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

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.

Fig. 1.

Growth behavior of K. lactis strains JA6 (solid circles), JA6Δrag5 (open triangles), and JA6Δrag5/pTSRAG5 (open circles) on different carbon sources. Cells were grown in YNB medium supplemented with the indicated carbon sources. Growth was monitored at 30 °C in a NEPHELOstar Galaxy laser-based microplate nephelometer. Cell density is given in relative nephelometric units (RNU). Data represent three independent experiments, each of which was performed in triplicate. For clarity, only one out of four data points is plotted.

Fig. 2.

KlHxk1 expression and glucose repression in K. lactis strains JA6, JA6Δrag5, and JA6Δrag5/pTSRAG5. A, immunodetection of KlHxk1 in protein extracts of strains JA6 (lane 1), JA6Δrag5 (lane 2), and JA6Δrag5/pTSRAG5 (lane 3) using rabbit polyclonal anti-KlHxk1 immunoglobulin. The cells were grown in YNB medium supplemented with 2% (w/v) galactose. 20 μg of protein were applied per lane. Primary antibody detection employed HRP-conjugated goat anti-rabbit IgG and Immobilon^TM Western Chemiluminescent HRP Substrate. B, β-galactosidase activity in strains JA6 (1), JA6Δrag5 (2), and JA6Δrag5/pTSRAG5 (3). Cells were grown in YNB medium supplemented with 2% (w/v) galactose, shifted into YNB medium containing 2% (w/v) glucose, washed with YNB medium without a carbon source, transferred into YNB medium containing 2% (w/v) of the indicated carbon source(s), and propagated until an OD (600 nm/1 cm) of 1 was reached. The activity of β-galactosidase in the protein extracts was determined using o-nitrophenyl-β-d-galactopyranoside as a substrate. One milliunit (mU) of β-galactosidase activity catalyzes the formation of one nanomole of o-nitrophenolate per minute at 30 °C.

Fig. 3.

Identification of unphosphorylated and serine-15-phosphorylated KlHxk1 in a two-dimensional image of the K. lactis proteome. The panels show sections of the fluorescence (A) and black and white (B) two-dimensional images of the Cy2-labeled internal standard proteome containing spike-ins of purified Cy3-labeled serine-15-phosphorylated (green) and Cy5-labeled unphosphorylated (red) KlHxk1. The coordinates of the two fluorescent KlHxk1 species in panels A and B are identical with the coordinates of spot proteins 968 (green) and 976 (red) in the two-dimensional image of the K. lactis proteome shown in Fig. 4. Mass spectrometric analysis of tryptic peptides employing selective reaction monitoring verified phosphorylation of spot protein 968 at serine-15 (supplemental Fig. S1 and supplemental Table S1). For further experimental details, see “Experimental Procedures.”

Fig. 4.

DIGE image of the K. lactis proteome (black and white image of the master gel) and annotation of hexokinase-dependent proteins. The identification of K. lactis proteins occurring during growth on glucose (2% initial concentration) at altered concentrations in the mutant proteome as a consequence of RAG5 disruption and concomitant hexokinase deficiency employed an AAR setting of ≥ 3/≤ −3 and a false discovery rate of 5% according to Ref. . The boundaries surround fluorescent spots containing hexokinase-dependent proteins or indicate their position in the two-dimensional gel when the absolute amount of the respective protein was low. The dot within each boundary marks the center of the protein mass, which generally represents the optimal picking location for protein identification. The proteins are annotated in accordance with the spot numbers used in supplemental Table S2, in which submitted or recommended names, molecular functions, and biological processes are additionally indicated. The positions of serine-15-phosphorylated (spot 968) and unphosphorylated KlHxk1 (spot 976) are labeled in green and red, respectively. For further experimental details, see “Experimental Procedures.”

Fig. 5.

Metabolic pathways in K. lactis affected by RAG5 disruption and concomitant KlHxk1 deficiency upon growth on glucose. Hexokinase-dependent proteins catalyzing central metabolic reactions are indicated by their EC numbers. Enzymes exhibiting enhanced levels in the rag5 null mutant proteome (AAR ≥ 3) are labeled in green, and enzymes exhibiting reduced levels (AAR ≤ −3) are labeled in red. Solid circles represent intermediates of the citric acid and 2-methylcitrate cycles. Open circles specifically indicate cis-aconitate and 2-methyl-cis-aconitate.

Fig. 6.

Assignment of hexokinase-dependent proteins of RAG5 wild-type strain JA6 of K. lactis to functional categories according to KEGG pathway and Gene Ontology (GO) biological process information. The categorized proteins were detected by DIGE at altered concentrations in the proteome of glucose-grown rag5 null mutant strain JA6Δrag5 as a consequence of RAG5 disruption and concomitant KlHxk1 deficiency (for a full list of hexokinase-dependent proteins, see supplemental Table S2). Categorization of hexokinase-dependent proteins with no explicit KEGG or GO annotation is based on the annotation of the closest yeast homolog. Proteins assigned more than one function are accordingly considered in more than one functional category. The sum of footprints of the categorized proteins is normalized to equal 100%.