Characteristics of Fps1-dependent and -independent glycerol transport in Saccharomyces cerevisiae

Abstract

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...

Analysis of the Pore of the Unusual Major Intrinsic Protein Channel, Yeast Fps1p

“…These observations establish that Fps1p primarily mediates glycerol diffusion out of the cell (11) with an energy-independent mechanism (8). Despite the fact that Fps1p can also mediate measurable glycerol influx into yeast cells, (7,8,12), this is probably not its physiological role because no growth defects have been found in fps1⌬ mutants cultivated with glycerol as the sole carbon source (8). Furthermore GUP1 and GUP2 have recently been proposed to be involved in active transport of glycerol in yeasts (13).…”

New insights on glycerol transport in Saccharomyces cerevisiae

“…To sustain this statement, ANOVA test was applied. All the K m values available from strains with W303-1A background from previous works [6,14] were used together with the ones here obtained. Values were not significantly different: p > 0:05 in all cases.…”

Intracellular Glycerol Levels Modulate the Activity of Sln1p, aSaccharomyces cerevisiae Two-component Regulator

“…Given the known characteristics of the fps1 mutant, two distinct but related stimuli can be postulated. Analysis of membrane composition in fps1 mutants revealed alterations in phospholipid and glycolipid fractions in the fps1 mutants (48), suggesting that membrane composition itself may mimic the effect of osmotic stress on membrane composition, fluidity, or relationship to the wall. Alternatively, the increase in intracellular glycerol levels in an fps1 mutant might provide the signal.…”