Molecular Characterization of GCV3, the Saccharomyces cerevisiae Gene Coding for the Glycine Cleavage System Hydrogen Carrier Protein

Nagarajan, Lakshmanan; Storms, Reginald

doi:10.1074/jbc.272.7.4444

Cited by 34 publications

(35 citation statements)

References 49 publications

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“…This may explain why in two standard Leishmania culture media the gcvP Ϫ mutant grew and differentiated normally. However, under conditions of excess glycine, growth of the gcvP Ϫ mutant was greatly inhibited (Table 2), as seen previously in plants or yeast (41,48,53). This may arise from the observation in other organisms that excess glycine contributes to decreased 5,10-CH 2 -THF production by SHMT (54), which would further reduce 5,10-CH 2 -THF levels in GCC-deficient mutants.…”

Section: Discussionmentioning

confidence: 47%

“…We attempted to assay GCC activity enzymatically in crude and fractionated parasite extracts but were unsuccessful. In other non-photosynthetic organisms where glycine cleavage activity has been measured, it is very low; 0.1-0.2 nmol/min/mg of protein for yeast and E. coli (48). Thus, we devised an alternative indirect in vivo assay for GCC activity after incorporation of label from [2-14 C]glycine through 5,10-CH 2 -THF into thymidylate and then DNA (Table 1).…”

Section: Discussionmentioning

confidence: 99%

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The Role of the Mitochondrial Glycine Cleavage Complex in the Metabolism and Virulence of the Protozoan Parasite Leishmania major

Scott

Hickerson

Vickers

et al. 2008

Journal of Biological Chemistry

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For the human pathogen Leishmania major, a key metabolic function is the synthesis of thymidylate, which requires 5,10-methylenetetrahydrofolate (5,10-CH 2 -THF). 5,10-CH 2 -THF can be synthesized from glycine by the mitochondrial glycine cleavage complex (GCC). Bioinformatic analysis revealed the four subunits of the GCC in the L. major genome, and the role of the GCC in parasite metabolism and virulence was assessed through studies of the P subunit (glycine decarboxylase (GCVP)). First, a tagged GCVP protein was expressed and localized to the parasite mitochondrion. Second, a gcvP ؊ mutant was generated and shown to lack significant GCC activity using an indirect in vivo assay after incorporation of label from [2-14 C]glycine into DNA. The gcvP ؊ mutant grew poorly in the presence of excess glycine or minimal serine; these studies also established that L. major promastigotes require serine for optimal growth. Although gcvP ؊ promastigotes and amastigotes showed normal virulence in macrophage infections in vitro, both forms of the parasite showed substantially delayed replication and lesion pathology in infections of both genetically susceptible or resistant mice. These data suggest that, as the physiology of the infection site changes during the course of infection, so do the metabolic constraints on parasite replication. This conclusion has great significance to the interpretation of metabolic requirements for virulence. Last, these studies call attention in trypanosomatid protozoa to the key metabolic intermediate 5,10-CH 2 -THF, situated at the junction of serine, glycine, and thymidylate metabolism. Notably, genome-based predictions suggest the related parasite Trypanosoma brucei is totally dependent on the GCC for 5,10-CH 2 -THF synthesis.

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Section: Discussionmentioning

confidence: 47%

Section: Discussionmentioning

confidence: 99%

The Role of the Mitochondrial Glycine Cleavage Complex in the Metabolism and Virulence of the Protozoan Parasite Leishmania major

Scott

Hickerson

Vickers

et al. 2008

Journal of Biological Chemistry

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“…Mutation in genes encoding a variety of cellular metabolic processes (supplemental Figure S6 at http://www.genetics. org/supplemental/) (Nagarajan and Storms 1997;Schneider et al 1997;Stoops et al 1997), such as the conversion of pyruvate into acetyl-CoA by pyruvate dehydrogenase complex (Pdx1p, Lpd1, Pdb1p, and Lat1p), the conversion of acetyl-CoA to malonyl-CoA by Hfa1p, fatty acid synthesis by fatty acid synthase complex (Oar1p, Mct1p, and Etr1p), glycine degradation (Gcv2p, Gcv3p, and Lpd1p), and a peptidase activity (Prd1p), resulted in a decrease of dipeptide utilization (Table 3). In these mutants the impairment of the dipeptide utilization appeared to be independent of Ptr2p transport activity since Ptr2p-GFP could be observed at the plasma membrane (supplemental Figure S3 at http://www.genetics.org/supplemental/).…”

