Dataset Information

Diversification of Paralogous ?-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae.

ABSTRACT: Production of ?-isopropylmalate (?-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeast Saccharomyces cerevisiae has two paralogous genes, LEU4 and LEU9, that encode ?-IPM synthase (?-IPMS) isozymes. Little is known about the biochemical differences between these two ?-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. The leu4? mutant, which expresses only the feedback-resistant Leu9 homodimer, grows slowly with either glucose or ethanol and accumulates elevated pools of leucine; this phenotype is alleviated by the addition of leucine. Transformation of the leu4? mutant with a centromeric plasmid carrying LEU4 restored the wild-type phenotype. Bimolecular fluorescent complementation analysis showed that Leu4-Leu9 heterodimeric isozymes are formed in vivo. Purification and kinetic analysis showed that the hetero-oligomeric isozyme has a distinct leucine sensitivity behavior. Determination of ?-IPMS activity in ethanol-grown cultures showed that ?-IPM biosynthesis and growth under these respiratory conditions depend on the feedback-sensitive Leu4 homodimer. We conclude that retention and further diversification of two yeast ?-IPMSs have resulted in a specific regulatory system that controls the leucine-?-IPM biosynthetic pathway by selective feedback sensitivity of homomeric and heterodimeric isoforms.

SUBMITTER: Lopez G

PROVIDER: S-EPMC4452578 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Diversification of Paralogous α-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae.

López Geovani G Quezada Héctor H Duhne Mariana M González James J Lezama Mijail M El-Hafidi Mohammed M Colón Maritrini M Martínez de la Escalera Ximena X Flores-Villegas Mirelle Citlali MC Scazzocchio Claudio C DeLuna Alexander A González Alicia A

Eukaryotic cell 20150403 6

Production of α-isopropylmalate (α-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeast Saccharomyces cerevisiae has two paralogous genes, LEU4 and LEU9, that encode α-IPM synthase (α-IPMS) isozymes. Little is known about the biochemical differences between these two α-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. The leu4Δ mutant, which exp ...[more]

PMID: 25841022

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Project description:BackgroundGene duplication and the subsequent divergence of paralogous pairs play a central role in the evolution of novel gene functions. S. cerevisiae possesses two paralogous genes (ALT1/ALT2) which presumably encode alanine aminotransferases. It has been previously shown that Alt1 encodes an alanine aminotransferase, involved in alanine metabolism; however the physiological role of Alt2 is not known. Here we investigate whether ALT2 encodes an active alanine aminotransferase.Principal findingsOur results show that although ALT1 and ALT2 encode 65% identical proteins, only Alt1 displays alanine aminotransferase activity; in contrast ALT2 encodes a catalytically inert protein. ALT1 and ALT2 expression is modulated by Nrg1 and by the intracellular alanine pool. ALT1 is alanine-induced showing a regulatory profile of a gene encoding an enzyme involved in amino acid catabolism, in agreement with the fact that Alt1 is the sole pathway for alanine catabolism present in S. cerevisiae. Conversely, ALT2 expression is alanine-repressed, indicating a role in alanine biosynthesis, although the encoded-protein has no alanine aminotransferase enzymatic activity. In the ancestral-like yeast L. kluyveri, the alanine aminotransferase activity was higher in the presence of alanine than in the presence of ammonium, suggesting that as for ALT1, LkALT1 expression could be alanine-induced. ALT2 retention poses the questions of whether the encoded protein plays a particular function, and if this function was present in the ancestral gene. It could be hypotesized that ALT2 diverged after duplication, through neo-functionalization or that ALT2 function was present in the ancestral gene, with a yet undiscovered function.ConclusionsALT1 and ALT2 divergence has resulted in delegation of alanine aminotransferase activity to Alt1. These genes display opposed regulatory profiles: ALT1 is alanine-induced, while ALT2 is alanine repressed. Both genes are negatively regulated by the Nrg1 repressor. Presented results indicate that alanine could act as ALT2 Nrg1-co-repressor.

Project description:How microbial cells leverage their phenotypic potential to survive in a changing environment is a complex biological problem, with important implications for pathogenesis and species evolution. Stochastic phenotype switching, a particularly fascinating adaptive approach observed in numerous species across the tree of life, introduces phenotypic diversity into a population through mechanisms which have remained difficult to define. Here we describe our investigations into the mechanistic basis of colony morphology phenotype switching which occurs in populations of a pathogenic isolate of Saccharomyces cerevisiae, YJM311. We observed that clonal populations of YJM311 cells produce variant colonies that display altered morphologies and, using whole genome sequence analysis, discovered that these variant clones harbored an exceptional collection of karyotypes newly altered by de novo structural genomic variations (SVs). Overall, our analyses indicate that copy number alterations, more often than changes in allelic identity, provide the causative basis of this phenotypic variation. Individual variants carried between 1 and 16 de novo copy number variations, most of which were whole chromosomal aneuploidies. Notably, we found that the inherent stability of the diploid YJM311 genome is comparable to that of domesticated laboratory strains, indicating that the collections of SVs harbored by variant clones did not arise by a chronic chromosomal instability (CIN) mechanism. Rather, our data indicate that these variant clones acquired such complex karyotypic configurations simultaneously, during stochastic and transient episodes of punctuated systemic genomic instability (PSGI). Surprisingly, we found that the majority of these highly altered variant karyotypes were propagated with perfect fidelity in long-term passaging experiments, demonstrating that high aneuploidy burdens can often be conducive with prolonged genomic integrity. Together, our results demonstrate that colony morphology switching in YJM311 is driven by a stochastic process in which genome stability and plasticity are integrally coupled to phenotypic heterogeneity. Consequently, this system simultaneously introduces both phenotypic and genomic variation into a population of cells, which can, in turn perpetuate population diversity for many generations thereafter.

Dataset Information

Diversification of Paralogous ?-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae.

Publications

Diversification of Paralogous α-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae.

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