Proteomic and functional consequences of hexokinase deficiency in glucose-repressible Kluyveromyces lactis

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 hexo-kinase 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 difference gel electrophoresis (DIGE). In total 2,851 fluorescent spots containing different protein species were detected in the master gel representing the entirety of 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, amino acid and protein turnover, but also to general stress response and chromatin remodeling which occurred as a consequence of KlHxk1 deficiency at minimum threefold 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 DIGE approach by post-lysis dimethyl labeling largely confirmed the abundance changes detected in the mutant proteome by the former method. Taking into consideration the high proportion of identified hexokinase-dependent proteins exhibiting increased proteomic levels, KlHxk1 is likely to exert a repressive function in a multitude of metabolic pathways. The entirety of proteomic alterations detected in the mutant classifies KlHxk1 as an enzyme with multiple functions and supports the view of an evolutionary conservation of dual-role hexokinases even in organisms that in comparison to S. cerevisiae are less specialized on glucose utilization

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SIGLEAvailable from TIB Hannover: F02B1472 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung und Forschung, Berlin (Germany)DEGerman