Milk Yeast Kluyveromyces Lactis Research Articles

N-terminal signals in the SNX-BAR paralogs Vps5 and Vin1 guide endosomal coat complex formation.

Endosomal coats incorporate membrane-binding subunits such as sorting nexin (SNX) proteins. The Saccharomyces cerevisiae SNX-BAR paralogs Vin1 and Vps5 are respective subunits of the endosomal VINE and retromer complexes whose dimerizing BAR domains are required for complex assembly and membrane association. However, a degree of promiscuity is predicted for yeast BAR-BAR pairings, and recent work has implicated the unstructured N-terminal domains of Vin1 and Vps5 in coat formation. Here, we map N-terminal signals in both SNX-BAR paralogs that contribute to the assembly and function of two distinct endosomal coats in vivo. Whereas Vin1 leverages a polybasic region and adjacent hydrophobic motif to bind Vrl1 and form VINE, the N-terminus of Vps5 interacts with the retromer subunit Vps29 at two sites, including a conserved hydrophobic pocket in Vps29 that engages other accessory proteins in humans. We also examined the sole isoform of Vps5 from the milk yeast Kluyveromyces lactis and found that ancestral yeasts may have used a nested N-terminal signal to form both VINE and retromer. Our results suggest that the specific assembly of Vps5-family SNX-BAR coats depends on inputs from unique N-terminal sequence features in addition to BAR domain coupling, expanding our understanding of endosomal coat biology.

Cellular Redox Metabolism Is Modulated by the Distinct Localization of Cyclic Nucleotide Phosphodiesterase 5A Isoforms.

3′-5′ cyclic nucleotide phosphodiesterases (PDEs) are a family of evolutionarily conserved cAMP and/or cGMP hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Among them, cGMP-specific PDE5—being a regulator of vascular smooth muscle contraction—is the molecular target of several drugs used to treat erectile dysfunction and pulmonary hypertension. Production of full-length murine PDE5A isoforms in the milk-yeast Kluyveromyces lactis showed that the quaternary assembly of MmPDE5A1 is a mixture of dimers and tetramers, while MmPDE5A2 and MmPDE5A3 only assembled as dimers. We showed that the N-terminal peptide is responsible for the tetramer assembly of MmPDE5A1, while that of the MmPDE5A2 is responsible for its mitochondrial localization. Overexpression of the three isoforms alters at different levels the cAMP/cGMP equilibrium as well as the NAD(P)+/NAD(P)H balance and induces a metabolic switch from oxidative to fermentative. In particular, the mitochondrial localization of MmPDE5A2 unveiled the existence of a cAMP-cGMP signaling cascade in this organelle, for which we propose a metabolic model that could explain the role of PDE5 in some cardiomyopathies and some of the side effects of its inhibitors.

Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis

The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.

Investigation of Heterologously Expressed Glucose-6-Phosphate Dehydrogenase Genes in a Yeast zwf1 Deletion.

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme of the oxidative part of the pentose phosphate pathway and serves as the major source of NADPH for metabolic reactions and oxidative stress response in pro- and eukaryotic cells. We here report on a strain of the model yeast Saccharomyces cerevisiae which lacks the G6PD-encoding ZWF1 gene and displays distinct growth retardation on rich and synthetic media, as well as a strongly reduced chronological lifespan. This strain was used as a recipient to introduce plasmid-encoded heterologous G6PD genes, synthesized in the yeast codon usage and expressed under the control of the native PFK2 promotor. Complementation of the hypersensitivity of the zwf1 mutant towards hydrogen peroxide to different degrees was observed for the genes from humans (HsG6PD1), the milk yeast Kluyveromyces lactis (KlZWF1), the bacteria Escherichia coli (EcZWF1) and Leuconostoc mesenteroides (LmZWF1), as well as the genes encoding three different plant G6PD isoforms from Arabidopsis thaliana (AtG6PD1, AtG6PD5, AtG6PD6). The plastidic AtG6PD1 isoform retained its redox-sensitive activity when produced in the yeast as a cytosolic enzyme, demonstrating the suitability of this host for determination of its physiological properties. Mutations precluding the formation of a disulfide bridge in AtG6PD1 abolished its redox-sensitivity but improved its capacity to complement the yeast zwf1 deletion. Given the importance of G6PD in human diseases and plant growth, this heterologous expression system offers a broad range of applications.

Chemical signaling and insect attraction is a conserved trait in yeasts.

