RESUMEN
Glutamate dehydrogenases (GDHs) are fundamental to cellular nitrogen and energy balance. Yet little is known about these enzymes in the oleaginous yeast Yarrowia lipolytica. The YALI0F17820g and YALI0E09603g genes, encoding potential GDH enzymes in this organism, were examined. Heterologous expression in gdh-null Saccharomyces cerevisiae and examination of Y. lipolytica strains carrying gene deletions demonstrate that YALI0F17820g (ylGDH1) encodes a NADP-dependent GDH whereas YALI0E09603g (ylGDH2) encodes a NAD-dependent GDH enzyme. The activity encoded by these two genes accounts for all measurable GDH activity in Y. lipolytica. Levels of the two enzyme activities are comparable during logarithmic growth on rich medium, but the NADP-ylGDH1p enzyme activity is most highly expressed in stationary and nitrogen starved cells by threefold to 12-fold. Replacement of ammonia with glutamate causes a decrease in NADP-ylGdh1p activity, whereas NAD-ylGdh2p activity is increased. When glutamate is both carbon and nitrogen sources, the activity of NAD-ylGDH2p becomes dominant up to 18-fold compared with that of NADP-ylGDH1p. Gene deletion followed by growth on different carbon and nitrogen sources shows that NADP-ylGdh1p is required for efficient nitrogen assimilation whereas NAD-ylGdh2p plays a role in nitrogen and carbon utilization from glutamate. Overexpression experiments demonstrate that ylGDH1 and ylGDH2 are not interchangeable. These studies provide a vital basis for future consideration of how these enzymes function to facilitate energy and nitrogen homeostasis in Y. lipolytica.
Asunto(s)
Glutamato Deshidrogenasa/metabolismo , Glutamatos/metabolismo , Yarrowia/enzimología , Yarrowia/crecimiento & desarrollo , Medios de Cultivo , Eliminación de Gen , Glutamato Deshidrogenasa/genética , Homeostasis , NAD/metabolismo , NADP/metabolismo , Nitrógeno/metabolismo , Saccharomyces cerevisiae/genética , Yarrowia/genéticaRESUMEN
The degree of severity of cardiomyopathy is inversely correlated with tissue levels of coenzyme Q (Q), suggesting that Q synthesis may impact the progression of the disease. It has been suggested that Q functions as an endogenously synthesized anti-oxidant, in addition to regenerating the potent anti-oxidants, vitamins E and C. However, very little is known about the mechanisms that regulate Q synthesis. Using the simple eukaryote Saccharomyces cerevisiae as a model, experiments have been designed to investigate the regulation of Q synthesis at the genetic level. To investigate the regulation of COQ5 gene expression by energy source, mRNA content was evaluated in yeast cells treated with dextrose, glycerol or oleic acid. After 1.5 h, more COQ5 mRNA is produced by oleic acid treated cells than by glycerol treated. Experiments performed using COQ5 promoter deletion/reporter constructs demonstrate a specific response to oleic acid. Additional promoter deletion analysis demonstrates that a non-fermentable carbon source element is also present, responding to both glycerol and oleic acid. The specific oleic acid response appears to be regulated by the Rtg family of transcription factors. This family of proteins is required for oleic acid-induced expression of genes of beta-oxidation and peroxisomal proliferation, and plays an important role in co-ordinating mitochondrial/peroxisomal/nuclear communication in response to oleic acid, as well as defects in cellular respiration.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Metabolismo Energético/fisiología , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Metiltransferasas/genética , Proteínas Represoras/genética , Elementos de Respuesta , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Glucosa/metabolismo , Glicerol/metabolismo , Metiltransferasas/metabolismo , Ácido Oléico/metabolismo , Regiones Promotoras GenéticasRESUMEN
In Saccharomyces cerevisiae, the glutamate dehydrogenase (GDH) enzymes play a pivotal role in glutamate biosynthesis and nitrogen assimilation. It has been proposed that, in GDH-deficient yeast, either the proline utilization (PUT) or the glutamine synthetase-glutamate synthase (GS/GOGAT) pathway serves as the alternative pathway for glutamate production and nitrogen assimilation to the exclusion of the other. Using a gdh-null mutant (gdh1Δ2Δ3Δ), this ambiguity was addressed using a combination of growth studies and pathway-specific enzyme assays on a variety of nitrogen sources (ammonia, glutamine, proline and urea). The GDH-null mutant was viable on all nitrogen sources tested, confirming that alternate pathways for nitrogen assimilation exist in the gdh-null strain. Enzyme assays point to GS/GOGAT as the primary alternative pathway on the preferred nitrogen sources ammonia and glutamine, whereas growth on proline required both the PUT and GS/GOGAT pathways. In contrast, growth on glucose-urea media elicited a decrease in GOGAT activity along with an increase in activity of the PUT pathway specific enzyme Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH). Together, these results suggest the alternative pathway for nitrogen assimilation in strains lacking the preferred GDH-dependent route is nitrogen source dependent and that neither GS/GOGAT nor PUT serves as the sole compensatory pathway.
