YCL047C/POF1 Is a Novel Nicotinamide Mononucleotide Adenylyltransferase (NMNAT) in Saccharomyces cerevisiae

NAD⁺ is an essential metabolic cofactor involved in various cellular biochemical processes. Nicotinamide riboside (NR) is an endogenously produced key pyridine metabolite that plays important roles in the maintenance of NAD⁺ pool. Using a NR-specific cell-based screen, we identified mutants that exhibit altered NR release phenotype. Yeast cells lacking the ORF YCL047C/POF1 release considerably more NR compared with wild type, suggesting that POF1 plays an important role in NR/NAD⁺ metabolism. The amino acid sequence of Pof1 indicates that it is a putative nicotinamide mononucleotide adenylyltransferase (NMNAT). Unlike other yeast NMNATs, Pof1 exhibits NMN-specific adenylyltransferase activity. Deletion of POF1 significantly lowers NAD⁺ levels and decreases the efficiency of NR utilization, resistance to oxidative stress, and NR-induced life span extension. We also show that NR is constantly produced by multiple nucleotidases and that the intracellular NR pools are likely to be compartmentalized, which contributes to the regulation of NAD⁺ homeostasis. Our findings may contribute to the understanding of the molecular basis and regulation of NAD⁺ metabolism in higher eukaryotes.     

Effects of excess succinate and retrograde control of metabolite accumulation in yeast tricarboxylic cycle mutants

Cellular and mitochondrial metabolite levels were measured in yeast TCA cycle mutants (sdh2Δ or fum1Δ) lacking succinate dehydrogenase or fumarase activities. Cellular levels of succinate relative to parental strain levels were found to be elevated ~8-fold in the sdh2Δ mutant and ~4-fold in the fum1Δ mutant, and there was a preferential increase in mitochondrial levels in these mutant strains. The sdh2Δ and fum1Δ strains also exhibited 3-4-fold increases in expression of Cit2, the cytosolic form of citrate synthase that functions in the glyoxylate pathway. Co-disruption of the SFC1 gene encoding the mitochondrial succinate/fumarate transporter resulted in higher relative mitochondrial levels of succinate and in substantial reductions of Cit2 expression in sdh2Δsfc1Δ and fum1Δsfc1Δ strains as compared with sdh2Δ and fum1Δ strains, suggesting that aberrant transport of succinate out of mitochondria mediated by Sfc1 is related to the increased expression of Cit2 in sdh2Δ and fum1Δ strains. A defect (rtg1Δ) in the yeast retrograde response pathway, which controls expression of several mitochondrial proteins and Cit2, eliminated expression of Cit2 and reduced expression of NAD-specific isocitrate dehydrogenase (Idh) and aconitase (Aco1) in parental, sdh2Δ, and fum1Δ strains. Concomitantly, co-disruption of the RTG1 gene reduced the cellular levels of succinate in the sdh2Δ and fum1Δ strains, of fumarate in the fum1Δ strain, and citrate in an idhΔ strain. Thus, the retrograde response is necessary for maintenance of normal flux through the TCA and glyoxylate cycles in the parental strain and for metabolite accumulation in TCA cycle mutants.     

N-acetylglucosamine (GlcNAc) induction of hyphal morphogenesis and transcriptional responses in Candida albicans are not dependent on its metabolism

N-acetylglucosamine (GlcNAc) stimulates important signaling pathways in a wide range of organisms. In the human fungal pathogen Candida albicans, GlcNAc stimulates hyphal cell morphogenesis, virulence genes, and the genes needed to catabolize GlcNAc. Previous studies on the GlcNAc transporter (NGT1) indicated that GlcNAc has to be internalized to induce signaling. Therefore, the role of GlcNAc catabolism was examined by deleting the genes required to phosphorylate, deacetylate, and deaminate GlcNAc to convert it to fructose-6-PO(4) (HXK1, NAG1, and DAC1). As expected, the mutants failed to utilize GlcNAc. Surprisingly, GlcNAc inhibited the growth of the nag1Δ and dac1Δ mutants in the presence of other sugars, suggesting that excess GlcNAc-6-PO(4) is deleterious. Interestingly, both hxk1Δ and an hxk1Δ nag1Δ dac1Δ triple mutant could be efficiently stimulated by GlcNAc to form hyphae. These mutants could also be stimulated to express GlcNAc-regulated genes. Because GlcNAc must be phosphorylated by Hxk1 to be catabolized, and also for it to enter the anabolic pathways that form chitin, N-linked glycosylation, and glycosylphosphatidylinositol anchors, the mutant phenotypes indicate that GlcNAc metabolism is not needed to induce signaling in C. albicans. Thus, these studies in C. albicans reveal a novel role for GlcNAc in cell signaling that may also regulate critical pathways in other organisms.     