Yeast volatiles attract insects, which apparently is of mutual benefit, for both yeasts and insects. However, it is unknown whether biosynthesis of metabolites that attract insects is a basic and general trait, or if it is specific for yeasts that live in close association with insects. Our goal was to study chemical insect attractants produced by yeasts that span more than 250 million years of evolutionary history and vastly differ in their metabolism and lifestyle. We bioassayed attraction of the vinegar fly Drosophila melanogaster to odors of phylogenetically and ecologically distinct yeasts grown under controlled conditions. Baker's yeast Saccharomyces cerevisiae, the insect‐associated species Candida californica, Pichia kluyveri and Metschnikowia andauensis, wine yeast Dekkera bruxellensis, milk yeast Kluyveromyces lactis, the vertebrate pathogens Candida albicans and Candida glabrata, and oleophilic Yarrowia lipolytica were screened for fly attraction in a wind tunnel. Yeast headspace was chemically analyzed, and co‐occurrence of insect attractants in yeasts and flowering plants was investigated through a database search. In yeasts with known genomes, we investigated the occurrence of genes involved in the synthesis of key aroma compounds. Flies were attracted to all nine yeasts studied. The behavioral response to baker's yeast was independent of its growth stage. In addition to Drosophila, we tested the basal hexapod Folsomia candida (Collembola) in a Y‐tube assay to the most ancient yeast, Y. lipolytica, which proved that early yeast signals also function on clades older than neopteran insects. Behavioral and chemical data and a search for selected genes of volatile metabolites underline that biosynthesis of chemical signals is found throughout the yeast clade and has been conserved during the evolution of yeast lifestyles. Literature and database reviews corroborate that yeast signals mediate mutualistic interactions between insects and yeasts. Moreover, volatiles emitted by yeasts are commonly found also in flowers and attract many insect species. The collective evidence suggests that the release of volatile signals by yeasts is a widespread and phylogenetically ancient trait, and that insect–yeast communication evolved prior to the emergence of flowering plants. Co‐occurrence of the same attractant signals in yeast and flowers suggests that yeast‐insect communication may have contributed to the evolution of insect‐mediated pollination in flowers.

Lack of the NAD+-dependent glycerol 3-phosphate dehydrogenase impairs the function of transcription factors Sip4 and Cat8 required for ethanol utilization in Kluyveromyces lactis

The NAD+-dependent glycerol 3-phosphate dehydrogenase (KlGpd1) is an important enzyme for maintenance of the cytosolic redox balance in the milk yeast Kluyveromyces lactis. The enzyme is localized in peroxisomes and in the cytosol, indicating its requirement for the oxidation of NADH in both compartments. Klgpd1 mutants grow more slowly on glucose than wild-type cells and do not grow on ethanol as a sole carbon source. We studied the molecular basis of the latter phenotype and found that Gpd1 is required for high expression of KlICL1 and KlMLS1 which encode the key enzymes of the glyoxylate pathway isocitrate lyase and malate synthase, respectively. This regulation is mediated by CSRE elements in the promoters of these genes and the Snf1-regulated transcription factors KlCat8 and KlSip4. To study the transactivation function of these factors we developed a modified yeast one-hybrid system for K. lactis, using the endogenous ß-galactosidase gene LAC4 as a reporter in a lac9 deletion background. In combination with ChIP analyses we discovered that Gpd1 controls both the specific binding of Cat8 and Sip4 to the target promoters and the capacity of these factors to activate the reporter gene expression. We propose a model in which KlGpd1 activity is required for maintenance of the redox balance. In its absence, genes which function in generating redox balance instabilities are not expressed. A comparison of mutant phenotypes further indicates, that this system not only operates in K. lactis, but also in Saccharomyces cerevisiae.

Cell wall synthesis and central carbohydrate metabolism are interconnected by the SNF1/Mig1 pathway in Kluyveromyces lactis

The trimeric AMP-activated kinase complex (AMPK) is conserved from yeast to humans and is best known for its role in balancing energy metabolism. Additional functions, including the regulation of cell wall biosynthesis, have been proposed for the SNF1 complex, the baker’s yeast homolog of AMPK. We here demonstrate that this function is conserved in the Crabtree-negative milk yeast Kluyveromyces lactis. Deletion mutants in the genes encoding the subunits of the trimeric complex (Klsnf1, Klgal83, Klsnf4) displayed increased sensitivities towards cell wall stress agents and a mutant lacking the kinase subunit had a thinner cell wall in transmission electron micrographs as compared to wild type. Epistasis analyses demonstrated that the KlSNF1 complex acts in parallel to cell wall integrity (CWI) signaling and stress sensitivities of Klsnf1 deletions can be suppressed by additional deletions in glycolytic genes (KlPFK1, KlPFK2, KlPGI1) or by a Klmig1 mutant. Western blot analyses of an HA-tagged KlMig1p revealed its phosphorylation on ethanol medium similar to its S. cerevisiae ortholog, but a substantial amount of protein remained phosphorylated even with high glucose concentrations. Application of cell wall stress shifted this equilibrium towards the non-phosphorylated fraction of KlMig1p. We conclude that KlMig1p may exert a negative regulatory function on cell wall biosynthesis.

