Heterologous complementation of the Klaac null mutation of Kluyveromyces lactis by the Saccharomyces cerevisiae AAC3 gene encoding the ADP/ATP carrier

The KlAAC gene, encoding the ADP/ATP carrier, has been assumed to be a single gene in Kluyveromyces lactis, an aerobic, petite-negative yeast species. The Klaac null mutation, which causes a respiratory-deficient phenotype, was fully complemented by AAC2, the Saccharomyces cerevisiae major gene for the ADP/ATP carrier and also by AAC1, a gene that is poorly expressed in S. cerevisiae. In this study, we demonstrate that the Klaac null mutation is partially complemented by the ScAAC3 gene, encoding the hypoxic ADP/ATP carrier isoform, whose expression in S. cerevisiae is prevented by oxygen. Once introduced into K. lactis, the AAC3 gene was expressed both under aerobic and under partial anaerobic conditions but did not support the growth of K. lactis under strict anaerobic conditions.     

The deletion of the succinate dehydrogenase gene KlSDH1 in Kluyveromyces lactis does not lead to respiratory deficiency

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose.     

Three target genes for the transcriptional activator Cat8p of Kluyveromyces lactis: acetyl coenzyme A synthetase genes KlACS1 and KlACS2 and lactate permease gene KlJEN1

The aerobic yeast Kluyveromyces lactis and the predominantly fermentative Saccharomyces cerevisiae share many of the genes encoding the enzymes of carbon and energy metabolism. The physiological features that distinguish the two yeasts appear to result essentially from different organization of regulatory circuits, in particular glucose repression and gluconeogenesis. We have isolated the KlCAT8 gene (a homologue of S. cerevisiae CAT8, encoding a DNA binding protein) as a multicopy suppressor of a fog1 mutation. The Fog1 protein is a homologue of the Snf1 complex components Gal83p, Sip1p, and Sip2p of S. cerevisiae. While CAT8 controls the key enzymes of gluconeogenesis in S. cerevisiae, KlCAT8 of K. lactis does not (I. Georis, J. J. Krijger, K. D. Breunig, and J. Vandenhaute, Mol. Gen. Genet. 264:193-203, 2000). We therefore examined possible targets of KlCat8p. We found that the acetyl coenzyme A synthetase genes, KlACS1 and KlACS2, were specifically regulated by KlCAT8, but very differently from the S. cerevisiae counterparts. KlACS1 was induced by acetate and lactate, while KlACS2 was induced by ethanol, both under the control of KlCAT8. Also, KlJEN1, encoding the lactate-inducible and glucose-repressible lactate permease, was found under a tight control of KlCAT8.     

A truncated form of KlLsm4p and the absence of factors involved in mRNA decapping trigger apoptosis in yeast

The LSM4 gene of Saccharomyces cerevisiae codes for an essential protein involved in pre-mRNA splicing and also in mRNA decapping, a crucial step for mRNA degradation. We previously demonstrated that the first 72 amino acids of the Kluyveromyces lactis Lsm4p (KlLsm4p), which contain the Sm-like domains, can restore cell viability in both K. lactis and S. cerevisiae cells not expressing the endogenous protein. However, the absence of the carboxy-terminal region resulted in a remarkable loss of viability in stationary phase cells (). Herein, we demonstrate that S. cerevisiae cells expressing the truncated LSM4 protein of K. lactis showed the phenotypic markers of yeast apoptosis such as chromatin condensation, DNA fragmentation, and accumulation of reactive oxygen species. The study of deletion mutants revealed that apoptotic markers were clearly evident also in strains lacking genes involved in mRNA decapping, such as LSM1, DCP1, and DCP2, whereas a slight effect was observed in strains lacking the genes DHH1 and PAT1. This is the first time that a connection between mRNA stability and apoptosis is reported in yeast, pointing to mRNA decapping as the crucial step responsible of the observed apoptotic phenotypes.     

Alterations of O-glycosylation, cell wall, and mitochondrial metabolism in Kluyveromyces lactis cells defective in KlPmr1p, the Golgi Ca(2+)-ATPase

In yeast the P-type Ca(2+)-ATPase of the Golgi apparatus, Pmr1p, is the most important player in calcium homeostasis. In Kluyveromyces lactis KlPMR1 inactivation leads to pleiotropic phenotypes, including reduced N-glycosylation and altered cell wall morphogenesis. To study the physiology of K. lactis when KlPMR1 was inactivated microarrays containing all Saccharomyces cerevisiae coding sequences were utilized. Alterations in O-glycosylation, consistent with the repression of KlPMT2, were found and a terminal N-acetylglucosamine in the O-glycans was identified. Klpmr1Delta cells showed increased expression of PIRs, proteins involved in cell wall maintenance, suggesting that responses to cell wall weakening take place in K. lactis. We found over-expression of KlPDA1 and KlACS2 genes involved in the Acetyl-CoA synthesis and down-regulation of KlIDP1, KlACO1, and KlSDH2 genes involved in respiratory metabolism. Increases in oxygen consumption and succinate dehydrogenase activity were also observed in mutant cells. The described approach highlighted the unexpected involvement of KlPMR1 in energy-yielding processes.     

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.