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Deletion of SIT4 phosphatase decreased the pyruvate decarboxylase activity, which is essential for directing the glucose flux to ethanol production. Concomitantly, a reduction in the fermentative capacity was observed. As pyruvate decarboxylase expression was not altered, its post-translational phosphorylation was studied. Immunoblot analyses using anti-phosphoserine antibodies against the affinity-purified Pdc1p showed that Pdc1p is a phosphoenzyme. Dephosphorylation of Pdc1p by alkaline phosphatase inhibited activity by 50%. Moreover, phosphorylation of Pdc1p was dependent on the growth phase, being hyperphosphorylated in the logarithmic phase, which showed to be dependent on the presence of SIT4. A comparison of the kinetic parameters of pyruvate decarboxylase in total protein extracts from WT yeast and the Δsit4 mutant revealed that the apparent Km values of the cofactor thiamin pyrophosphate (TPP) were 81 and 205 μM, respectively, with Vmax values of 0.294 and 0.173 μmol mg−1 min−1, respectively. Treatment of the purified enzyme with alkaline phosphatase increased the Km for TPP from 20 to 84 μM and for pyruvate from 2.3 to 4.6 mM, while the Vmax changed from 0.806 to 0.673 μmol mg−1 min−1. These results suggest that the Pdc1p phosphorylation dependent on SIT4 occurs at residues that change the apparent affinity for TPP and pyruvate. [ABSTRACT FROM AUTHOR]

We studied the effect of the loss of the Ser- Thr protein phosphatase Sit4, an important post-translational regulator, on the steady-state levels of the low-affinity glucose transporter Hxt1p and observed a delay in its appearance after high glucose induction, slow growth, and diminished glucose consumption. By analyzing the known essential pathway necessary to induce Hxt1p, we observed a partial inhibition of casein kinase I activity. In both WT and sit4Δ strains, the transcript was induced with no significant difference at 15 min of glucose induction; however, after 45 min, a clear difference in the level of expression was observed being 45% higher in WT than in sit4Δ strain. As at early time of induction, the HXT1 transcript was present but not the protein in the sit4Δ strain we analyzed association of HXT1 with ribosomes, which revealed a significant difference in the association profile; in the mutant strain, the HXT1 transcript associated with a larger set of ribosomal fractions than it did in the WT strain, suggesting also a partial defect in protein synthesis. Overexpression of the translation initiation factor TIF2/ eIF4A led to an increase in Hxt1p abundance in the WT strain only. It was concluded that Sit4p ensures that HXT1 transcript is efficiently transcribed and translated thus increasing protein levels of Hxt1p when high glucose levels are present. [ABSTRACT FROM AUTHOR]

