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2005). hyperosmotic shock entails a redundant pair of regulators, Rgc1 (regulator of the glycerol channel 1) and Rgc2, and the MAPK Hog1 (high-osmolarity glycerol response 1). However, the mechanism by which these factors influence channel activity is usually unknown. We show that Rgc2 maintains Fps1 in the open channel state in the absence of osmotic stress by binding to its C-terminal cytoplasmic domain name. This interaction entails a tripartite pleckstrin homology (PH) domain name within Rgc2 and a partial PH domain name within Fps1. Activation of Hog1 in response to hyperosmotic shock induces the quick eviction of Rgc2 from Fps1 and consequent channel closure. Hog1 was recruited to the N-terminal cytoplasmic domain name of Fps1, which it uses as a platform from which to multiply phosphorylate Rgc2. Thus, these results reveal the mechanism by which Hog1 regulates Fps1 in response to hyperosmotic shock. Under conditions of high osmolarity stress, many fungal species, includingSaccharomyces cerevisiae, maintain osmotic equilibrium by generating and retaining high concentrations of glycerol as a compatible solute (Nevoigt and Stahl 1997). Intracellular glycerol concentration is usually regulated inS. cerevisiaein part by the Fps1 plasma membrane glycerol channel (Luyten et al. 1995;Sutherland et al. 1997;Tams et al. 1999). Increased external osmolarity induces Fps1 closure, whereas decreased osmolarity causes channel opening, both within seconds of the switch in external osmolarity (Tams et al. 1999). This channel, which functions as a homotetramer (Beese-Sims et al. 2011), is required for survival of a hypo-osmotic shock, when yeast cells must export glycerol rapidly to prevent bursting (Luyten et al. 1995;Tams et al. 1999). Fps1 is also required for controlling turgor pressure during fusion of mating yeast cells (Philips and Herskowitz 1997). Fps1 is usually a member of the major intrinsic protein (MIP) family of channel proteins. The MIP family is usually subdivided into users that are selectively permeable to water (aquaporins) and those permeated by glycerol and to a lesser extent by water, called aquaglyceroporins or glycerol facilitators (Borgnia and Agre 2001;Agre et al. 2002). Relative to nonfungal aquaglyceroporins, Fps1 possesses N-terminal and C-terminal cytoplasmic extensions that are important MPH1 for its regulation (Tams Norepinephrine hydrochloride et al. 2003;Hedfalk et al. 2004). The pathway responsible for regulation of Fps1 in response to changes in osmolarity has not been fully delineated but entails the MAPK Hog1 (high-osmolarity glycerol response 1) (Tams et al. 1999;Hohmann 2009;Ahmadpour et al. 2013), a homolog of the mammalian p38 MAPK, which binds to the N-terminal cytoplasmic domain of Fps1 (Mollapour and Piper 2007). Hog1 is usually Norepinephrine hydrochloride activated in response to hyperosmotic stress to mediate both the biosynthesis of glycerol and its retention within the cell (Hohmann 2009). Although Hog1 plays a role in glycerol retention by control of Fps1, the mechanism by which it influences Fps1 activity is usually unknown. Hog1 function has been mainly associated with the regulation of transcriptional events (Reiser et al. 1999;Rep et al. 1999;de Nadal and Posas 2010;de Nadal et al. 2011;Saito and Posas 2012), although a recent quantitative mass spectrometry Norepinephrine hydrochloride (MS) analysis has revealed several new candidate substrates of Hog1 (Reiter et al. 2012). Fps1 activity is also controlled by a pair of redundant positive regulators, named Rgc1 (regulator of the glycerol channel 1) and Rgc2 (YPR115WandYGR097W, respectively) (Beese et al. 2009). Additional genetic analyses suggested that Hog1 is usually a negative regulator of Rgc1 and Rgc2, and electrophoretic bandshift assays revealed that Rgc2 is usually phosphorylated in response to hyperosmotic shock in a manner partially dependent on Hog1, suggesting that Hog1 may regulate Fps1 activity indirectly through Rgc1 and Rgc2 (Beese et al. 2009). However, the mechanism by which Rgc1 and Rgc2 control Fps1 activity and its relationship to Hog1 activity remain unclear. Loss of eitherFPS1orRGC1andRGC2function results in excess turgor pressure and consequent cell wall stress (Beese et al. 2009). Additional cell wall stress imposed on these mutants Norepinephrine hydrochloride by, for example, growth at elevated temperature results in cell lysis. Although the fungal kingdom is replete with Rgc orthologs, they are not represented in metazoans, suggesting that the RgcFps pathway may be an attractive target for antifungal drug development. Indeed, loss of the Fps glycerol channels in the fungal pathogenCandida glabratasensitizes cells to antifungal agents that target the cell wall (Beese-Sims et al. 2012). In this study, we explore the mechanisms by which Hog1 and Rgc1/2 control Fps1 channel activity in response to hyperosmotic shock. We demonstrate that Rgc2 maintains Fps1 in an open channel state through an association between the pleckstrin homology (PH) domain of Rgc2 and a partial PH domain within the C-terminal domain of Fps1. We identify several Hog1 phosphorylation sites on Rgc2 by mass.