基因的转录激活降低约三倍，并出现与ada2缺失平行的生长变化。微阵列分析进一步表明，ada2-RLR改变了受ada2缺失影响的一部分基因的表达。ada2-RLR影响Gcn5功能的证据是，在体外，Ada2-RLR导致Gcn5介导的组蛋白乙酰化水平降低至野生型Ada2的约40%。此外，在ada2-RLR菌株中，Ada2调控的启动子处组蛋白H2B的K16位点的体内乙酰化几乎完全消除，而组蛋白H3的K9和K18位点的乙酰化水平降低至野生型水平的约40%。我们还表明，Ada2的中心区域与磷脂相互作用。由于磷脂酰丝氨酸结合与Ada2功能平行，我们认为脂质结合可能在SAGA复合物的功能或调控中发挥作用。
