Dataset Information

Proteomic consequences of TDA1 deficiency in Saccharomyces cerevisiae — Protein kinase Tda1 is essential for Hxk1 and Hxk2 serine 15 phosphorylation

ABSTRACT: Hexokinase 2 (Hxk2) of Saccharomyces cerevisiae is a dual function hexokinase, acting as a glycolytic enzyme and being involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by phosphorylation of Hxk2 at serine 15, which has been attributed to the protein kinase Tda1. To explore the role of Tda1 beyond Hxk2 phosphorylation the proteomic consequences of TDA1 deficiency were investigated by difference gel electrophoresis (2D-DIGE) comparing a wild type and a Δtda1 deletion mutant. Both were grown at 2 % and 0.1 % (w/v) glucose to additionally regard glucose repression/derepression. A total of eight spots exhibiting a minimum 2-fold enhanced or reduced fluorescence upon TDA1 deficiency was detected and identified by mass spectrometry. These include – besides the expected Hxk2 – two proteoforms of hexokinase 1 (Hxk1). Targeted proteomics analyses in conjunction with 2D-DIGE demonstrated that TDA1 is indispensable for Hxk2 and Hxk1 phosphorylation at serine 15. 35 glucose-dependent proteins were identified. To improve spot quantification, a simple method approximating spots as rotationally symmetric solids is presented along with new data on the quantities of Hxk1 and Hxk2 and their serine 15 phosphorylated forms at high and low glucose growth conditions. The Δtda1 deletion mutant exhibited no altered growth comparing high or low glucose conditions or on alternative carbon sources. Also, invertase activity, serving as a reporter for glucose derepression, was not significantly altered. Instead, an involvement of Tda1 in oxidative stress response is suggested.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Culture

SUBMITTER: Marc Gentzel

LAB HEAD: Dr. Karina Kettner

PROVIDER: PXD030105 | Pride | 2023-02-06

REPOSITORIES: Pride

ACCESS DATA

Publications

Proteomic consequences of TDA1 deficiency in Saccharomyces cerevisiae: Protein kinase Tda1 is essential for Hxk1 and Hxk2 serine 15 phosphorylation.

Müller Henry H Lesur Antoine A Dittmar Gunnar G Gentzel Marc M Kettner Karina K

Scientific reports 20221027 1

Hexokinase 2 (Hxk2) of Saccharomyces cerevisiae is a dual function hexokinase, acting as a glycolytic enzyme and being involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by phosphorylation of Hxk2 at serine 15, which has been attributed to the protein kinase Tda1. To explore the role of Tda1 beyond Hxk2 phosphorylation, the proteomic consequences of TDA1 deficiency were investigated by difference gel electrophoresis (2D-DIGE) co ...[more]

PMID: 36302925

Similar Datasets

Project description:Plant cells are characterized by a high degree of compartmentalization and a diverse proteome and metabolome. Only a very limited number of studies has addressed combined subcellular proteomics and metabolomics which strongly limits biochemical and physiological interpretation of large-scale omics data. Our study presents a methodological combination of non-aqueous fractionation, shotgun proteomics, enzyme kinetics and metabolomics to reveal subcellular diurnal dynamics of plant metabolism. Subcellular marker protein sets were identified and enzymatically validated to resolve metabolism in a 4-compartment model comprising chloroplasts, cytosol, vacuole and mitochondria. These marker sets are now available for future studies which aim to monitor subcellular metabolome and proteome dynamics. Comparing subcellular dynamics in wild type plants and (hexokinase) HXK1-deficient gin2-1 mutants revealed a strong impact of HXK1 activity on metabolome dynamics in multiple compartments. Glucose accumulation in the cytosol of gin2-1 was accompanied by diminished vacuolar glucose levels. Subcellular dynamics of pyruvate, succinate and fumarate concentrations were significantly affected in gin2-1 which coincided with differential mitochondrial proteome dynamics. Lowered mitochondrial glycine and serine concentrations in gin2-1 together with reduced abundance of photorespiratory proteins indicated an effect of the gin2-1 mutation on photorespiratory capacity. Our findings highlight the necessity to resolve plant metabolism to a subcellular level in order to provide a causal relationship between metabolites, proteins and metabolic pathway regulation. --- In an attempt to draw a full picture of abiotic stress responses a complete set of time resolved, compartmentalized metabolomics and proteomics data under different stress conditions was generated. To investigate interactions of stress responses with primary metabolism, the gin2-1 mutant, was compared to its wildtype Landsberg erecta (Ler) under high irradiance and at low temperature. Chlorophyll fluorescence parameters proved the well-known sensitivity of the gin2-1 mutant to high irradiance. Dynamics of subcellular metabolite redistribution upon stress were delayed in gin2-1, and this correlated with massive reduction in proteins belonging to the ATP producing electron transport chain. Evidence for compounds specifically protecting compartments, e.g. maltose in plastids and myo-inositol in mitochondria, was obtained in Ler, but gin2-1 failed to respond or responded only slowly to high irradiance, while no delay was obtained in the cold. Whole-cell concentrations of the amino acids glycine, and serine, provided strong evidence for an important role of the photorespiratory pathway during stress exposure and may contribute to the slow growth of the gin2-1 mutant under high irradiance.

Dataset Information

Proteomic consequences of TDA1 deficiency in Saccharomyces cerevisiae — Protein kinase Tda1 is essential for Hxk1 and Hxk2 serine 15 phosphorylation

Publications

Proteomic consequences of TDA1 deficiency in Saccharomyces cerevisiae: Protein kinase Tda1 is essential for Hxk1 and Hxk2 serine 15 phosphorylation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure