Enolase activates homotypic vacuole fusion and protein transport to the vacuole in yeast

Membrane fusion and protein trafficking to the vacuole are complex processes involving many proteins and lipids. Cytosol from Saccharomyces cerevisiae contains a high Mr activity, which stimulates the in vitro homotypic fusion of isolated yeast vacuoles. Here we purify this activity and identify it as enolase (Eno1p and Eno2p). Enolase is a cytosolic glycolytic enzyme, but a small portion of enolase is bound to vacuoles. Recombinant Eno1p or Eno2p stimulates in vitro vacuole fusion, as does a catalytically inactive mutant enolase, suggesting a role for enolase in fusion that is separate from its glycolytic function. Either deletion of the non-essential ENO1 gene or diminished expression of the essential ENO2 gene causes vacuole fragmentation in vivo, reflecting reduced fusion. Combining an ENO1 deletion with ENO2-deficient expression causes a more severe fragmentation phenotype. Vacuoles from enolase 1 and 2-deficient cells are unable to fuse in vitro. Immunoblots of vacuoles from wild type and mutant strains reveal that enolase deficiency also prevents normal protein sorting to the vacuole, exacerbating the fusion defect. Band 3 has been shown to bind glycolytic enzymes to membranes of mammalian erythrocytes. Bor1p, the yeast band 3 homolog, localizes to the vacuole. Its loss results in the mislocalization of enolase and other vacuole fusion proteins. These studies show that enolase stimulates vacuole fusion and that enolase and Bor1p regulate selective protein trafficking to the vacuole.     

Connection between the Rag4 glucose sensor and the KlRgt1 repressor in Kluyveromyces lactis

The RAG4 gene encodes for the sole transmembrane glucose sensor of Kluyveromyces lactis. A rag4 mutation leads to a fermentation-deficient phenotype (Rag- phenotype) and to a severe defect in the expression of the major glucose transporter gene RAG1. A recessive extragenic suppressor of the rag4 mutation has been identified. It encodes a protein (KlRgt1) 31% identical to the Saccharomyces cerevisiae Rgt1 regulator of the HXT genes (ScRgt1). The Klrgt1 null mutant displays abnormally high levels of RAG1 expression in the absence of glucose but still presents an induction of RAG1 expression in the presence of glucose. KlRgt1 is therefore only a repressor of RAG1. As described for ScRgt1, the KlRgt1 repressor function is controlled by phosphorylation in response to high glucose concentration and this phosphorylation is dependent on the sensor Rag4 and the casein kinase Rag8. However, contrary to that observed with ScRgt1, KlRgt1 is always bound to the RAG1 promoter. This article reveals that the key components of the glucose-signaling pathway are conserved between S. cerevisiae and K. lactis, but points out major differences in Rgt1 regulation and function that might reflect different carbon metabolism of these yeasts.     

A phosphoglucose isomerase gene is involved in the Rag phenotype of the yeast Kluyveromyces lactis

Summary The rag2 mutant of Kluyveromyces lactis cannot grow on glucose when mitochondrial functions are blocked by various mitochondrial inhibitors, suggesting the presence of a defect in the fermentation pathway. The RAG2 gene has been cloned from a K. lactis genomic library by complementation of the rag2 mutation. The amino acid sequence of the RAG2 protein deduced from the nucleotide sequence of the cloned RAG2 gene shows homology to the sequences of known phosphoglucose isomerases (PGI and PHI). In vivo complementation of the pgi1 mutation in Saccharomyces cerevisiae by the cloned RAG2 gene, together with measurements of specific PGI activities and the detection of PGI proteins, confirm that the RAG2 gene of K. lactis codes for the phosphoglucose isomerase enzyme. Complete loss of PGI activity observed when the coding sequence of RAG2 was disrupted leads us to conclude that RAG2 is the only gene that codes for phosphoglucose isomerase in K. lactis. The RAG2 gene of K. lactis is expressed constitutively, independently of the growth substrates (glycolytic or gluconeogenic). Unlike the pgi1 mutants of S. cerevisiae, the K. lactis rag2 mutants can still grow on glucose, however they do not produce ethanol.     

