We have cloned three genes for protein phosphatases in the yeast Saccharomyces cerevisiae. Two of the genes, PPH21 and PPH22, encode highly similar proteins that are homologs of the mammalian protein phosphatase 2A (PP2A), while the third gene, PPH3, encodes a new PP2A-related protein. Disruptions of either PPH21 or PPH22 had no effects, but spores disrupted for both genes produced very small colonies with few surviving cells. We conclude that PP2A performs an important function in yeast cells. A disruption of the third gene, PPH3, did not in itself affect growth, but it completely prevented growth of spores disrupted for both PPH21 and PPH22. Thus, PPH3 provides some PP2A-complementing activity which allows for a limited growth of PP2A-deficient cells. Strains were constructed in which we could study the phenotypes caused by either excess PP2A or total PP2A depletion. We found that the level of PP2A activity has dramatic effects on cell shape. PP2A-depleted cells develop an abnormal pear-shaped morphology which is particularly pronounced in the growing bud. In contrast, overexpression of PP2A produces more elongated cells, and high-level overexpression causes a balloonlike phenotype with huge swollen cells filled by large vacuoles.The eukaryotic cell has a large number of protein kinases that are involved in signal transduction, metabolic regulation, and cell cycle control (24). This profusion of kinases is matched by a rapidly expanding number of protein phosphatases. Most of these belong to a superfamily of serine/ threonine phosphatases that includes the type 1, type 2A, and type 2B enzymes (8, 9, 11). The type 1 and 2A phosphatases are more closely related to each other than to the type 2B enzyme (9). The type 2A-related phosphatases comprise mammalian protein phosphatase 2A (PP2A) and its homologs in other organisms, but also more distantly related proteins, such as the yeast PPH1/SIT4 (2), rabbit PPX (11), and Drosophila PPV phosphatases (11).The type 1 and 2A phosphatases have broad substrate specificities and are thought to be involved in a number of cellular processes (8). In particular, they have been implicated in cell cycle control (12). Thus, a type 1 phosphatase is necessary for completion of mitosis in both fungi and Drosophila sp. (3,5,15,39), and experiments in Xenopus sp. have shown that PP2A negatively regulates cdc2 kinase, a key effector in the cell cycle (17, 28). In fission yeast cells, PP2A is an essential enzyme encoded by two duplicated genes (25). Disruption of one of the genes reduces the cell size, which was interpreted as evidence of premature mitosis (25). PP2A has also been implicated in the control of cyclin degradation (30). Further evidence that PP2A plays an important role in growth control comes from experiments with simian virus 40 and polyomavirus. Thus, PP2A binds to the middle T and small t antigens, and it is thought that this interaction is necessary for their functions in transformation and viral replication (40, 52). PP2A also regulates the function of simian virus 4...

