Abstract
The yeast Fps1 protein is an aquaglyceroporin that functions as the major facilitator of glycerol transport in response to changes in extracellular osmolarity. Although the High Osmolarity Glycerol pathway is thought to have a function in at least basal control of Fps1 activity, its mode of regulation is not understood. We describe the identification of a pair of positive regulators of the Fps1 glycerol channel, Rgc1 (Ypr115w) and Rgc2 (Ask10). An rgc1/2Δ mutant experiences cell wall stress that results from osmotic pressure associated with hyper-accumulation of glycerol. Accumulation of glycerol in the rgc1/2Δ mutant results from a defect in Fps1 activity as evidenced by suppression of the defect through Fps1 overexpression, failure to release glycerol upon hypo-osmotic shock, and resistance to arsenite, a toxic metalloid that enters the cell through Fps1. Regulation of Fps1 by Rgc1/2 appears to be indirect; however, evidence is presented supporting the view that Rgc1/2 regulate Fps1 channel activity, rather than its expression, folding, or localization. Rgc2 was phosphorylated in response to stresses that lead to regulation of Fps1. This stress-induced phosphorylation was partially dependent on the Hog1 MAPK. Hog1 was also required for basal phosphorylation of Rgc2, suggesting a mechanism by which Hog1 may regulate Fps1 indirectly.
Highlights
Under conditions of high osmolarity stress, many fungal species, including Saccharomyces cerevisiae, maintain osmotic equilibrium by producing and retaining high concentrations of glycerol as a compatible solute [1,2]
We identify a pair of proteins (Rgc1 and Rgc2) that function as positive regulators of Fps1 activity
The double rgc1/2D mutant, but not the single mutants, displayed a temperature-sensitive growth defect (37uC; Figure 1A) accompanied by cell lysis, as judged by the presence of non-refractile ‘‘ghosts.’’ This result is in contrast to that reported by Cohen et al [20], who found that deletion of ASK10 (RGC2) alone conferred a temperature-sensitive phenotype in the same strain background
Summary
Under conditions of high osmolarity stress, many fungal species, including Saccharomyces cerevisiae, maintain osmotic equilibrium by producing and retaining high concentrations of glycerol as a compatible solute [1,2]. Increased external osmolarity induces Fps closure, whereas decreased osmolarity causes channel opening, both within seconds of the change in external osmolarity [5]. This channel is required for survival of a hypo-osmotic shock when yeast cells have to rapidly export glycerol to prevent bursting [3,5], and is required for controlling turgor pressure during fusion of mating yeast cells [6]. Hog is activated in response to hyper-osmotic stress to mediate the biosynthesis of glycerol and perhaps its retention as well through inhibition of Fps channel activity. A hog1D mutant displays an elevated rate of glycerol uptake in the absence of osmotic stress, it is not impaired for Fps closure in response to hyper-osmotic stress [5], suggesting that Hog regulates the basal activity of Fps
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