Investigation of the role of four mitotic septins and chitin synthase 2 for cytokinesis in Kluyveromyces lactis

Septins are key components of the cell division machinery from yeast to humans. The model yeast Saccharomyces cerevisiae has five mitotic septins, Cdc3, Cdc10, Cdc11, Cdc12, and Shs1. Here we characterized the five orthologs from the genetically less-redundant milk yeast Kluyveromyces lactis. We found that except for KlSHS1 all septin genes are essential. Klshs1 deletions displayed temperature-sensitive growth and morphological defects. Heterologous complementation analyses revealed that all five K. lactis genes encode functional orthologs of their S. cerevisiae counterparts. Fluorophore-tagged versions of the K. lactis septins localized to a ring at the incipient bud site and split into two separate rings at the bud neck later in cytokinesis. One of the key proteins recruited to the bud neck by septins in S. cerevisiae is the chitin synthase Chs2, which synthesizes the primary septum. KlCHS2 was found to be essential and deletions showed cytokinetic defects upon spore germination. KlChs2-GFP also localized to the bud neck and to punctate structures in K. lactis. We conclude that cytokinesis in K. lactis is similar to S. cerevisiae and chimeric septin complexes are fully functional in both yeasts. In contrast to some S. cerevisiae strains, KlChs2 and KlCdc10 were found to be essential.

13C NMR based profiling unveils different α-ketoglutarate pools involved into glutamate and lysine synthesis in the milk yeast Kluyveromyces lactis

BackgroundThe construction of efficient cell factories for the production of metabolites requires the rational improvement/engineering of the metabolism of microorganisms. The subject of this paper is directed towards the quantitative understanding of the respiratory/fermentative Kluyveromyces lactis yeast metabolism and its rag8 casein kinase mutant, taken as a model for all rag gene mutations. Methods13C NMR spectroscopy and [1,2-13C2]glucose were used as metabolic stable-isotope tracer to define the metabolic profiling of a K. lactis yeast and its derivative mutants. ResultsRag8 showed a decrease of all 13C glutamate fractional enrichments, except for [4-13C]glutamate that was higher than wild type ones. A decrease of TCA cycle flux in rag8 mutants and a contribution of a [4-13C]ketoglutarate pool not originating from mitochondria were suggested.13C lysine enrichments confirmed the presence of two compartmentalized α-ketoglutarate (α-KG) pools participating to glutamate and lysine synthesis.Moreover, an increased transaldolase, as compared to transketolase activity, was observed in the rag8 mutant by 13C NMR isotopomer analysis of alanine. Conclusions13C NMR-based isotopomer analysis showed the existence of different α-KG metabolic pools for glutamate and lysine biosynthesis. In the rag8 mutant, 13C labeled pentose phosphate intermediates participated in the synthesis of this compartmentalized α-KG pool. General significanceA compartmentalization of the α-KG pools involved in lysine biosynthesis has been revealed for the first time in K. lactis. Given its great impact in metabolic engineering field, its existence should be validated/compared with other yeasts and/or fungal species.

Regulation of cytokinesis in the milk yeast Kluyveromyces lactis

Cytokinesis in yeast and mammalian cells is a highly coordinated process mediated by the constriction of an actomyosin ring. In yeasts, it is accompanied by the formation of a chitinous primary septum. Although much is known about the regulation of cytokinesis in budding yeast, overlapping functions of redundant genes complicates genetic analyses. Here, we investigated the effects of various deletion mutants on cytokinesis in the milk yeast Kluyveromyces lactis. To determine the spatiotemporal parameters of cytokinesis components, live-cell imaging of fluorophor-tagged KlMyo1 and a new Lifeact probe for KlAct1 was employed. In contrast to Saccharomyces cerevisiae, where deletion of ScMYO1 is lethal, Klmyo1 deletion was temperature-sensitive. Transmission and scanning electron microscopy demonstrated that the Klmyo1 deletion cells had a defect in the formation of the primary septum and in cell separation; this result was confirmed by FACS analyses. Deletion of KlCYK3 was lethal, whereas in S. cerevisiae a cyk3 deletion is synthetically lethal with hof1 deletion. Growth of Klhof1 mutants was osmoremedial at 25°C, as it is in S. cerevisiae. CYK3 and HOF1 genes cross-complemented in both species, suggesting that they are functional homologs. Inn1, a common interactor for these two regulators, was essential in both yeasts and the encoding genes did not cross-complement. The C2 domain of the Inn1 homologs conferred species specificity. Thus, our work establishes K. lactis as a model yeast to study cytokinesis with less genetic redundancy than S. cerevisiae. The viability of Klmyo1 deletions provides an advantage over budding yeast to study actomyosin-independent cytokinesis. Moreover, the lethality of Klcyk3 null mutants suggests that there are fewer functional redundancies with KlHof1 in K. lactis.

