Wood Matthew J, Andrade Erika C, Storz Gisela
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-5430, USA.
Biochemistry. 2003 Oct 21;42(41):11982-91. doi: 10.1021/bi035003d.
The subcellular localization of the Saccharomyces cerevisiae transcription factor Yap1p is regulated by oxidation and reduction. We purified Yap1p from yeast and characterized its properties in vitro. Electrophoretic mobility shift assays showed that the purified protein can specifically bind the TRX2 target promoter. Yap1p was purified under reducing conditions, but removal of reducing agents resulted in the formation of an oxidized Yap1p species with properties similar to in vivo oxidized Yap1p. MALDI-TOF mass spectrometry analysis revealed that the oxidized form of Yap1p contains two disulfide bonds between C303-C598 and C310-C629. A stable domain of approximately 15 kDa was detected upon limited proteolysis of oxidized but not reduced Yap1p. This Yap1p protease resistant domain was purified, and MALDI-TOF mass spectrometry analysis showed that it was comprised of two separate cysteine-containing peptides of Yap1p. These peptides are separated by 250 amino acids and are joined by the C303-C598 and C310-C629 disulfide bonds. Taken together, these data suggest that the domain that controls Yap1p subcellular localization is modular and contains a redox center comprised of four cysteine residues.
酿酒酵母转录因子Yap1p的亚细胞定位受氧化还原调控。我们从酵母中纯化了Yap1p并在体外对其性质进行了表征。电泳迁移率变动分析表明，纯化的蛋白能特异性结合TRX2靶启动子。Yap1p是在还原条件下纯化的，但去除还原剂会导致形成一种氧化型Yap1p，其性质与体内氧化型Yap1p相似。基质辅助激光解吸电离飞行时间质谱分析显示，氧化型Yap1p在C303 - C598和C310 - C629之间含有两个二硫键。对氧化型而非还原型Yap1p进行有限蛋白酶解后，检测到一个约15 kDa的稳定结构域。纯化了这个抗Yap1p蛋白酶的结构域，基质辅助激光解吸电离飞行时间质谱分析表明它由Yap1p的两个单独的含半胱氨酸肽段组成。这些肽段相隔250个氨基酸，并通过C303 - C598和C310 - C629二硫键相连。综上所述，这些数据表明控制Yap1p亚细胞定位的结构域是模块化的，且包含一个由四个半胱氨酸残基组成的氧化还原中心。
