Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.
PLoS One. 2019 Jan 25;14(1):e0211380. doi: 10.1371/journal.pone.0211380. eCollection 2019.
To cope with increased extracellular osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK), which controls a variety of adaptive responses. Hog1 is activated through the high-osmolarity glycerol (HOG) pathway, which consists of a core MAPK cascade and two independent upstream branches (SHO1 and SLN1 branches) containing distinct osmosensing machineries. In the SHO1 branch, a homo-oligomer of Sho1, the four-transmembrane (TM) osmosensor, interacts with the transmembrane co-osmosensors, Hkr1 and Msb2, and the membrane anchor protein Opy2, through their TM domains, and activates the Ste20-Ste11-Pbs2-Hog1 kinase cascade. In this study, we isolated and analyzed hyperactive mutants of Sho1 and Opy2 that harbor mutations within their TM domains. Several hyperactive mutations enhanced the interaction between Sho1 and Opy2, indicating the importance of the TM-mediated interaction between Sho1 and Opy2 for facilitating effective signaling. The interaction between the TM domains of Sho1 and Opy2 will place their respective cytoplasmic binding partners Pbs2 and Ste11 in close proximity. Indeed, genetic analyses of the mutants showed that the Sho1-Opy2 interaction enhances the activation of Pbs2 by Ste11, but not Hog1 by Pbs2. Some of the hyperactive mutants had mutations at the extracellular ends of either Sho1 TM4 or Opy2 TM, and defined the Sho1-Opy2 binding site 1 (BS1). Chemical crosslinking and mutational analyses revealed that the cytoplasmic ends of Sho1 TM1 and Opy2 TM also interact with each other, defining the Sho1-Opy2 binding site 2 (BS2). A geometric consideration constrains that one Opy2 molecule must interact with two adjacent Sho1 molecules in Sho1 oligomer. These results raise a possibility that an alteration of the conformation of the Sho1-Opy2 complex might contributes to the osmotic activation of the Hog1 MAPK cascade.
为了应对细胞外渗透压的增加，出芽酵母酿酒酵母激活了 Hog1 丝裂原活化蛋白激酶（MAPK），该激酶控制着各种适应性反应。Hog1 通过高渗透压甘油（HOG）途径激活，该途径由一个核心 MAPK 级联和两个独立的上游分支（SHO1 和 SLN1 分支）组成，包含不同的渗透压感应机制。在 SHO1 分支中，四跨膜（TM）渗透压传感器 Sho1 的同源寡聚物通过其 TM 结构域与跨膜共渗透压传感器 Hkr1 和 Msb2 以及膜锚定蛋白 Opy2 相互作用，并激活 Ste20-Ste11-Pbs2-Hog1 激酶级联。在这项研究中，我们分离并分析了其 TM 结构域内存在突变的 Sho1 和 Opy2 的超活性突变体。一些超活性突变增强了 Sho1 和 Opy2 之间的相互作用，表明 Sho1 和 Opy2 之间 TM 介导的相互作用对于促进有效的信号传递非常重要。Sho1 和 Opy2 的 TM 结构域之间的相互作用将使它们各自的细胞质结合伴侣 Pbs2 和 Ste11 紧密接近。事实上，突变体的遗传分析表明，Sho1-Opy2 相互作用增强了 Ste11 对 Pbs2 的激活，但不能增强 Pbs2 对 Hog1 的激活。一些超活性突变体在 Sho1 TM4 或 Opy2 TM 的细胞外末端存在突变，并定义了 Sho1-Opy2 结合位点 1（BS1）。化学交联和突变分析表明，Sho1 TM1 和 Opy2 TM 的细胞质末端也相互作用，定义了 Sho1-Opy2 结合位点 2（BS2）。几何考虑限制了 Sho1 寡聚物中一个 Opy2 分子必须与两个相邻的 Sho1 分子相互作用。这些结果提出了一种可能性，即 Sho1-Opy2 复合物构象的改变可能有助于 Hog1 MAPK 级联的渗透激活。
