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The accumulation of compatible solutes, such as glycerol, in the yeast Saccharomyces cerevisiae, is a ubiquitous mechanism in cellular osmoregulation. Here, we demonstrate that yeast cells control glycerol accumulation in part via a regulated, Fps1p-mediated export of glycerol. Fps1p is a member of the MIP family of channel proteins most closely related to the bacterial glycerol facilitators. The protein is localized in the plasma membrane. The physiological role of Fps1p appears to be glycerol export rather than uptake. Fps1Δ mutants are sensitive to hypo-osmotic shock, demonstrating that osmolyte export is required for recovery from a sudden drop in external osmolarity. In wild-type cells, the glycerol transport rate is decreased by hyperosmotic shock and increased by hypo-osmotic shock on a subminute time scale. This regulation seems to be independent of the known yeast osmosensing HOG and PKC signalling pathways. Mutants lacking the unique hydrophilic N-terminal domain of Fps1p, or certain parts thereof, fail to reduce the glycerol transport rate after a hyperosmotic shock. Yeast cells carrying these constructs constitutively release glycerol and show a dominant hyperosmosensitivity, but compensate for glycerol loss after prolonged incubation by glycerol overproduction. Fps1p may be an example of a more widespread class of regulators of osmoadaptation, which control the cellular content and release of compatible solutes.
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It is likely that all types of cells, even those of multicellular organisms, have developed mechanisms to adapt to changes of the external osmolarity and hence are able to respond to altered water availability. Osmotic adaptation not only plays a role in the response to osmotic stress but is also central to many developmental and morphogenic processes (Dale, 1986; Harold, 1990; Howard and Valent, 1996). A ubiquitous strategy in osmoregulation probably used by all cells is the accumulation of one or several compatible solutes to control the osmolarity of the cytosol (Yancey et al., 1982). The solutes used are species specific and range from ions to amino acids and their derivatives, as well as sugars and sugar alcohols. The unicellular eukaryotic model organism Saccharomyces cerevisiae (baker's yeast) uses glycerol (Brown, 1976).
The accumulation of glycerol by yeasts is controlled at different levels. In S. cerevisiae the regulation of glycerol production appears to play a central role (Larsson et al., 1993; Albertyn et al., 1994; Hohmann, 1997), whereas in other, more osmotolerant yeasts active uptake of glycerol from the medium also seems to be important (Van Zyl et al., 1990; Blomberg and Adler, 1992). Glycerol is produced from the glycolytic intermediate dihydroxyacetone phosphate in two steps catalysed by NAD+-dependent glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Both enzymes are encoded by two similar isogenes, GPD1 plus GPD2 and GPP1 plus GPP2 respectively (Larsson et al., 1993; Albertyn et al., 1994; Eriksson et al., 1995; Norbeck et al., 1996). Expression of GPD1 and GPP2 is induced by high osmolarity (Albertyn et al., 1994; Hirayama et al., 1995; Norbeck et al., 1996), whereas that of GPD2 and GPP1 is stimulated under anaerobic conditions. In the absence of oxygen, glycerol production is required for redox-regulation to reoxidize excess NADH (Albers et al., 1996; Nordström, 1966; Ansell et al., 1997; A.-K. Påhlman and L. Adler, unpublished).
Although very little is known about mechanisms controlling gene expression under anaerobic conditions, several signalling pathways have been reported to be stimulated by altered medium osmolarity. Osmotic induction of GPD1 and GPP2 expression is controlled partly by the high osmolarity glycerol (HOG) response pathway (Brewster et al., 1993; Albertyn et al., 1994; Hirayama et al., 1995; Norbeck et al., 1996; Rep et al., 1999). Another MAP kinase pathway (Treisman, 1996), the protein kinase C (PKC) pathway, can be stimulated by hypo-osmotic shock (Davenport et al., 1995), but does not appear to be involved in the control of GPD1 expression (Rep et al., 1999). Recently, it has been shown that a hypo-osmotic shock leads to a rapid, transient increase in the intracellular calcium level, but the pathway(s) controlling this effect is unknown (Batiza et al., 1996).