Yeast on the milky way: genetics, physiology and biotechnology of Kluyveromyces lactis

The milk yeast Kluyveromyces lactis has a life cycle similar to that of Saccharomyces cerevisiae and can be employed as a model eukaryote using classical genetics, such as the combination of desired traits, by crossing and tetrad analysis. Likewise, a growing set of vectors, marker cassettes and tags for fluorescence microscopy are available for manipulation by genetic engineering and investigating its basic cell biology. We here summarize these applications, as well as the current knowledge regarding its central metabolism, glucose and extracellular stress signalling pathways. A short overview on the biotechnological potential of K. lactis concludes this review.

Solid-state NMR spectroscopy reveals anomer specific transport of galactose in the milk yeast Kluyveromyces lactis

Genetic evidence indicates that only the β-anomer of galactose is transported to Kluyveromyces lactis cells by galactose/glucose transporter Hgt1p, and that aldose-1-epimerase encoded by GAL10 is a prerequisite for growth on galactose. Minor aldose-1-epimerases other than Gal10p also exist in K.lactis. Using a mutant defective in both aldose-1-epimerases, we show by solid-state nuclear magnetic resonance spectroscopy that only β-anomer is transported in the cell and stays without or with a slow rate of conversion to α-anomer. Signals due to intracellular β-galactose appeared at two positions, both of which were shifted towards higher magnetic fields than that of β-galactose in aqueous solution, suggesting that incorporated galactose binds to cellular components, probably proteins.

Milk and sugar: Regulation of cell wall synthesis in the milk yeast Kluyveromyces lactis

The milk yeast Kluyveromyces lactis is an alternative model yeast to the well established Saccharomyces cerevisiae. The cell wall of these fungi consists of polysaccharides (i.e. long chains of β-1,3- and β-1,6-linked sugar chains and some chitin) and mannoproteins, both of which are continually adapted to environmental conditions in terms of their abundance and organization. This implies the need to perceive signals at the cell surface and to transform them into a proper cellular response. The signal transduction cascade involved in this process is generally referred to as the cell wall integrity (CWI) pathway. CWI signaling and cell wall composition have been extensively studied in the Baker's yeast S. cerevisiae and are also of interest in other yeast species with commercial potential, such as K. lactis. We here summarize the results obtained in the past years on CWI signaling in K. lactis and use a comparative approach to the findings obtained in S. cerevisiae to highlight special adaptations to their natural environments.

KlHsl1 is a component of glycerol response pathways in the milk yeast Kluyveromyces lactis

In Saccharomyces cerevisiae, HSL1 (NIK1) encodes a serine-threonine protein kinase involved in cell cycle control and morphogenesis. Deletion of its putative orthologue in Kluyveromyces lactis, KlHSL1, gives rise to sensitivity to the respiratory inhibitor antimycin A (AA). Resistance to AA on glucose (Rag+ phenotype) is associated with genes (RAG) required for glucose metabolism/glycolysis. To understand the relationship between RAG and KlHSL1, rag and Klhsl1Δ mutant strains were investigated. The analysis showed that all the mutants contained a phosphorylated form of Hog1 and displayed an inability to synthesize/accumulate glycerol as a compatible solute. In addition, rag mutants also showed alterations in both cell wall and membrane fatty acids. The pleiotropic defects of these strains indicate that a common pathway regulates glucose utilization and stress response mechanisms, suggesting impaired adaptation of the plasma membrane/cell wall during the respiratory-fermentative transition. KlHsl1 could be the link between these adaptive pathways and the morphogenetic checkpoint.

