Wotton D, Freeman K, Shore D
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
J Biol Chem. 1996 Feb 2;271(5):2717-23. doi: 10.1074/jbc.271.5.2717.
Rap1p is a transcriptional regulator of Saccharomyces cerevisiae, which plays roles in both transcriptional activation and silencing. To identify proteins involved in Rap1p-dependent regulation of transcription, we used the two-hybrid system to screen for Rap1p-interacting proteins. Two of the clones isolated from this screen encode a truncated protein with homology to small heat shock proteins (HSPs). Here we present an analysis of this novel S. cerevisiae HSP, which we name Hsp42p. Expression of HSP42 is regulated by a range of stress conditions similar to S. cerevisiae HSP26, with which Hsp42p shares most homology. However, HSP42 expression is more sensitive to increased salt concentration and to starvation and, in contrast to HSP26 is expressed in unstressed cells. Hsp42p interacts with itself in the two-hybrid assay. This interaction is dependent on a hydrophobic region which is conserved among small HSPs. Using bacterially expressed Hsp42p fusion proteins. we demonstrate that this is a direct interaction. Fractionation of yeast protein extracts by size demonstrates that all of the Hsp42p in these extracts is present in complexes with a molecular mass of greater than 200 kDa, suggesting that Hsp42p exists in high molecular mass complexes.
Rap1p是酿酒酵母的一种转录调节因子，在转录激活和沉默过程中均发挥作用。为了鉴定参与Rap1p依赖的转录调控的蛋白质，我们利用双杂交系统筛选与Rap1p相互作用的蛋白质。从该筛选中分离出的两个克隆编码一种与小热休克蛋白（HSPs）具有同源性的截短蛋白。在此，我们对这种新的酿酒酵母HSP进行分析，将其命名为Hsp42p。HSP42的表达受一系列应激条件调控，这与酿酒酵母HSP26类似，Hsp42p与HSP26具有大部分同源性。然而，HSP42的表达对盐浓度升高和饥饿更为敏感，并且与HSP26不同，它在未受应激的细胞中表达。在双杂交试验中，Hsp42p与自身相互作用。这种相互作用依赖于小HSPs中保守的疏水区域。使用细菌表达的Hsp42p融合蛋白，我们证明这是一种直接相互作用。通过大小对酵母蛋白提取物进行分级分离表明，这些提取物中的所有Hsp42p都存在于分子量大于200 kDa的复合物中，这表明Hsp42p以高分子量复合物形式存在。
