Binding of the extracellular eight-cysteine motif of Opy2 to the putative osmosensor Msb2 is essential for activation of the yeast High-Osmolarity Glycerol pathway
Binding of the extracellular eight-cysteine motif of Opy2 to the putative osmosensor Msb2 is essential for activation of the yeast High-Osmolarity Glycerol pathway
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Opy2 的胞外八个半胱氨酸基序与推定的渗透传感器 Msb2 的结合对于激活酵母高渗透甘油途径至关重要
DOI：
10.1128/mcb.00853-15
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
and Saito H
中科院分区：
文献类型：
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作者：
Yamamoto K;Tatebayashi K;and Saito H
为了适应环境的高渗透压，出芽酵母酿酒酵母激活Hog1丝裂原激活蛋白激酶，调节多种渗透适应反应。Hog1通过高渗透压甘油（high- osmoarity glycerol， HOG）通路被激活，该通路由独立的上游信号通路SLN1分支和SHO1分支组成。在这里，我们报道了跨膜锚定蛋白Opy2的细胞外富含半胱氨酸（CR）结构域与假定的渗透传感器Msb2的Hkr1-Msb2同源性（HMH）结构域结合，并且Opy2-Msb2复合物的形成是通过SHO1分支的Msb2亚分支渗透激活Hog1的必要条件。通过分析Opy2半胱氨酸-丙氨酸突变突变体的表型，我们推断出CR结构域形成了四个分子内二硫键。为了探究渗透胁迫对Opy2-Msb2复合物构象变化的潜在诱导作用，我们构建了Opy2 CR结构域的Cys- To - ala突变位点突变体和Msb2 HMH结构域的Cys取代突变体。每个突变体的半胱氨酸含量都降低了。然后使用不同长度的化学交联剂对这些突变体进行组合交联。Opy2 Cys48和Msb2 Cys1023之间的交联对渗透变化敏感，表明渗透胁迫引起了构象变化。因此，我们提出Opy2-Msb2复合物可能作为一种渗透传感器。
To adapt to environmental high osmolarity, the budding yeastSaccharomyces cerevisiaeactivates the Hog1 mitogen-activated protein kinase, which regulates diverse osmoadaptive responses. Hog1 is activated through the high-osmolarity glycerol (HOG) pathway, which consists of independent upstream signaling routes termed the SLN1 branch and the SHO1 branch. Here, we report that the extracellular cysteine-rich (CR) domain of the transmembrane-anchor protein Opy2 binds to the Hkr1-Msb2 homology (HMH) domain of the putative osmosensor Msb2 and that formation of the Opy2-Msb2 complex is essential for osmotic activation of Hog1 through the MSB2 subbranch of the SHO1 branch. By analyzing the phenotypes of mutants with Opy2 cysteine-to-alanine mutations, we deduced that the CR domain forms four intramolecular disulfide bonds. To probe for the potential induction of conformational changes in the Opy2-Msb2 complex by osmostress, we constructed mutants with a site-specific Cys-to-Ala mutation of the Opy2 CR domain and mutants with a Cys substitution of the Msb2 HMH domain. Each of these mutants had a reduced cysteine. These mutants were then combinatorially cross-linked using chemical cross-linkers of different lengths. Cross-linking between Opy2 Cys48 and Msb2 Cys1023 was sensitive to osmotic changes, suggesting that osmostress induced a conformational change. We therefore propose that the Opy2-Msb2 complex might serve as an osmosensor.