Multidrug resistance in Saccharomyces cerevisiae is frequently associated with gain-of-function mutations in zinc finger-containing transcription factors Pdr1p and Pdr3p. These regulatory proteins activate the expression of several ATP-binding cassette transporter genes, leading to elevated drug resistance. Here, we report that loss of the type 2A-related serine/threonine protein phosphatase Sit4p renders yeast cells sensitive to cycloheximide, azoles, daunorubicin and rhodamine 6G. This effect is a consequence of the decreased transcriptional levels of mainly PDR3 and its target genes, PDR5, SNQ2 and YOR1, which encode multidrug efflux pumps. The multidrug sensitivity of sit4 mutant cells is suppressed by the PDR1- 3 mutant allele, which encodes a hyperactive form of Pdr1p. Sit4p is known to associate with regulatory proteins Sap155p, Sap4p, Sap185p and Sap190p. We found that the sap155 mutant strain is sensitive to azoles, but not to cycloheximide, while the sap155sap4 and sap185sap190 mutant strains are sensitive to both drugs. This finding indicates that the Sit4p–Sap protein complex subtly modulates the expression of drug efflux pumps. Drug resistance conferred by the expression of the Candida albicans CDR1 gene, an ortholog of PDR5 in S. cerevisiae, is also positively modulated by Sit4p. These data uncover a new regulatory pathway that connects multidrug resistance to Sit4p function. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae, lithium induces a ‘galactosemia-like’ phenotype as a consequence of inhibition of phosphoglucomutase, a key enzyme in galactose metabolism. Induced galactose toxicity is prevented by deletion of GAL4, which inhibits the transcriptional activation of genes involved in galactose metabolism and by deletion of the galactokinase ( GAL1), indicating that galactose-1-phosphate, a phosphorylated intermediate of the Leloir pathway, is the toxic compound. As an alternative to inhibiting entry and metabolism of galactose, we investigated whether deviation of galactose metabolism from the Leloir pathway would also overcome the galactosemic effect of lithium. We show that cells overexpressing the aldose reductase GRE3, which converts galactose to galactitol, are more tolerant to lithium than wild-type cells when grown in galactose medium and they accumulate more galactitol and less galactose-1-phosphate. Overexpression of GRE3 also suppressed the galactose growth defect of the ‘galactosemic’ gal7- and gal10-deleted strains, which lack galactose-1-P-uridyltransferase or UDP-galactose-4-epimerase activities, respectively. Furthermore, the effect of GRE3 was independent of the inositol monophosphatases INM1 and INM2. We propose that lithium induces a galactosemic state in yeast and that inhibition of the Leloir pathway before the phosphorylation step or stimulation of galactitol production suppresses lithium-induced galactose toxicity. [ABSTRACT FROM AUTHOR]

Hyphal development in Candida albicans contributes to virulence, and inhibition of filamentation is a target for the development of antifungal agents. Lithium is known to impair Saccharomyces cerevisiae growth in galactose-containing media by inhibition of phosphoglucomutase, which is essential for galactose metabolism. Lithium-mediated phosphoglucomutase inhibition is reverted by Mg2+. In this study we have assessed the effect of lithium upon C. albicans and found that growth is inhibited preferentially in galactose-containing media. No accumulation of glucose-1-phosphate or galactose-1-phosphate was detected when yeasts were grown in the presence of galactose and 15 mM LiCl, though we observed that in vitro lithium-mediated phosphoglucomutase inhibition takes place with an IC50 of 2 mM. Furthermore, growth inhibition by lithium was not reverted by Mg2+. These results show that lithium-mediated inhibition of growth in a galactose-containing medium is not due to inhibition of galactose conversion to glucose-6-phosphate but is probably due to inhibition of a signaling pathway. Deletion of the Ser-Thr protein phosphatase SIT4 and treatment with rapamycin have been shown to inhibit filamentous differentiation. We observed that C. albicans filamentation was inhibited by lithium in solid medium containing either galactose as the sole carbon source or 10% fetal bovine serum. These results suggest that suppression of hyphal outgrowth by lithium could be related to inhibition of the target of rapamycin (TOR) pathway. [ABSTRACT FROM AUTHOR]

In this study, we found that cell cycle arrest induced by alpha-factor mating pheromone (G1), hydroxyurea (S) or nocodazole (G2/M) was associated to increased lipid droplet (LD) content. To identify novel cell cycle genes involved in LD homeostasis, we screened a deletion library for strains with altered LD levels. Among the mutants related to mitotic cell cycle, we found 24 hits that displayed a significantly higher LD content. Ontology mapping showed that neither a biological process nor a specific cell cycle phase was enriched among the hits. We decided to further study the role of SWI4 on LD homeostasis as it is involved in G1/S transition, a stage where lipolysis is active. The high LD content of swi4Δ mutant was not due to inhibition of lipolysis, but due to an increase in triacylglycerol (TAG) synthesis. In addition, deletion of the AMP kinase gene SNF1 or inhibition of TORC1 activity, both known regulators of LD homeostasis, further increased the LD content of a swi4Δ mutant. These findings highlight a role of the cell cycle regulator SWI4 in the coordination of lipid metabolism which is independent of the TORC1 and SNF1/AMPK pathways. [ABSTRACT FROM AUTHOR]