Claudio A. Masuda

Regulation of monovalent ion homeostasis and pH by the Ser-Thr protein phosphatase SIT4 in Saccharomyces cerevisiae.

A gene, SIT4, was identified as corresponding to a serine/threonine protein phosphatase and when overexpressed confers lithium tolerance in galactose medium to the budding yeast Saccharomyces cerevisiae. This gene has been previously identified as a regulator of the cell cycle and involved in nitrogen sensing. It is shown that the transcription levels ofSIT4 are induced by low concentrations of Li+in a time-dependent manner. Na+ and K+ at high concentrations, but not sorbitol, also induce transcription. As a response to Na+ or Li+stress, yeast cells lower the intracellular K+ content. This effect is enhanced in cells overexpressing SIT4, which also increase 86Rb efflux after the addition of Na+ or Li+ to the extracellular medium. Another feature of SIT4-overexpressing cells is that they maintain a more alkaline pH of 6.64 compared with 6.17 in the wild type cells. It has been proposed that the main pathway of salt tolerance in yeast is mediated by a P-type ATPase, encoded by PMR2A/ENA1. However, our results show that in a sit4 strain, expression of ENA1 is still induced by monovalent cations, and overexpression of SIT4 does not alter the amount ofENA1 transcript. These results show that SIT4acts in a parallel pathway not involving induction of transcription ofENA1 and suggest a novel function for SIT4 in response to salt stress.

A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism

In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

TORC1 Inhibition Induces Lipid Droplet Replenishment in Yeast

ABSTRACT Lipid droplets (LDs) are intracellular structures that regulate neutral lipid homeostasis. In mammals, LD synthesis is inhibited by rapamycin, a known inhibitor of the mTORC1 pathway. In Saccharomyces cerevisiae, LD dynamics are modulated by the growth phase; however, the regulatory pathways involved are unknown. Therefore, we decided to study the role of the TORC1 pathway on LD metabolism in S. cerevisiae. Interestingly, rapamycin treatment resulted in a fast LD replenishment and growth inhibition. The discovery that osmotic stress (1 M sorbitol) also induced LD synthesis but not growth inhibition suggested that the induction of LDs in yeast is not a secondary response to reduced growth. The induction of LDs by rapamycin was due to increased triacylglycerol but not sterol ester synthesis. Induction was dependent on the TOR downstream effectors, the PP2A-related phosphatase Sit4p and the regulatory protein Tap42p. The TORC1-controlled transcriptional activators Gln3p, Gat1p, Rtg1p, and Rtg3p, but not Msn2p and Msn4p, were required for full induction of LDs by rapamycin. Furthermore, we show that the deletion of Gln3p and Gat1p transcription factors, which are activated in response to nitrogen availability, led to abnormal LD dynamics. These results reveal that the TORC1 pathway is involved in neutral lipid homeostasis in yeast.

The unfolded protein response has a protective role in yeast models of classic galactosemia.

Classic galactosemia is a human autosomal recessive disorder caused by mutations in the GALT gene (GAL7 in yeast), which encodes the enzyme galactose-1-phosphate uridyltransferase. Here we show that the unfolded protein response pathway is triggered by galactose in two yeast models of galactosemia: lithium-treated cells and the gal7Δ mutant. The synthesis of galactose-1-phosphate is essential to trigger the unfolded protein response under these conditions because the deletion of the galactokinase-encoding gene GAL1 completely abolishes unfolded protein response activation and galactose toxicity. Impairment of the unfolded protein response in both yeast models makes cells even more sensitive to galactose, unmasking its cytotoxic effect. These results indicate that endoplasmic reticulum stress is induced under galactosemic conditions and underscores the importance of the unfolded protein response pathway to cellular adaptation in these models of classic galactosemia.

Pyruvate decarboxylase activity is regulated by the Ser/Thr protein phosphatase Sit4p in the yeast Saccharomyces cerevisiae

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 K(m) values of the cofactor thiamin pyrophosphate (TPP) were 81 and 205 μM, respectively, with V(max) values of 0.294 and 0.173 μmol mg⁻¹ min⁻¹, respectively. Treatment of the purified enzyme with alkaline phosphatase increased the K(m) for TPP from 20 to 84 μM and for pyruvate from 2.3 to 4.6 mM, while the V(max) 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.

Lithium‐mediated suppression of morphogenesis and growth in Candida albicans

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 Mg(2+). 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 IC(50) of 2 mM. Furthermore, growth inhibition by lithium was not reverted by Mg(2+). 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.