Protein kinase inhibitors block the stimulation of the AMP-activated protein kinase by 5-amino-4-imidazolecarboxamide riboside

The AMP-activated protein kinase (AMPK) is the central component of a protein kinase cascade that plays a major role in energy sensing. AMPK is activated pharmacologically by 5-amino-4-imidazolecarboxamide (AICA) riboside monophosphate (ZMP), which mimics the effects of AMP on the AMPK cascade. Here we show that uptake of AICA riboside into cells, mediated by the adenosine transport system, is blocked by a number of protein kinase inhibitors. Under these conditions, ZMP does not accumulate to sufficient levels to stimulate AMPK. Our results demonstrate that careful interpretation is required when using AICA riboside in conjunction with protein kinase inhibitors to investigate the physiological role of AMPK.     

Purine deoxynucleosides and adenosine dialdehyde decrease 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis in cultured T and B lymphoblasts.

Deoxyadenosine (dAdo) and deoxyguanosine (dGuo) decrease methionine synthesis from homocysteine in cultured lymphoblasts; because of the possible trapping of 5-methyltetrahydrofolate this could lead to decreased purine nucleotide synthesis. Since purine deoxynucleosides could also inhibit purine synthesis de novo at an early step not involving folate metabolism, we measured in azaserine-treated cells 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis using [14C]formate. In the T lymphoblasts, Z-base-dependent purine nucleotide synthesis was decreased 26% by 0.3 microM-dAdo, 21% by 1 microM-dGuo and 28% by 1 microM-adenosine dialdehyde, a potent S-adenosylhomocysteine hydrolase inhibitor; homocysteine fully reversed the inhibitions. The B lymphoblasts were considerably less sensitive to the deoxynucleoside-induced decrease in Z-base-dependent purine nucleotide synthesis, with 100 microM-dAdo required for significant inhibition and no inhibition by dGuo at this concentration; homocysteine partly reversed the inhibition by dAdo. The observed decrease in Z-base-dependent purine nucleotide synthesis could not be attributed either to dUMP depletion changing the folate pools or to decreased ATP availability because dUrd was without effect and during the experimental period the intracellular ATP concentration did not change significantly. Cells with 5,10-methylenetetrahydrofolate reductase deficiency were relatively resistant to inhibition of Z-base-dependent purine nucleotide synthesis by dAdo and adenosine dialdehyde. Our results suggest that deoxynucleosides decrease purine nucleotide synthesis by trapping 5-methyltetrahydrofolate.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with an associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-¹⁴C]-hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-¹⁴C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-¹⁴C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-¹⁴C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-¹⁴C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-³H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the ‘salvage’ pathways and de novo synthesis of purines and pyrimidines.     

Homocysteine Methyltransferases Mht1 and Sam4 Prevent the Accumulation of Age-damaged (R,S)-AdoMet in the Yeast Saccharomyces cerevisiae

The biological methyl donor S-adenosyl-l-methionine (AdoMet) is spontaneously degraded by inversion of its sulfonium center to form the R,S diastereomer. Unlike its precursor, (S,S)-AdoMet, (R,S)-AdoMet has no known cellular function and may have some toxicity. Although the rate of (R,S)-AdoMet formation under physiological conditions is significant, it has not been detected at substantial levels in vivo in a wide range of organisms. These observations imply that there are mechanisms that either dispose of (R,S)-AdoMet or convert it back to (S,S)-AdoMet. Previously, we identified two homocysteine methyltransferases (Mht1 and Sam4) in yeast capable of recognizing and metabolizing (R,S)-AdoMet. We found similar activities in worms, plants, and flies. However, it was not established whether these activities could prevent R,S accumulation. In this work, we show that both the Mht1 and Sam4 enzymes are capable of preventing R,S accumulation in Saccharomyces cerevisiae grown to stationary phase; deletion of both genes results in significant (R,S)-AdoMet accumulation. To our knowledge, this is the first time that such an accumulation of (R,S)-AdoMet has been reported in any organism. We show that yeast cells can take up (R,S)-AdoMet from the medium using the same transporter (Sam3) used to import (S,S)-AdoMet. Our results suggest that yeast cells have evolved efficient mechanisms not only for dealing with the spontaneous intracellular generation of the (R,S)-AdoMet degradation product but for utilizing environmental sources as a nutrient.     

CgCYN1, a plasma membrane cystine-specific transporter of Candida glabrata with orthologues prevalent among pathogenic yeast and fungi

We describe a novel plasma membrane cystine transporter, CgCYN1, from Candida glabrata, the first such transporter to be described from yeast and fungi. C. glabrata met15Δ strains, organic sulfur auxotrophs, were observed to utilize cystine as a sulfur source, and this phenotype was exploited in the discovery of CgCYN1. Heterologous expression of CgCYN1 in Saccharomyces cerevisiae met15Δ strains conferred the ability of S. cerevisiae strains to grow on cystine. Deletion of the CgCYN1 ORF (CAGL0M00154g) in C. glabrata met15Δ strains caused abrogation of growth on cystine with growth being restored when CgCYN1 was reintroduced. The CgCYN1 protein belongs to the amino acid permease family of transporters, with no similarity to known plasma membrane cystine transporters of bacteria and humans, or lysosomal cystine transporters of humans/yeast. Kinetic studies revealed a K(m) of 18 ± 5 μM for cystine. Cystine uptake was inhibited by cystine, but not by other amino acids, including cysteine. The structurally similar cystathionine, lanthionine, and selenocystine alone inhibited transport, confirming that the transporter was specific for cystine. CgCYN1 localized to the plasma membrane and transport was energy-dependent. Functional orthologues could be demonstrated from other pathogenic yeast like Candida albicans and Histoplasma capsulatum, but were absent in Schizosaccharomyces pombe and S. cerevisiae.     

Cytotoxic mechanism of selenomethionine in yeast

Although selenium is an essential element, its excessive uptake is detrimental to living organisms. The significance of selenium for living organisms has been exploited for various purposes. However, the molecular basis of selenium toxicity is not completely understood. Here, we applied a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach to analysis of yeast cells treated with selenomethionine. The data indicated that intracellular thiol compounds are significantly decreased, and diselenide and selenosulfide compounds are increased in selenomethionine-treated cells. The growth defect induced by selenomethionine was recovered by extracellular addition of cysteine and by genetic modification of yeast cells that have an additional de novo synthetic pathway for cysteine. Because cysteine is an intermediate of thiol compounds, these results suggested that the loss of a reduced form of thiol compounds due to selenomethionine causes a growth defect of yeast cells.     

Methanol metabolism in mutants of the methylotrophic yeast Hansenula polymorpha

Abstract Three types of Hansenula polymorpha 356 (leu⁻) mutants unable to grow on methanol were isolated and characterized. The first type of mutants, M8, M14, and M41, were deficient in the alcohol oxidase activity (MOX⁻). The dihydroxyacetone synthase activity appeared after incubation of the strains in the medium with glycerol and methylamine but not with methanol. One of the mutants (W218) with the reduced activity of alcohol oxidase lacked the formate dehydrogenase activity (FDH⁻). All these mutants produced a low level of extracellular formaldehyde from methanol.
The second and third types of mutants were deficient in dihydroxyacetone synthase (DAS⁻; 349, 409, 450), and dihydroxyacetone kinase (DAK⁻; 4D1, 4D3, 4D16) activities, respectively. DAK⁻ mutants showed both the high activities of alcohol oxidase and NADH-dependent reduction of CH₂O catalyzed by alcohol dehydrogenase. This indicated the possibility that NADH, generated in the oxidation of formaldehyde to CO₂, may be oxidized by molecular oxygen via a futile cycle composed of the alcohol oxidase and alcohol dehydrogenase.