Proteomic and Functional Consequences of Hexokinase Deficiency in Glucose-repressible Kluyveromyces lactis

Nadia Mates,Karina Kettner,Falk Heidenreich,Theresia Pursche,Rebekka Migotti,Günther Kahlert,Eberhard Kuhlisch,Karin D Breunig,Wolfgang Schellenberger,Gunnar Dittmar,Bernard Hoflack,Thomas M Kriegel

doi:10.1074/mcp.m113.032714

Abstract

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.

Highlights

From the *Technische Universitat Dresden, Medizinische Fakultat Carl Gustav Carus, Institute of Physiological Chemistry, D-01307 Dresden, Germany; ¶Technische Universitat Dresden, Biotechnology Center, Proteomics Group, D-01307 Dresden, Germany; ʈMax Delbruck Center for Molecular Medicine, Mass Spectrometry Platform, D-13125 Berlin, Germany; **Technische Universitat Dresden, Medizinische Fakultat Carl Gustav Carus, Institute for Medical Informatics and Biometry, D-01307 Dresden, Germany; ‡‡Martin Luther University Halle-Wittenberg, Science Faculty I, Institute of Biology, Department of Genetics, D-06120 Halle, Germany; §§University of Leipzig, Medical Faculty, Institute of Biochemistry, D-04103 Leipzig, Germany
The rate of phosphorylation of glucose and fructose determined according to Ref. 12 with extracts of glucose- and galactose-grown JA6⌬rag5 was less than 3% compared with JA6 control extracts
In accordance with above data, strain JA6⌬rag5 was considered appropriate for analyzing the proteomic consequences of KlHxk1 deficiency in glucose-repressible K. lactis

Summary

EXPERIMENTAL PROCEDURES

Strains—The strains used in the present work were JA6 (MAT␣, ade 600 adeT-600 trp ura3–12 [35]) and its derivatives JA6⌬rag (rag5::ScURA3, Wesolowski-Louvel laboratory strain collection, University of Lyon, France), JA6⌬rag (rag5::Scura, Breunig laboratory strain collection, University of Halle-Wittenberg, Germany), and JA6⌬rag5/pTSRAG5. Difference Gel Electrophoresis—Equal amounts of protein extracted from 10 cultures of wild-type and mutant cells grown in media containing 2% (w/v) glucose, 2% (w/v) galactose, or 2% (w/v) glycerol (60 samples in total) were mixed to set up an internal standard for multiplex matching of DIGE images, spot normalization, and calculation of abundance changes. Experimental Determination of pI and Mr Marker proteins for twodimensional electrophoresis (Sigma-Aldrich, Taufkirchen, Germany) were labeled with Cy3 and added to the Cy2-labeled internal standard Their DIGE coordinates were used to determine experimental pI values (pI(exp)) by employing an algorithm integrated in DeCyder Software Version 7.0 (GE Healthcare, Munich, Germany). The proteins present in the discs cut out of the preparative twodimensional gels at the DIGE coordinates of the fluorescently labeled unphosphorylated and serine-15-phosphorylated KlHxk were converted to peptides according to an in-gel digestion protocol using sequencing-grade trypsin (Promega, Mannheim, Germany) [42]. All other reagents used were from SigmaAldrich (Taufkirchen, Germany)

RESULTS

Gene locus

DISCUSSION

Phosphoglucomutase Pyruvate decarboxylase