The isolation and characterization of a Saccharomyces mutant deficient in Δ1-pyrroline-5-carboxylate dehydrogenase activity

Lundgren, Dan; Ogur, Maurice; Yuen, Sylvia

doi:10.1016/0304-4165(72)90271-1

Cited by 8 publications

(2 citation statements)

References 5 publications

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“…One of the earliest findings regarding proline utilization in S. cerevisiae was the observation that vegetative petite (rho-) strains failed to utilize proline as the sole source of nitrogen (7,20). rho-strains were shown to have the second Put enzyme, P5C dehydrogenase, but were lacking detectable levels of proline oxidase (9).…”

Section: Molecularmentioning

confidence: 99%

Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT1 gene.

Wang¹,

Brandriss²

1986

Mol. Cell. Biol.

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The PUT) gene was isolated by functional complementation of a put) (proline oxidase-deficient) mutation in Saccharomyces cerevisiae. Three independent clones with overlapping inserts of 6.8, 10.5, and 11 kilobases (kb) were isolated from S. cerevisiae genomic libraries in YEp24 (2,um) and YCp5O (CEN) plasmids. The identity of the PUT) gene was determined by a gene disruption technique, and Southern hybridization and genetic analyses confirmed that the bona fide gene had been cloned. Plasmids containing the PUT) gene restored regulated levels of proline oxidase activity to put) recipient strains. The PUT) DNA was present in a single copy in the yeast genome and encoded a transcript of ca. 1.5 kb. Si nuclease protection experiments were used to determine the direction of transcription of the PUT) message and to localize its 5' and 3' termini within a subcloned 3-kb DNA fragment. Approximately 50-fold more PUT)-specific mRNA was detected in induced (proline-grown) cells than in uninduced (ammonia-grown) cells. A yeast strain carrying the previously identified put3 regulatory mutation that caused constitutive levels of proline oxidase activity was found to have sevenfold elevated PUT) mRNA levels under noninducing conditions. The absence of a functional electron transport system in vegetative petite (rho-) strains interfered with their ability to use proline as a nitrogen source. Although these strains were Put-and made no detectable proline oxidase activity, PUT) message was detected under inducing conditions. The PUT) gene was mapped distal to the GAL2 gene on chromosome XII by tetrad analysis.Metabolic conversion of proline to glutamate in Saccharomyces cerevisiae cells permits them to grow on a medium containing proline as the sole source of nitrogen. This two-step pathway occurs inside mitochondria and is catalyzed by the nuclearly encoded, cytoplasmically synthesized, and mitochondrially imported enzymes, proline oxidase and A'-pyrroline-5-carboxylate (P5C) dehydrogenase (7,8,10). The activity levels of both enzymes are induced by proline, while the two transport systems that bring proline into the cell, the proline-specific (PUT4) permease and the general amino acid permease, are regulated not by proline induction but by nitrogen catabolite repression (18).Several mutations in nuclear genes that affect proline utilization have been identified and characterized. Mutations in the PUT] gene result in a deficiency in proline oxidase activity, and those in the PUT2 gene cause a reduction in P5C dehydrogenase activity (7). The put3 mutation results in inducer-independent expression of both of these enzymes (8).The PUT2 gene has been cloned and characterized (6), sequenced (17), and recently identified as the structural gene for P5C dehydrogenase (J. Kaput and M. C. Brandriss, unpublished results). The gene is regulated at the level of RNA by proline, and the increase in enzyme activity seen in the put3 mutant is correlated with an increase in PUT2-specific mRNA in that strain (6).This report describes the isolati...

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Section: Molecularmentioning

confidence: 99%

Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT1 gene.

Wang¹,

Brandriss²

1986

Mol. Cell. Biol.

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“…showed that the PUT pathway could serve as a source of glutamate, as the put2-glt 1-1 mutant they isolated was no longer able to utilize proline as a nitrogen source (Lundgren et al ., 1972). In contrast with the results from Lundgren et al ., Avendano and colleagues asserted that the GS/GOGAT pathway, in combination with the GDHs, represents the sole pathway for glutamate biosynthesis in S. cerevisiae (Avendano et al ., 1997).…”

Section: Introductionmentioning

confidence: 99%

Differential contribution of the proline and glutamine pathways to glutamate biosynthesis and nitrogen assimilation in yeast lacking glutamate dehydrogenase

Sieg

Trotter

2014

Microbiological Research

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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 Δ1-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.

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Regulation of Allantoin Catabolism in Saccharomyces cerevisiae

Cooper¹

1996

Biochemistry and Molecular Biology

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The isolation and characterization of a Saccharomyces mutant deficient in Δ1-pyrroline-5-carboxylate dehydrogenase activity

Cited by 8 publications

References 5 publications

Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT1 gene.

Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT1 gene.

Differential contribution of the proline and glutamine pathways to glutamate biosynthesis and nitrogen assimilation in yeast lacking glutamate dehydrogenase

Regulation of Allantoin Catabolism in Saccharomyces cerevisiae

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