Section: Gene Namementioning

confidence: 99%

Genomewide Screen Reveals a Wide Regulatory Network for Di/Tripeptide Utilization inSaccharomyces cerevisiae

et al. 2006

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Small peptides of two to six residues serve as important sources of amino acids and nitrogen required for growth by a variety of organisms. In the yeast Saccharomyces cerevisiae, the membrane transport protein Ptr2p, encoded by PTR2, mediates the uptake of di/tripeptides. To identify genes involved in regulation of dipeptide utilization, we performed a systematic, functional examination of this process in a haploid, nonessential, single-gene deletion mutant library. We have identified 103 candidate genes: 57 genes whose deletion decreased dipeptide utilization and 46 genes whose deletion enhanced dipeptide utilization. On the basis of Ptr2p-GFP expression studies, together with PTR2 expression analysis and dipeptide uptake assays, 42 genes were ascribed to the regulation of PTR2 expression, 37 genes were involved in Ptr2p localization, and 24 genes did not apparently affect Ptr2p-GFP expression or localization. The 103 genes regulating dipeptide utilization were distributed among most of the Gene Ontology functional categories, indicating a very wide regulatory network involved in transport and utilization of dipeptides in yeast. It is anticipated that further characterization of how these genes affect peptide utilization should add new insights into the global mechanisms of regulation of transport systems in general and peptide utilization in particular.

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“…The fourth subunit, lipoamide dehydrogenase, is present in several other multienzyme complexes (11). These genes are up-regulated following addition of glycine to the medium (12)(13)(14) via a glycine response element with the consensus sequence CATCN 7 CTTCTT (15). Previously, it was demonstrated that this glycine effect is a response to a decrease in cytosolic 5,10-CH 2 -H 4 folate levels rather than directly to glycine (16).…”

mentioning

confidence: 99%

Identification of a Novel One-carbon Metabolism Regulon in Saccharomyces cerevisiae

Gelling

Piper²,

Hong³

et al. 2004

Journal of Biological Chemistry

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Glycine specifically induces genes encoding subunits of the glycine decarboxylase complex (GCV1, GCV2, and GCV3), and this is mediated by a fall in cytoplasmic levels of 5,10-methylenetetrahydrofolate caused by inhibition of cytoplasmic serine hydroxymethyltransferase. Here it is shown that this control system extends to genes for other enzymes of one-carbon metabolism and de novo purine biosynthesis. Northern analysis of the response to glycine demonstrated that the induction of the GCV genes and the induction of other amino acid metabolism genes are temporally distinct. The genomewide response to glycine revealed that several other genes are rapidly co-induced with the GCV genes, including SHM2, which encodes cytoplasmic serine hydroxymethyltransferase. These results were refined by examining transcript levels in an shm2⌬ strain (in which cytoplasmic 5,10-methylenetetrahydrofolate levels are reduced) and a met13⌬ strain, which lacks the main methylenetetrahydrofolate reductase activity of yeast and is effectively blocked at consumption of 5,10-methylene tetrahydrofolate for methionine synthesis. Glycine addition also caused a substantial transient disturbance to metabolism, including a sequence of changes in induction of amino acid biosynthesis and respiratory chain genes. Analysis of the glycine response in the shm2⌬ strain demonstrated that apart from the one-carbon regulon, most of these transient responses were not contingent on a disturbance to onecarbon metabolism. The one-carbon response is distinct from the Bas1p purine biosynthesis regulon and thus represents the first example of transcriptional regulation in response to activated one-carbon status.

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Molecular Characterization of GCV3, the Saccharomyces cerevisiae Gene Coding for the Glycine Cleavage System Hydrogen Carrier Protein

Cited by 34 publications

References 49 publications

The Role of the Mitochondrial Glycine Cleavage Complex in the Metabolism and Virulence of the Protozoan Parasite Leishmania major

The Role of the Mitochondrial Glycine Cleavage Complex in the Metabolism and Virulence of the Protozoan Parasite Leishmania major

Genomewide Screen Reveals a Wide Regulatory Network for Di/Tripeptide Utilization inSaccharomyces cerevisiae

Identification of a Novel One-carbon Metabolism Regulon in Saccharomyces cerevisiae

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