Asunto(s)
Glutamato Deshidrogenasa/deficiencia , Glutamatos/biosíntesis , Glutamina/metabolismo , Nitrógeno/metabolismo , Prolina/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Medios de Cultivo/química , Eliminación de Gen , Análisis de Flujos Metabólicos , Redes y Vías Metabólicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Glutamate dehydrogenases (GDH) interconvert α-ketoglutarate and glutamate. In yeast, NADP-dependent enzymes, encoded by GDH1 and GDH3, are reported to synthesize glutamate from α-ketoglutarate, while an NAD-dependent enzyme, encoded by GDH2, catalyzes the reverse. Cells were grown in acetate/raffinose (YNAceRaf) to examine the role(s) of these enzymes during aerobic metabolism. In YNAceRaf the doubling time of wild type, gdh2Δ, and gdh3Δ cells was comparable at â¼4 h. NADP-dependent GDH activity (Gdh1p+Gdh3p) in wild type, gdh2Δ, and gdh3Δ was decreased â¼80% and NAD-dependent activity (Gdh2p) in wild type and gdh3Δ was increased â¼20-fold in YNAceRaf as compared to glucose. Cells carrying the gdh1Δ allele did not divide in YNAceRaf, yet both the NADP-dependent (Gdh3p) and NAD-dependent (Gdh2p) GDH activity was â¼3-fold higher than in glucose. Metabolism of [1,2-(13)C]-acetate and analysis of carbon NMR spectra were used to examine glutamate metabolism. Incorporation of (13)C into glutamate was nearly undetectable in gdh1Δ cells, reflecting a GDH activity at <15% of wild type. Analysis of (13)C-enrichment of glutamate carbons indicates a decreased rate of glutamate biosynthesis from acetate in gdh2Δ and gdh3Δ strains as compared to wild type. Further, the relative complexity of (13)C-isotopomers at early time points was noticeably greater in gdh3Δ as compared to wild type and gdh2Δ cells. These in vivo data show that Gdh1p is the primary GDH enzyme and Gdh2p and Gdh3p play evident roles during aerobic glutamate metabolism.
Asunto(s)
Isótopos de Carbono/metabolismo , Glutamato Deshidrogenasa/genética , Ácido Glutámico/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Medios de Cultivo/química , Eliminación de Gen , Glutamato Deshidrogenasa/metabolismo , Ácidos Cetoglutáricos/metabolismo , Espectroscopía de Resonancia Magnética , Saccharomyces cerevisiae/metabolismoRESUMEN
Utilization of fatty acids such as oleic acid as sole carbon source by the yeast Saccharomyces cerevisiae requires coordinated function of peroxisomes, where the fatty acids are degraded, and the mitochondria, where oxidation is completed. We identified two mitochondrial oxodicarboxylate transporters, Odc1p and Odc2p, as important in efficient utilization of oleic acid in yeast [Tibbetts et al., Arch. Biochem. Biophys. 406 (2002) 96-104]. Yet, the growth phenotype of odc1delta odc2delta strains indicated that additional transporter(s) were also involved. Here, we identify two putative transporter genes, YMC1 and YMC2, as able to suppress the odc1delta odc2delta growth phenotype. The mRNA levels for both are elevated in the presence of glycerol or oleic acid, as compared to glucose. Ymc1p and Ymc2p are localized to the mitochondria in oleic acid-grown cells. Deletion of all four transporters (quad mutant) prevents growth on oleic acid as sole carbon source, while growth on acetate is retained. It is known that the glutamate-sensitive retrograde signaling pathway is important for upregulation of peroxisomal function in response to oleic acid and the oxodicarboxylate alpha-ketoglutarate is transported out of the mitochondria for synthesis of glutamate. So, citric acid cycle function and glutamate synthesis were examined in transporter mutants. The quad mutant has significantly decreased citrate synthase activity and whole cell alpha-ketoglutarate levels, while isocitrate dehydrogenase activity is unaffected and glutamate dehydrogenase activity is increased 10-fold. Strains carrying only two or three transporter deletions exhibit intermediate affects. 13C NMR metabolic enrichment experiments confirm a defect in glutamate biosynthesis in the quad mutant and, in double and triple mutants, suggest increased cycling of the glutamate backbone in the mitochondria before export. Taken together these studies indicate that these four transporters have overlapping activity, and are important not only for utilization of oleic acid, but also for glutamate biosynthesis.
Asunto(s)
Ácido Glutámico/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Ácido Oléico/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetatos/metabolismo , Secuencia de Bases , Transporte Biológico , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Genes Fúngicos , Proteínas de Transporte de Membrana/genética , Datos de Secuencia Molecular , Mutación , Oxidación-Reducción , Saccharomyces cerevisiae/genética , Transducción de Señal , Regulación hacia ArribaRESUMEN
The yeast genes ODC1 and ODC2 encode members of the Saccharomyces cerevisiae family of mitochondrial transport proteins that transport oxodicarboxylates. In these studies, the ODC1 gene was identified as able, in low-copy, to rescue a yeast strain that is unable to grow on oleic acid but can grow on other nonfermentable carbon sources. ODC2 was shown to be a high-copy suppressor of this mutant. Odc1delta odc2delta double mutants are unable to grow on oleic acid at 36 degrees C. ODC1 mRNA and protein expression is elevated in oleic acid medium as compared to glucose or glycerol. The ODC1 promoter contains sequences required for the oleic acid response. However, regulation of ODC1 does not require the transcription factors Oaf1p and Pip2p, known to mediate oleic acid induction of other genes. These studies provide the first link between these mitochondrial transporters and peroxisomal beta-oxidation.