Although the regulation of glycerol production can account, at least partly, for the accumulation of the compatible solute after a hyperosmotic shock, additional levels of control are needed when the external osmolarity decreases and the cell has to dispose of osmolytes to avoid excessive turgor pressure. For example, we have shown previously that yeast cells rapidly release accumulated glycerol after a hypo-osmotic shock (Luyten et al., 1995). Rapid export mechanisms of compatible solutes have also been described for bacteria and for mammalian cells (Koo et al., 1991; Strange et al., 1994; Kwon and Handler, 1995; Ruffert et al., 1997; Kirk and Strange, 1998), and appear to be involved in the process of regulated volume decrease after the initial cell swelling caused by a hypo-osmotic shock. Although proteins such as MscL in Escherichia coli and ClC-2 in mammalian cells are candidate solute exporters, the molecular nature of the efflux pathways is unknown (Kirk and Strange, 1998). The rapid glycerol release by yeast after a hypo-osmotic shock has been shown to be dependent on Fps1p (Luyten et al., 1995).
The FPS1 gene was isolated as a suppressor of the growth defect of a mutation affecting the control of yeast glycolysis (Van Aelst et al., 1991), and it is most closely related to bacterial glycerol facilitators such as GlpF from E. coli (Heller et al., 1980; Muramatsu and Mizuno, 1989). We have shown that Fps1p mediates both uptake and export of glycerol from yeast cells (Luyten et al., 1995; Sutherland et al., 1997). In this work, we demonstrate that the physiological role of Fps1p is to regulate export rather than uptake of glycerol by describing growth phenotypes associated to the lack of proper glycerol export in fps1Δ mutants. Mutants lacking Fps1p are sensitive to hypo-osmotic shock, a phenotype which demonstrates that rapid solute release is an essential step in the recovery from a sudden drop in medium osmolarity. The same mutants also grow poorly under anaerobic conditions when glycerol production is stimulated for redox regulation.
More than 150 MIP channels have been identified in all five of the organism kingdoms (Park and Saier, 1996; Froger et al., 1998). Most of the animal and plant MIPs are aquaporin water channels (Engel et al., 1994; Nielsen and Agre, 1995; Park and Saier, 1996; Weig et al., 1997). Humans seem to possess at least nine aquaporins (Ishibashi et al., 1998) supposed to be involved in water flux through many tissues and organs such as the kidney (Nielsen and Agre, 1995; Nielsen et al., 1996). Aquaporins are abundant in plants too: Arabidopsis thaliana apparently possesses 23 such proteins both in the plasma membrane and in the tonoplast (Weig et al., 1997). Microbial MIP channels transport small polyols such as glycerol, but, recently, water channels have also been described in E. coli (Calamita et al., 1995; 1998) and in S. cerevisiae (André, 1995; Bonhivers et al., 1998; V. Laizé, S. Hohmann, P. Ripoche and F. Tacnet, in preparation).
Relatively little is known about the molecular mechanisms of MIP channel regulation. Kidney AQP2, which is required for the concentration of primary urine, appears to be controlled by vasopressin and protein kinase A at the level of membrane localization (Knepper and Inoue, 1997; Mulders et al., 1997a). Plant aquaporins are regulated at the level of gene expression by drought stress and developmental cues (Yamada et al., 1995; Kaldenhoff et al., 1996). In addition, evidence has been presented suggesting that the transport activity of at least some aquaporins might be directly regulated via phosphorylation, as shown in studies involving heterologous expression in Xenopus oocytes (Maurel et al., 1995) and recently also in native tissue (Johansson et al., 1998).
In this work, we demonstrate that Fps1p-mediated glycerol export in S. cerevisiae is rapidly regulated by external osmolarity. We also describe a domain N-terminal to the first transmembrane domain, which is required for closing of the channel. Deletion of this domain renders transport function constitutive, resulting in loss of glycerol from the cell and sensitivity to high osmolarity.