The unique hexokinase of Kluyveromyces lactis. Molecular and functional characterization and evaluation of a role in glucose signaling

The Crabtree-negative yeast Kluyveromyces lactis is capable of adjusting its glycolytic flux to the requirements of respiration by tightly regulating glucose uptake. RAG5 encoding the only glucose and fructose phosphorylating enzyme present in K. lactis is required for the up-regulation of glucose transport and also for glucose repression. To understand the significance of the molecular identity and specific function(s) of the corresponding kinase to glucose signaling, RAG5 was overexpressed and its gene product KlHxk1 (Rag5p) isolated and characterized. Stopped-flow kinetics and sedimentation analysis indicated a monomer-homodimer equilibrium of KlHxk1 in a condition of catalysis, i.e. in the presence of substrates and products. The kinetic constants of ATP-dependent glucose phosphorylation identified a 53-kDa monomer as the high affinity/high activity form of the novel enzyme for both glycolytic substrates suggesting a control of glucose phosphorylation at the level of dimer formation and dissociation. In contrast to the highly homologous hexokinase isoenzyme 2 of Saccharomyces cerevisiae (ScHxk2), KlHxk1 was not inhibited by free ATP in a physiological range of nucleotide concentration. Mass spectrometric sequencing of tryptic peptides of KlHxk1 identified unmodified serine at amino acid position 156. The corresponding amino acid in ScHxk2 is serine 157, which represents the autophosphorylation-inactivation site. KlHxk1 did not display, however, the typical pattern of inactivation under the respective in vitro conditions and maintained a high residual glucose phosphorylating activity. The biophysical and functional data are discussed with respect to a possible regulatory role of KlHxk1 in glucose metabolism and signaling in K. lactis.     

Glycolysis Controls Plasma Membrane Glucose Sensors To Promote Glucose Signaling in Yeasts

Sensing of extracellular glucose is necessary for cells to adapt to glucose variation in their environment. In the respiratory yeast Kluyveromyces lactis, extracellular glucose controls the expression of major glucose permease gene RAG1 through a cascade similar to the Saccharomyces cerevisiae Snf3/Rgt2/Rgt1 glucose signaling pathway. This regulation depends also on intracellular glucose metabolism since we previously showed that glucose induction of the RAG1 gene is abolished in glycolytic mutants. Here we show that glycolysis regulates RAG1 expression through the K. lactis Rgt1 (KlRgt1) glucose signaling pathway by targeting the localization and probably the stability of Rag4, the single Snf3/Rgt2-type glucose sensor of K. lactis. Additionally, the control exerted by glycolysis on glucose signaling seems to be conserved in S. cerevisiae. This retrocontrol might prevent yeasts from unnecessary glucose transport and intracellular glucose accumulation.     

The oxygen level determines the fermentation pattern in Kluyveromyces lactis

Yeasts belonging to the lineage that underwent whole-genome duplication (WGD) possess a good fermentative potential and can proliferate in the absence of oxygen. In this study, we analyzed the pre-WGD yeast Kluyveromyces lactis and its ability to grow under oxygen-limited conditions. Under these conditions, K. lactis starts to increase the glucose metabolism and accumulates ethanol and glycerol. However, under more limited conditions, the fermentative metabolism decreases, causing a slow growth rate. In contrast, Saccharomyces cerevisiae and Saccharomyces kluyveri in anaerobiosis exhibit almost the same growth rate as in aerobiosis. In this work, we showed that in K. lactis , under oxygen-limited conditions, a decreased expression of RAG1 occurred. The activity of glucose-6-phosphate dehydrogenase also decreased, likely causing a reduced flux in the pentose phosphate pathway. Comparison of related and characterized yeasts suggests that the behavior observed in K. lactis could reflect the lack of an efficient mechanism to maintain a high glycolytic flux and to balance the redox homeostasis under hypoxic conditions. This could be a consequence of a recent specialization of K. lactis toward living in a niche where the ethanol accumulation at high oxygen concentrations and the ability to survive at a low oxygen concentration do not represent an advantage.     

Galactokinase encoded by GAL1 is a bifunctional protein required for induction of the GAL genes in Kluyveromyces lactis and is able to suppress the gal3 phenotype in Saccharomyces cerevisiae.

We have analyzed a GAL1 mutant (gal1-r strain) of the yeast Kluyveromyces lactis which lacks the induction of beta-galactosidase and the enzymes of the Leloir pathway in the presence of galactose. The data show that the K. lactis GAL1 gene product has, in addition to galactokinase activity, a function required for induction of the lactose system. This regulatory function is not dependent on galactokinase activity, as it is still present in a galactokinase-negative mutant (gal1-209). Complementation studies in Saccharomyces cervisiae show that K. lactis GAL1 and gal1-209, but not gal1-r, complement the gal3 mutation. We conclude that the regulatory function of GAL1 in K. lactis soon after induction is similar to the function of GAL3 in S. cerevisiae.     

Improved production of heterologous proteins by a glucose repression-defective mutant of Kluyveromyces lactis

The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1beta compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.