The PH084 gene specifies Pi-transport in Saccharomyces cerevisiae. A DNA fragment bearing the PH084 gene was cloned by its ability to complement constitutive synthesis of repressible acid phosphatase of pho84 mutant cells. Its nucleotide sequence predicted a protein of 596 amino acids with a sequence homologous to that of a superfamily of sugar transporters. Hydropathy analysis suggested that the secondary structure of the PH084 protein consists of two blocks of six transmembrane domains separated by 74 amino acid residues. The cloned PH084 DNA restored the Pi transport activity of pho84 mutant cells. The PH084 transcription was regulated by P1 like those of the PHOS, PHO8, and PHO81 genes. A PH084-lacZ fusion gene produced j}-galactosidase activity under the regulation of Pi, and the activity was suggested to be bound to a membrane fraction. Gene disruption of PH084 was not lethal. By comparison of nucleotide sequences and by tetrad analysis with GAL80 as a standard, the PH084 locus was mapped at a site beside the TUB3 locus on the left arm of chromosome XIII.

A higher plant homologue to the highaffinity phosphate transporter gene of yeast (Saccharomyces cerevisiae) PHO84 was isolated from Arabidopsis thaliana. Expression of the Arabidopsis gene PHT1 at high levels in tobacco-cultured cells increased the rate of phosphate uptake. The uptake activity attributable to the transgene was inhibited by protonophores, suggesting an H ؉ cotransport mechanism of phosphate uptake, and had a K m of 3.1 M which is within limits characteristic of high-affinity transport mechanisms. These results indicate that PHT1 encodes a high-affinity phosphate transporter. The transgenic cells exhibited increased biomass production when the supply of phosphate was limited, establishing gene engineering of phosphate transport as one approach toward enhancing plant cell growth.

During fermentation, yeast cells are exposed to a number of stresses -- such as high alcohol concentration, high osmotic pressure, and temperature fluctuation - so some overlap of mechanisms involved in the response to these stresses has been suggested. To identify the genes required for tolerance to alcohol (ethanol, methanol, and 1-propanol), heat, osmotic stress, and oxidative stress, we performed genome-wide screening by using 4828 yeast deletion mutants. Our screens identified 95, 54, 125, 178, 42, and 30 deletion mutants sensitive to ethanol, methanol, 1-propanol, heat, NaCl, and H2O2, respectively. These deleted genes were then classified based on their cellular functions, and cross-sensitivities between stresses were determined. A large number of genes involved in vacuolar H(+)-ATPase (V-ATPase) function, cytoskeleton biogenesis, and cell wall integrity, were required for tolerance to alcohol, suggesting their protective role against alcohol stress. Our results revealed a partial overlap between genes required for alcohol tolerance and those required for thermotolerance. Genes involved in cell wall integrity and the actin cytoskeleton are required for both alcohol tolerance and thermotolerance, whereas the RNA polymerase II mediator complex seems to be specific to heat tolerance. However, no significant overlap of genes required for osmotic stress and oxidative stress with those required for other stresses was observed. Interestingly, although mitochondrial function is likely involved in tolerance to several stresses, it was found to be less important for thermotolerance. The genes identified in this study should be helpful for future research into the molecular mechanisms of stress response.

Transcriptional coactivators play a crucial role in gene expression by communicating between regulatory factors and the basal transcription machinery. The coactivator multiprotein bridging factor 1 (MBF1) was originally identified as a bridging molecule that connects the Drosophila nuclear receptor FTZ-F1 and TATAbinding protein (TBP). The MBF1 sequence is highly conserved across species from Saccharomyces cerevisiae to human. Here we provide evidence acquired in vitro and in vivo that yeast MBF1 mediates GCN4-dependent transcriptional activation by bridging the DNA-binding region of GCN4 and TBP. These findings indicate that the coactivator MBF1 functions by recruiting TBP to promoters where DNA-binding regulators are bound.Studies on transcriptional control in eukaryotes have revealed many regulatory proteins that bind to control elements on DNA (17,25). Typical regulators such as GAL4 and GCN4 consist of two domains (29), a DNA-binding domain that binds to a control element in a sequence-specific fashion and a transcriptional activation domain that somehow stimulates the basal transcription machinery. A variety of observations have led to the proposal that some transactivation domains may facilitate binding of TATA-binding protein (TBP) to a promoter (32, 37 see reference 35 for a review). In many cases, a member of another class of transcription factors, termed coactivators, adapters, or mediators, is necessary to connect a regulatory protein and a component of the basal transcription machinery (see references 23 and 30 for reviews). Recent studies demonstrated the importance of these non-DNA-binding transcription factors in gene expression (1,4,5,11,14,19,20,27,31).In in vitro transcription studies, it was found that an insect coactivator, multiprotein bridging factor 1 (MBF1), can recruit TBP to a promoter carrying the FTZ-F1-binding site by interconnecting 34). A homology search of the databases showed that the MBF1 sequence is highly conserved across species from Saccharomyces cerevisiae to human (34). A key question concerning MBF1, and its close relatives, is its in vivo function. To address this issue, we have started genetic studies of MBF1. We report here that MBF1 serves as a coactivator of GCN4 in the yeast S. cerevisiae. MATERIALS AND METHODSYeast strains and plasmids. The GCN4 disruptant used in this study was KY803 (trp1-⌬1 ura3-52 leu2-P1 gcn4-⌬1) (13). Wild-type strain KT130 (trp1-⌬1 ura3-52 leu2-P1) was constructed by homologous recombination after introduction of GCN4 genomic DNA into KY803. KT130 was used to generate the ⌬mbf1 strain by replacement of the sequence encoding amino acid residues 64 to 131 of yeast MBF1 (yMBF1) (34) with a LEU2 selectable marker.To construct pMBF1, the 3-kb EcoRI genomic fragment encompassing the entire MBF1 regulatory and coding regions was subcloned into YCplac33 (9). YCp88 (13), a centromeric vector containing the constitutive DED1 promoter, was used to express GCN4 or its mutants in KY803. pYCGCN4, pYCGCN4⌬bZIP, and pYCGCN4bZIP carry the entire coding regi...

In Saccharomyces cerevisiae, the phosphate signal transduction pathway (PHO pathway) is known to regulate the expression of several phosphate-responsive genes, such as PHO5 and PHO84. However, the fundamental issue of whether cells sense intracellular or extracellular phosphate remains unresolved. To address this issue, we have directly measured intracellular phosphate concentrations by 31 P NMR spectroscopy. We find that PHO5 expression is strongly correlated with the levels of both intracellular orthophosphate and intracellular polyphosphate and that the signaling defect in the ⌬pho84 strain is likely to result from insufficient intracellular phosphate caused by a defect in phosphate uptake. Furthermore, the ⌬phm1⌬phm2, ⌬phm3, and ⌬phm4 strains, which lack intracellular polyphosphate, have higher intracellular orthophosphate levels and lower expression of PHO5 than the wild-type strain. By contrast, the ⌬phm5 strain, which has lower intracellular orthophosphate and higher polyphosphate levels than the wild-type strain, shows repressed expression of PHO5, similar to the wildtype strain. These observations suggest that PHO5 expression is under the regulation of intracellular orthophosphate, although orthophosphate is not the sole signaling molecule. Moreover, the disruption of PHM3, PHM4, or of both PHM1 and PHM2 in the ⌬pho84 strain suppresses, although not completely, the PHO5 constitutive phenotype by increasing intracellular orthophosphate, suggesting that Pho84p affects phosphate signaling largely by functioning as a transporter.Inorganic phosphate is an essential nutrient needed in large amounts for nucleic acid and phospholipid biosynthesis, as well as for energy metabolism. It is therefore essential that organisms have appropriate regulatory mechanisms to respond and adapt rapidly to changes in phosphate availability (1). In Saccharomyces cerevisiae, the phosphate signal transduction pathway (PHO pathway) regulates the expression of several phosphate-responsive genes that are involved in the scavenging and specific uptake of phosphate from extracellular sources, including PHO5, which encodes repressible acid phosphatase (rAPase), 1 and PHO84, which encodes a high affinity phosphate transporter that acts in response to phosphate levels (2-4). Under high phosphate conditions, the transcription factor Pho4p is phosphorylated by the cyclin-dependent kinase (CDK) complex Pho80p-Pho85p (5) and exported from the nucleus to the cytoplasm (6), thereby turning off expression of the PHO5 and PHO84 genes. When yeast cells are starved of phosphate, the CDK inhibitor Pho81p inactivates 8). Under these conditions, Pho4p is unphosphorylated and active (9), leading to the induction of PHO5 and PHO84 expression to scavenge phosphate from the environment (2-4, 10). Although many components and the molecular mechanism of the PHO pathway have been extensively studied, fundamental questions, such as whether cells sense intracellular or extracellular phosphate and what phosphate metabolites function as signals for regulating t...