Med5 (Nut1) is identified here as a component of the Mediator tail region. Med5 is positioned peripherally to Med16 (Sin4) together with the three members of the putative Gal11 module, Med15 (Gal11), Med2, and Med3 (Pgd1). The biochemical analysis receives support from genetic interactions between med5⌬ and med15⌬ deletions. The med5⌬ and med16⌬ deletion strains share many phenotypes, including effects on mitochondrial function with enhanced growth on nonfermentable carbon sources, increased citrate synthase activity, and increased oxygen consumption. Deletion of the MED5 gene leads to increased transcription of nuclear genes encoding components of the oxidative phosphorylation machinery, whereas mitochondrial genes encoding components of the same machinery are down-regulated. We discuss a possible role for Med5 in coordinating nuclear and mitochondrial gene transcription.The multiprotein Mediator complex is required for basal and regulated expression of nearly all RNA polymerase II (pol II) 3 -dependent genes in the Saccharomyces cerevisiae genome. Mediator conveys regulatory information from enhancers and other control elements to the promoter (1). The functional activities identified for Mediator include stimulation of basal transcription, support of activated transcription, and enhancement of phosphorylation of the C-terminal domain of pol II by the transcription factor IIH kinase (2, 3). S. cerevisiae Mediator also contains a histone acetyltransferase (HAT) activity, which is not found in other eukaryotic Mediator complexes (4). The HAT activity was localized to Med5 (Nut1), a S. cerevisiae-specific protein, which lacks homologues in higher eukaryotes (4, 5). The MED5 gene was originally isolated in a screen for mutants that would suppress the Swi4/Swi6 dependence of a synthetic reporter gene containing part of the HO promoter (6). Several other genes encoding Mediator proteins were identified in the same screen, including MED10 (NUT2), MED16 (SIN4), MED19 (ROX3), MED12 (SRB8), MED13 (SRB9), CDK8 (SRB10), and CYCC (SRB11). The MED5 gene is nonessential in yeast. A deletion of MED5 relieves repression at the URS2 element in the HO promoter but only in combination with a mutant allele of either MED10 or CCR4 (6). These effects on the HO promoter were seen with a lacZ reporter gene but not at the endogenous HO gene locus. The in vivo role of Med5 in Mediator-dependent gene expression therefore remains an open question.In the presence of RNA pol II, Mediator adopts an extended conformation that embraces the globular pol II core complex (7). The extended structure reveals three distinct submodules of Mediator. Direct contacts are formed between pol II and the head and middle region (7,8). The largest part of Mediator is made up of an elongated tail region, which does not appear to contact pol II. Structural analysis of mutant Mediator complex has demonstrated that the tail region contains the Med2, Med3, Med15 (Gal11), and Med16, proteins, which are involved in interactions with a number of different activators,...

BTF3 is a human protein that is thought to be involved in transcription by RNA polymerase II [Zheng et al., Cell 50, 361-368, 1987]. A yeast homologue of BTF3, Egd1p, has been identified by its ability to enhance DNA binding of the Gal4p activator [Parthun et al., Mol. Cell. Biol. 12, 5683-5689, 1992]. We have cloned a second yeast gene, BTT1, which also encodes a BTF3 homologue. Btt1p and Egd1p are highly similar in sequence, which suggests that they are duplicated proteins with similar functions. Gene disruptions were used to investigate the function of the two proteins. Consistent with published results, we found that loss of EGD1 causes a minor defect in GAL gene induction. Loss of BTT1 has little if any effect. Surprisingly, we found that cells which lack both genes instead express the GAL1 and GAL10 mRNAs at much higher levels than wild type cells. This suggests that BTF3 really plays a negative role in GAL gene expression. Further experiments revealed that expression of the ACT1 and SSO1 genes also is elevated in cells that lack EGD1 and BTT1. In contrast, expression of rRNA and tRNA was not affected. We conclude that Btt1p and Egd1p have redundant functions in vivo, and that they exert a negative effect on the expression of several genes that are transcribed by RNA polymerase II.

5-Fluorouracil (5-FU) is an anticancer drug and pyrimidine analogue. A problem in 5-FU therapy is acquired resistance to the drug. To find out more about the mechanisms of resistance, we screened a plasmid library in yeast for genes that confer 5-FU resistance when overexpressed. We cloned five genes: CPA1, CPA2, HMS1, HAM1 and YJL055W. CPA1 and CPA2 encode a carbamoyl phosphate synthase involved in arginine biosynthesis and HMS1 a helix-loop-helix transcription factor. Our results suggest that CPA1, CPA2, and HMS1 confer 5-FU resistance by stimulating pyrimidine biosynthesis. Thus, they are unable to confer 5-FU resistance in a ura2 mutant, and inhibit the uptake and incorporation into RNA of both uracil and 5-FU. In contrast, HAM1 and YJL055W confer 5-FU resistance in a ura2 mutant, and selectively inhibit incorporation into RNA of 5-FU but not uracil. HAM1 is the strongest resistance gene, but it partially depends on YJL055W for its function. This suggests that HAM1 and YJL055W function together in mediating resistance to 5-FU. Ham1p encodes an inosine triphosphate pyrophosphatase that has been implicated in resistance to purine analogues. Our results suggest that Ham1p could have a broader specificity that includes 5-FUTP and other pyrimidine analogoue triphosphates.