An NH2-terminal deleted plasma membrane H+-ATPase is a dominant negative mutant and is sequestered in endoplasmic reticulum derived structures

The NH2-terminus of the plasma membrane H+-ATPase is one of the least conserved segments of this protein among fungi. We constructed and expressed a mutant H+-ATPase from Saccharomyces cerevisiae deleted at an internal peptide within the cytoplasmic NH2-terminus (D44-F116). When the enzyme was subjected to limited trypsinolysis it was digested more rapidly than wild type H+-ATPase. Membrane fractionation experiments and immunofluorescence microscopy, using antibodies against H+-ATPase showed that the mutant ATPase is retained in the endoplasmic reticulum. The pattern observed in the immunofluorescence microscopy resembled structures similar to Russell bodies (modifications of the endoplasmic reticulum membranes) recently described in yeast. When the wild type H+-ATPase was co-expressed with the mutant, wild type H+-ATPase was also retained in the endoplasmic reticulum. Co-expression of both ATPases in a wild type yeast strain was lethal, demonstrating that this is a dominant negative mutant.

A Fluorescent Mutant of the NM Domain of the Yeast Prion Sup35 Provides Insight into Fibril Formation and Stability

The Sup35 protein of Saccharomyces cerevisiae forms a prion that generates the [PSI(+)] phenotype. Its NM region governs prion status, forming self-seeding amyloid fibers in vivo and in vitro. A tryptophan mutant of Sup35 (NM(F117W)) was used to probe its aggregation. Four indicators of aggregation, Trp 117 maximum emission, Trp polarization, thio-T binding, and light scattering increase, revealed faster aggregation at 4 degrees C than at 25 degrees C, and all indicators changed in a concerted fashion at the former temperature. Curiously, at 25 degrees C the changes were not synchronized; the first two indicators, which reflect nucleation, changed more quickly than the last two, which reflect fibril formation. These results suggest that nucleation is insensitive to temperature, whereas fibril extension is temperature dependent. As expected, aggregation is accelerated when a small fraction (5%) of the nuclei produced at 4 or 25 degrees C are added to a suspension containing the soluble NM domain, although these nuclei do not seem to propagate any structural information to the growing fibrils. Fibrils grown at 4 degrees C were less stable in GdmCl than those grown at higher temperature. However, they were both resistant to high pressure; in fact, both sets of fibrils responded to high pressure by adopting an altered conformation with a higher capacity for thio-T binding. From these data, we calculated the change in volume and free energy associated with this conformational change. AFM revealed that the fibrils grown at 4 degrees C were statistically smaller than those grown at 25 degrees C. In conclusion, the introduction of Trp 117 allowed us to more carefully dissect the effects of temperature on the aggregation of the Sup35 NM domain.

Structural and Functional Study of Yer067w, a New Protein Involved in Yeast Metabolism Control and Drug Resistance

The genome of Saccharomyces cerevisiae is arguably the best studied eukaryotic genome, and yet, it contains approximately 1000 genes that are still relatively uncharacterized. As the majority of these ORFs have no homologs with characterized sequence or protein structure, traditional sequence-based approaches cannot be applied to deduce their biological function. Here, we characterize YER067W, a conserved gene of unknown function that is strongly induced in response to many stress conditions and repressed in drug resistant yeast strains. Gene expression patterns of YER067W and its paralog YIL057C suggest an involvement in energy metabolism. We show that yeast lacking YER067W display altered levels of reserve carbohydrates and a growth deficiency in media that requires aerobic metabolism. Impaired mitochondrial function and overall reduction of ergosterol content in the YER067W deleted strain explained the observed 2- and 4-fold increase in resistance to the drugs fluconazole and amphotericin B, respectively. Cell fractionation and immunofluorescence microscopy revealed that Yer067w is associated with cellular membranes despite the absence of a transmembrane domain in the protein. Finally, the 1.7 Å resolution crystal structure of Yer067w shows an alpha-beta fold with low similarity to known structures and a putative functional site. YER067Ws involvement with aerobic energetic metabolism suggests the assignment of the gene name RGI1, standing for respiratory growth induced 1. Altogether, the results shed light on a previously uncharacterized protein family and provide basis for further studies of its apparent role in energy metabolism control and drug resistance.

The serine/threonine protein phosphatase Sit4p activates multidrug resistance in Saccharomyces cerevisiae

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.