Osmosensing and scaffolding functions of the oligomeric four-transmembrane domain osmosensor Sho1

Abstract

The yeast high osmolarity glycerol (HOG) pathway activates the Hog1 MAP kinase, which coordinates adaptation to high osmolarity conditions. Here we demonstrate that the four-transmembrane (TM) domain protein Sho1 is an osmosensor in the HKR1 sub-branch of the HOG pathway. Crosslinking studies indicate that Sho1 forms planar oligomers of the dimers-of-trimers architecture by dimerizing at the TM1/TM4 interface and trimerizing at the TM2/TM3 interface. High external osmolarity induces structural changes in the Sho1 TM domains and Sho1 binding to the cytoplasmic adaptor protein Ste50, which leads to Hog1 activation. Besides its osmosensing function, the Sho1 oligomer serves as a scaffold. By binding to the TM proteins Opy2 and Hkr1 at the TM1/TM4 and TM2/TM3 interface, respectively, Sho1 forms a multi-component signalling complex that is essential for Hog1 activation. Our results illuminate how the four TM domains of Sho1 dictate the oligomer structure as well as its osmosensing and scaffolding functions.

Scaffold Protein Ahk1, Which Associates with Hkr1, Sho1, Ste11, and Pbs2, Inhibits Cross Talk Signaling from the Hkr1 Osmosensor to the Kss1 Mitogen-Activated Protein Kinase

“…Thus, we examined whether any constitutively active mutant required Hkr1-cyto to induce osmostress-induced Hog1 activation. Overexpression of Opy2-F96I A104V (22) activated Hog1 in the presence of WT Hkr1 but not in the presence of Hkr1 cytoplasmic deletion mutants (Fig. 2C), suggesting that Hkr1-cyto was required downstream of active Opy2 for activation of Hog1.…”

“…Sho1, which has four TM segments (Fig. 5A), homodimerizes at the TM1/TM4 interface and homotrimerizes at the TM2/TM3 interface (22). The Sho1 TM1/TM4 interface binds to Opy2, and the TM2/TM3 interface binds to Hkr1.…”

“…Thus, osmostress must activate a signaling step that occurs after Ste11 is phosphorylated by Ste20. Our recent finding that osmostress induces Ste50-Sho1 binding might explain this activation step (22). Because Ste50 is constitutively bound to Ste11 (27,28), and Sho1 is constitutively bound to Pbs2 (19), an enhanced Ste50-Sho1 interaction will inevitably lead to an increased Ste11-Pbs2 interaction.…”

“…The major osmosensor for the SHO1 branch is the four-transmembrane (four-TM) protein Sho1 (19,22). In the SHO1 branch, osmostress activates the Ste11 MAPKKK (23), which then sequentially activates Pbs2 and Hog1 (24).…”

“…The HMH domain has a positive regulatory function, because deletion of the HMH domain renders both Hkr1 and Msb2 incapable of activating Hog1 in response to osmostress (29,30,33). We have recently shown that the HMH domains bind to the extracellular Cys-rich region of Opy2 (22,29). Opy2 is a single-path transmembrane protein whose cytoplasmic tail binds to the Ste50 adaptor protein (36,37).…”