Abstract
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.
Highlights
Extreme environmental osmotic conditions are major threats to their survival for free living single-celled organisms such as the budding yeast Saccharomyces cerevisiae
MAP kinase (MAPK) modules are evolutionarily conserved three-kinase cascades composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK)
We describe our findings concerning the possible mechanism by which the signaling efficiency of the Ste11-Pbs2-Hog1 MAPK cascade is regulated by the interaction between the transmembrane domains of Sho1 and Opy2
Summary
Extreme environmental osmotic conditions are major threats to their survival for free living single-celled organisms such as the budding yeast Saccharomyces cerevisiae. To cope with increased external osmolarity, yeast initiates coordinated adaptive responses that include the synthesis, uptake, and intracellular retention of the compatible osmolyte glycerol [1,2,3,4,5], changes in the global pattern of gene expression and protein synthesis [6,7,8], and temporary arrest of the cell cycle at multiple phases to gain time for adaptation [9,10,11] These adaptive responses are governed by the Hog MAP kinase (MAPK). The activated MAPKK phosphorylates and activates a cognate MAPK [15]
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