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Glycerol and other polyols are used as osmoprotectants by many organisms. Several yeasts and other fungi can take up glycerol by proton symport. To identify genes involved in active glycerol uptake in Saccharomyces cerevisiae we screened a deletion mutant collection comprising 321 genes encoding proteins with 6 or more predicted transmembrane domains for impaired growth on glycerol medium. Deletion of STL1, which encodes a member of the sugar transporter family, eliminates active glycerol transport. Stl1p is present in the plasma membrane in S. cerevisiae during conditions where glycerol symport is functional. Both the Stl1 protein and the active glycerol transport are subject to glucose-induced inactivation, following identical patterns. Furthermore, the Stl1 protein and the glycerol symporter activity are strongly but transiently induced when cells are subjected to osmotic shock. STL1 was heterologously expressed in Schizosaccharomyces pombe, a yeast that does not contain its own active glycerol transport system. In S. pombe, STL1 conferred the ability to take up glycerol against a concentration gradient in a proton motive force-dependent manner. We conclude that the glycerol proton symporter in S. cerevisiae is encoded by STL1. INTRODUCTIONGlycerol, a C 3 polyalcohol, is an important intermediate in energy metabolism in both prokaryotes and eukaryotes. It has long been used for therapeutic and industrial processes. Aspects of glycerol metabolism are also important in biotechnology, e.g., for bio-alcohol production yields or wine smoothness. Essential roles of glycerol in basic biochemical aspects have been extensively studied in several yeasts and fungi. These include biosynthesis of glycerophospholipid and triacylglycerol from glycerol 3-phosphate and dihydroxyacetone phosphate (Kohlwein et al., 1996;Mü llner and Daum, 2004), redox balance (Ansell et al., 1997;Valadi et al., 2004), osmoadaptation (reviewed by Hohmann, 2002, oxidative stress protection (Påhlman et al., 2001), and response to heat shock (Siderius et al., 2000). Responses to elevated temperatures and high osmolarity involve several signaling pathways including the protein kinase C pathway and the HOG pathway, which regulates intracellular levels of glycerol (Hohmann, 2002;Wojda et al., 2003).In cells ranging from mammals (Lang et al., 1998) to archea (Kempf and Bremer, 1998), osmolytes play an important role in the response to osmotic stress caused by low water availability in environments as diverse as poorly irrigated soils or high-sugar musts. In eukaryotic microorganisms like algae or yeasts, polyols, primarily glycerol, act as osmolytes (reviewed by Brown, 1977 andWang et al., 2001). Their production, consumption and retention are consequently tightly regulated and dynamic processes (reviewed by Hohmann, 2002). Magnaporthe grisea (rice blast) a phytopathogenic fungus with a strong impact on world economy, accumulates glycerol, which allows the penetration of the appressorium into the plant host cell (Thines et al., 2000). Glycerol has also...

Fructans play an important role in assimilate partitioning and possibly in stress tolerance in many plant families. Sucrose:fructan 6-fructosyltransferase (6-SFT), an enzyme catalyzing the formation and extension of P-2,6-linked

The yeast mitochondrial outer membrane contains a major 70 kd protein with an amino‐terminal hydrophobic membrane anchor and a hydrophilic 60 kd domain exposed to the cytosol. We now show that this protein (which we term MAS70) accelerates the mitochondrial import of many (but not all) precursor proteins. Anti‐MAS70 IgGs or removal of MAS70 from the mitochondria by either mild trypsin treatment or by disrupting the nuclear MAS70 gene inhibits import of the F1‐ATPase beta‐subunit, the ADP/ATP translocator, and of several other precursors into isolated mitochondria by up to 75%, but has little effect on the import of porin. Intact cells of a mas70 null mutant import the F1‐ATPase alpha‐subunit and beta‐subunits, cytochrome c1 and other precursors at least several fold more slowly than wild‐type cells. Removal of MAS70 from wild‐type mitochondria inhibits binding of the ADP/ATP translocator to the mitochondrial surface, indicating that MAS70 mediates one of the earliest import steps. Several precursors are thus imported by a pathway in which MAS70 functions as a receptor‐like component. MAS70 is not essential for import of these precursors, but only accelerates this process.

The Saccharomyces cerevisiae deletion collection was screened for impaired growth on glucose-based complex medium containing 6% ethanol. Forty-six mutants were found. Genes encoding proteins involved in vacuolar function, the cell integrity pathway, mitochondrial function, subunits of the co-chaperone complex GimC and components of the SAGA transcription factor complex were in this way found to be important for the growth of wild-type Saccharomyces yeast in the presence of ethanol. Several mutants were also sensitive to Calcofluor white (14 mutants), sorbic acid (9), increased temperature (5) and NaCl (3). The transcription factors Msn2p and Ars1p, tagged with green fluorescent protein, were translocated to the nucleus upon ethanol stress. Only one of the genes that contain STRE elements in the promoter was important under ethanol stress; this was TPS1, encoding trehalose 6-phosphate synthase. The map kinase of the cell integrity pathway, Slt2p, was phosphorylated when cells were treated with 6% ethanol. Two out of three mutants tested fermented 20% glucose more slowly than the wild-type.