Ubiquitination and localization of Ssk1.

<p>(A) Wild-type cells transformed with vector control (pRS415) or with a plasmid expressing Myc-Ssk1 were exponentially grown in HC-Leu (Exp), and subjected to glucose starvation for 30 min (-Glc). Myc-Ssk1 was immunoprecipitated with anti-Myc antibody and ubiquitinated Ssk1 was detected by Western blotting using anti-Ub antibodies. Equal loading of each lane was confirmed by reprobing with anti-Myc antibodies and by immunoblotting with anti-G6pd antibodies. * nonspecific band. (B) Localization of Ssk1-GFP. Wild-type cells expressing Ssk1-GFP were exponentially grown in HC-Leu (EXP), and then starved for glucose for 30 min (-Glc) or subjected to osmotic stress (+NaCl) for 5 min. A strain expressing Hog1-GFP was used as positive control. Intracellular localization of Ssk1-GFP and Hog1-GFP was determined by fluorescence microscopy.</p

Ssk1 is stabilized during glucose starvation.

<p>Wild-type (A) and <i>sln1Δhog1Δ</i> (C) cells expressing Myc-Ssk1 were exponentially grown in HC-Leu (EXP), and then starved for glucose (-Glc) or subjected to osmotic stress (+NaCl). Cycloheximide was added to stop protein synthesis and cells were collected at the indicated times. Levels of unphosphorylated Ssk1 were monitored using SDS-PAGE followed by immunoblotting with anti-Myc antibody. Glucose-6-phosphate dehydrogenase (G6pd) was detected as the loading control. The relative quantities of unphosphorylated Ssk1 in wild-type (B) and <i>sln1Δhog1Δ</i> cells (D) were normalized using G6pd levels. E—<i>snf1Δ</i> cells expressing Myc-Ssk1 were exponentially grown in HC-Leu (EXP), and then starved for glucose (-Glc) or subjected to osmotic stress (+NaCl). Cycloheximide was added to stop protein synthesis and cells were collected at the indicated times. Levels of unphosphorylated Ssk1 were monitored using SDS-PAGE followed by immunoblotting with anti-Myc antibody. Glucose-6-phosphate dehydrogenase (G6pd) was detected as the loading control.</p

Activation of Hog1 MAPK during glucose starvation is dependent of Ssk1.

<p>(A) Schematic diagram of the yeast Hog1 pathway—Osmotic stress can be transduced by Sho1 and Sln1 activation branches. Proteins important for Hog1 phosphorylation during glucose starvation are highlighted. (B) Wild-type (wt), <i>ssk1</i>Δ and <i>sho1</i>Δ strains were exponentially grown and subjected to 0.4 M NaCl for 5 min (+NaCl) or starved for glucose for 45 min (-Glc). Cells were collected at the indicated times and equal amounts of cell extracts were analyzed by SDS-PAGE and by immunoblotting using anti-phospho-p38 antibody. Glucose-6-phosphate dehydrogenase (G6pd) was probed as the loading control. Levels of phosphorylated Hog1 were normalized using G6pd levels and then the relative quantification was normalized by time 0.</p

Detection of phosphorylated and unphosphorylated Ssk1.

<p>(A) Schematic diagram of Sln1 branch. Under normal osmotic conditions, Sln1 is autophosphorylated by an intrinsic His kinase activity. The phosphate group is transferred to Ssk1 via a phosphorelay system that involves the transfer protein Ypd1. Phosphorylated Ssk1 is unable to bind Ssk2/Ssk22 and keeps the pathway inactive. During osmotic stress Sln1 kinase activity is inhibited and unphosphorylated Ssk1 activates the Hog1 pathway and is ubiquitinated via Ubc7 and degraded by the proteasome. (B) Wild-type (wt) and sln1Δhog1Δ cells transformed with Myc-Ssk1 plasmid (pMV06) or wt cells containing the pRS415 vector (control) were grown as indicated. Cell extracts were analyzed by SDS-PAGE followed by immunoblotting with anti-Myc antibody. The presence of phosphorylated Ssk1 (Myc-Ssk1-P) was determined by shift of electrophoretic mobility after treatment with alkaline phosphatase CIP performed according to manufacturer’s instructions. Glucose-6-phosphate dehydrogenase (G6pd) was detected as the loading control. (C) Effects of proteasome inhibition by MG132 on degradation of Myc-Ssk1 and phosphorylation of Hog1. Wild-type yeast cells expressing Myc-Ssk1 were exponentially grown (EXP) and treated with the proteasome inhibitor MG132 or DMSO (control), containing cycloheximide to stop protein synthesis. The degradation of unphosphorylated Ssk1 was analyzed on 7% Phos-tag gel containing 50μM Phos-tag acrylamide followed by immunoblotting with anti-Myc antibody. Levels of phosphorylated Hog1 were analyzed on normal SDS-PAGE followed by immunoblotting with anti-phospho-p38 antibody. Glucose-6-phosphate dehydrogenase (G6pd) was detected as the loading control. Levels of phosphorylated Hog1 and unphosphorylated Ssk1 were normalized using G6pd levels and then the relative quantification was normalized using the DMSO sample at time 0.</p