SummaryThe 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. Fps1⌬ 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.

Eadie-Hofstee plots of glycerol uptake in wild-type Saccharomyces cerevisiae W303-1A grown on glucose showed the presence of both saturable transport and simple diffusion, whereas an fps1⌬ mutant displayed only simple diffusion. Transformation of the fps1⌬ mutant with the glpF gene, which encodes glycerol transport in Escherichia coli, restored biphasic transport kinetics. Yeast extract-peptone-dextrose-grown wild-type cells had a higher passive diffusion constant than the fps1⌬ mutant, and ethanol enhanced the rate of proton diffusion to a greater extent in the wild type than in the fps1⌬ mutant. In addition, the lipid fraction of the fps1⌬ mutant contained a lower percentage of phospholipids and a higher percentage of glycolipids than that of the wild type. Fps1p, therefore, may be involved in the regulation of lipid metabolism in S. cerevisiae, affecting membrane permeability in addition to fulfilling its specific role in glycerol transport. Simultaneous uptake of glycerol and protons occurred in both glycerol-and ethanol-grown wild-type and fps1⌬ cells and resulted in the accumulation of glycerol at an inside-to-outside ratio of 12:1 to 15:1. Carbonyl cyanide m-chlorophenylhydrazone prevented glycerol accumulation in both strains and abolished transport in the fps1⌬ mutant grown on ethanol. Likewise, 2,4-dinitrophenol inhibited transport in glycerol-grown wild-type cells. These results indicate the presence of an Fps1p-dependent facilitated diffusion system in glucose-grown cells and an Fps1p-independent proton symport system in derepressed cells.Glycerol crosses all biological membranes by passive diffusion due to its lipophilic nature. In addition, specific transport proteins are frequently produced by microorganisms, resulting in more rapid transport of glycerol across the membrane. Active glycerol transport systems requiring the expenditure of metabolic energy have been identified in Zygosaccharomyces rouxii, Debaryomyces hansenii and Pichia sorbitophila (21,23,36), whereas glycerol crosses the Escherichia coli cytoplasmic membrane via a proteinaceous pore mechanism which is encoded by glpF (15).It has been assumed that glycerol is taken up by Saccharomyces cerevisiae by passive diffusion only. Recently FPS1, which encodes a protein belonging to the MIP family, has been shown to affect the movement of glycerol across the membrane of S. cerevisiae (24). The FPS1 gene was isolated as a multicopy suppressor of the growth defect on fermentable sugars of a yeast fdp1 (FDP1 is also known as CIF1 and GGS1) mutant (33). Fps1p seems to play an important role in glycerol efflux, since mutants lacking FPS1 fail to rapidly release excess glycerol when hyperosmotic stress is relieved and during glycerol overproduction (reference 24 and unpublished results).The MIP family is a group of channel proteins present in organisms ranging from bacteria to humans (28). Most of these proteins are around 250 to 280 amino acids long and consist of six membrane-spanning segments. Fps1p differs from most members of the MIP family b...

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