Millson Stefan H, Truman Andrew W, King Victoria, Prodromou Chrisostomos, Pearl Laurence H, Piper Peter W
Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, United Kingdom.
Eukaryot Cell. 2005 May;4(5):849-60. doi: 10.1128/EC.4.5.849-860.2005.
The Hsp90 chaperone cycle catalyzes the final activation step of several important eukaryotic proteins (Hsp90 "clients"). Although largely a functional form of Hsp90, an Hsp90-Gal4p DNA binding domain fusion (Hsp90-BD) displays no strong interactions in the yeast two-hybrid system, consistent with a general transience of most Hsp90-client associations. Strong in vivo interactions are though detected when the E33A mutation is introduced into this bait, a mutation that should arrest Hsp90-client complexes at a stage where the client is stabilized, yet prevented from attaining its active form. This E33A mutation stabilized the two-hybrid interactions of the Hsp90-BD fusion with approximately 3% of the Saccharomyces cerevisiae proteome in a screen of the 6,000 yeast proteins expressed as fusions to the Gal4p activation domain (AD). Among the detected interactors were the two stress-activated mitogen-activated protein (MAP) kinases of yeast, Hog1p and Slt2p (Mpk1p). Column retention experiments using wild-type and mutant forms of Hsp90 and Slt2p MAP kinase, as well as quantitative measurements of the effects of stress on the two-hybrid interaction of mutant Hsp90-BD and AD-Slt2p fusions, revealed that Hsp90 binds exclusively to the dually Thr/Tyr-phosphorylated, stress-activated form of Slt2p [(Y-P,T-P)Slt2p] and also to the MAP kinase domain within this (Y-P,T-P)Slt2p. Phenotypic analysis of a yeast mutant that expresses a mutant Hsp90 (T22Ihsp82) revealed that Hsp90 function is essential for this (Y-P,T-P)Slt2p to activate one of its downstream targets, the Rlm1p transcription factor. The interaction between Hsp90 and (Y-P,T-P)Slt2p, characterized in this study, is probably essential in this Hsp90 facilitation of the Rlm1p activation by Slt2p.
热休克蛋白90（Hsp90）伴侣循环催化几种重要真核蛋白（Hsp90“客户蛋白”）的最终激活步骤。尽管Hsp90主要以功能形式存在，但Hsp90-半乳糖凝集素4p（Gal4p）DNA结合结构域融合体（Hsp90-BD）在酵母双杂交系统中未显示出强烈相互作用，这与大多数Hsp90-客户蛋白关联的普遍短暂性一致。然而，当将E33A突变引入该诱饵时，在体内检测到了强烈的相互作用，该突变应使Hsp90-客户蛋白复合物停滞在客户蛋白稳定但无法达到其活性形式的阶段。在对作为与Gal4p激活结构域（AD）融合表达的6000种酵母蛋白进行筛选时，这种E33A突变稳定了Hsp90-BD融合体与约3%的酿酒酵母蛋白质组的双杂交相互作用。在检测到的相互作用蛋白中有酵母的两种应激激活的丝裂原活化蛋白（MAP）激酶，Hog1p和Slt2p（Mpk1p）。使用野生型和突变型Hsp90以及Slt2p MAP激酶进行的柱保留实验，以及对应激对突变型Hsp90-BD和AD-Slt2p融合体双杂交相互作用影响的定量测量，表明Hsp90仅与双苏氨酸/酪氨酸磷酸化的应激激活形式的Slt2p[（Y-P，T-P）Slt2p]结合，并且还与该（Y-P，T-P）Slt2p内的MAP激酶结构域结合。对表达突变型Hsp90（T22Ihsp82）的酵母突变体进行的表型分析表明，Hsp90功能对于该（Y-P，T-P）Slt2p激活其下游靶标之一Rlm1p转录因子至关重要。本研究中表征的Hsp90与（Y-P，T-P）Slt2p之间的相互作用可能在Hsp90促进Slt2p激活Rlm1p过程中至关重要。
