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Enolase and glycolytic flux play a role in the regulation of the glucose permease gene RAG1 of Kluyveromyces lactis.

ABSTRACT: We isolated a mutant, rag17, which is impaired in glucose induction of expression of the major glucose transporter gene RAG1. The RAG17 gene encodes a protein 87% identical to S. cerevisiae enolases (Eno1 and Eno2). The Kleno null mutant showed no detectable enolase enzymatic activity and has severe growth defects on glucose and gluconeogenic carbon sources, indicating that K. lactis has a single enolase gene. In addition to RAG1, the transcription of several glycolytic genes was also strongly reduced in the DeltaKleno mutant. Moreover, the defect in RAG1 expression was observed in other mutants of the glycolytic pathway (hexokinase and phosphoglycerate kinase). Therefore, it seems that the enolase and a functional glycolytic flux are necessary for induction of expression of the Rag1 glucose permease in K. lactis.

SUBMITTER: Lemaire M

PROVIDER: S-EPMC1448853 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

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Enolase and glycolytic flux play a role in the regulation of the glucose permease gene RAG1 of Kluyveromyces lactis.

Lemaire Marc M Wésolowski-Louvel Micheline M

Genetics 20041001 2

We isolated a mutant, rag17, which is impaired in glucose induction of expression of the major glucose transporter gene RAG1. The RAG17 gene encodes a protein 87% identical to S. cerevisiae enolases (Eno1 and Eno2). The Kleno null mutant showed no detectable enolase enzymatic activity and has severe growth defects on glucose and gluconeogenic carbon sources, indicating that K. lactis has a single enolase gene. In addition to RAG1, the transcription of several glycolytic genes was also strongly r ...[more]

PMID: 15514048

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Project description:The analysis of glucose signaling in the Crabtree-positive eukaryotic model organism Saccharomyces cerevisiae has disclosed a dual role of its hexokinase ScHxk2, which acts as a glycolytic enzyme and key signal transducer adapting central metabolism to glucose availability. In order to identify evolutionarily conserved characteristics of hexokinase structure and function, the cellular response of the Crabtree-negative yeast Kluyveromyces lactis to rag5 null mutation and concomitant deficiency of its unique hexokinase KlHxk1 was analyzed by means of difference gel electrophoresis. In total, 2,851 fluorescent spots containing different protein species were detected in the master gel representing all of the K. lactis proteins that were solubilized from glucose-grown KlHxk1 wild-type and mutant cells. Mass spectrometric peptide analysis identified 45 individual hexokinase-dependent proteins related to carbohydrate, short-chain fatty acid and tricarboxylic acid metabolism as well as to amino acid and protein turnover, but also to general stress response and chromatin remodeling, which occurred as a consequence of KlHxk1 deficiency at a minimum 3-fold enhanced or reduced level in the mutant proteome. In addition, three proteins exhibiting homology to 2-methylcitrate cycle enzymes of S. cerevisiae were detected at increased concentrations, suggesting a stimulation of pyruvate formation from amino acids and/or fatty acids. Experimental validation of the difference gel electrophoresis approach by post-lysis dimethyl labeling largely confirmed the abundance changes detected in the mutant proteome via the former method. Taking into consideration the high proportion of identified hexokinase-dependent proteins exhibiting increased proteomic levels, KlHxk1 is likely to have a repressive function in a multitude of metabolic pathways. The proteomic alterations detected in the mutant classify KlHxk1 as a multifunctional enzyme and support the view of evolutionary conservation of dual-role hexokinases even in organisms that are less specialized than S. cerevisiae in terms of glucose utilization.

Dataset Information

Enolase and glycolytic flux play a role in the regulation of the glucose permease gene RAG1 of Kluyveromyces lactis.

Publications

Enolase and glycolytic flux play a role in the regulation of the glucose permease gene RAG1 of Kluyveromyces lactis.

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