Identification of Positive Regulators of the Yeast Fps1 Glycerol Channel

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. Author Summary When challenged by changes in extracellular osmolarity, many fungal species regulate their intracellular glycerol concentration to modulate their internal osmotic pressure. Maintenance of osmotic homeostasis prevents either cellular collapse under hyper-osmotic stress or cell rupture under hypo-osmotic stress. In baker's yeast, the Fps1 glycerol channel functions as the main vent for glycerol. Proper regulation of Fps1 is critical to the maintenance of osmotic homeostasis. In this study, we identify a pair of proteins (Rgc1 and Rgc2) that function as positive regulators of Fps1 activity. Their absence results in hyper-accumulation of glycerol and consequent cell lysis due to impaired Fps1 channel activity. Additionally, we found that these glycerol channel regulators function between the Hog1 (High Osmolarity Glycerol response) signaling kinase and Fps1, defining a signaling pathway for control of glycerol efflux. Because members of the Rgc1/2 family are found among pathogenic fungal species, but not in humans, they represent potentially attractive targets for antifungal drug development.National Institues of Health (GM48533