Galactose transporters discriminate steric anomers at the cell surface in yeast

Aldose-1-epimerase or mutarotase (EC 5.1.3.3) catalyzes interconversion of alpha/beta-anomers of aldoses, such as glucose and galactose, and is distributed in a wide variety of organisms from bacteria to humans. Nevertheless, the physiological role of this enzyme has been elusive in most cases, because the alpha-form of aldoses in the solid state spontaneously converts to the beta-form in an aqueous solution until an equilibrium of alpha : beta=36.5 : 63.5 is reached. A gene named GAL10 encodes this enzyme in yeast. Here, we show that the GAL10-encoded mutarotase is necessary for utilization of galactose in the milk yeast Kluyveromyces lactis, and that this condition is presumably created by the presence of the beta-specific galactose transporter, which excludes the alpha-anomer from the alpha/beta-mixture in the medium at the cell surface. Thus, we found that a mutarotase-deficient mutant of K. lactis failed to grow on medium, in which galactose was the sole carbon source, but, surprisingly, that the growth failure is suppressed by concomitant expression of the Saccharomyces cerevisiae-derived galactose transporter Gal2p, but not by that of the K. lactis galactose transporter Hgt1p. We also suggest the existence of another mutarotase in K. lactis, whose physiological role remains unknown, however.

Differential control of isocitrate lyase gene transcription by non-fermentable carbon sources in the milk yeast Kluyveromyces lactis

The KlICL1 gene, encoding isocitrate lyase in Kluyveromyces lactis, is essential for ethanol utilization. Deletion analyses identified two functional promoter elements, CSRE-A and CSRE-B. Transcription is activated on ethanol, but not on glucose, glycerol or lactate. Expression depends on the KlCat8p transcription factor and KlSip4p binds to the promoter elements. Glycerol diminishes KlICL1 expression and a single carbon source responsive element (CSRE) sequence is both necessary and sufficient to mediate this regulation. The glycerol effect is less pronounced in Saccharomyces cerevisiae than in K. lactis. Mutants lacking KlGUT2 (which encodes the glycerol 3-phosphate dehydrogenase) still show reduced expression in glycerol, whereas mutants deficient in glycerol kinase ( Klgut1) do not. We conclude that a metabolite of glycerol is required for this regulation.

Characterization of KLBCK1, encoding a MAP kinase kinase kinase of Kluyveromyces lactis

The cellular integrity and response to hypoosmotic conditions in the yeast Saccharomyces cerevisiae are ensured by a MAP kinase signal transduction pathway mediated by the yeast homolog of mammalian protein kinase C. Bck1p functions as the MAP kinase kinase kinase of this pathway. Here we report on the cloning and analysis of the BCK1 homolog from the milk yeast Kluyveromyces lactis ( KlBCK1). The deduced protein sequences display three highly conserved domains with the serine/threonine kinase domain containing 89 % identical amino acid residues. Interestingly, a region identified in KlBck1p as a putative SAM domain, mediating protein-protein interactions, is also conserved in ScBck1p. Yet, two-hybrid analyses indicate that this region may not be involved in dimerization of KlBck1p in contrast to its S. cerevisiae counterpart. Expression of KlBCK1 fully complements the defects in a Scbck1 null mutant and is capable of activating the pathway as indicated by a reporter system based on the transcription factor Rlm1p. However, deletion from the haploid K. lactis genome does not result in a loss of cellular integrity under a variety of conditions tested. Thus, despite the functional conservation in this component of the MAP kinase pathway in both yeast, cellular integrity in K. lactis may depend at least in part on different signalling mechanisms when compared with S. cerevisiae.

The molecular genetics of hexose transport in yeasts.

Transport across the plasma membrane is the first, obligatory step of hexose utilization. In yeast cells the uptake of hexoses is mediated by a large family of related transporter proteins. In baker's yeast Saccharomyces cerevisiae the genes of 20 different hexose transporter-related proteins have been identified. Six of these transmembrane proteins mediate the metabolically relevant uptake of glucose, fructose and mannose for growth, two others catalyze the transport of only small amounts of these sugars, one protein is a galactose transporter but also able to transport glucose, two transporters act as glucose sensors, two others are involved in the pleiotropic drug resistance process, and the functions of the remaining hexose transporter-related proteins are not yet known. The catabolic hexose transporters exhibit different affinities for their substrates, and expression of their corresponding genes is controlled by the glucose sensors according to the availability of carbon sources. In contrast, milk yeast Kluyveromyces lactis contains only a few different hexose transporters. Genes of other monosaccharide transporter-related proteins have been found in fission yeast Schizosaccharomyces pombe and in the xylose-fermenting yeast Pichia stipitis. However, the molecular genetics of hexose transport in many other yeasts remains to be established. The further characterization of this multigene family of hexose transporters should help to elucidate the role of transport in yeast sugar metabolism.

Coregulation of theKluyveromyces lactislactose permease and β-galactoidase genes is achieved by interaction of multiple LAC9 binding sites in a 2.6 kbp divergnent promoter

The coregulated genes LAC4 and LAC12 encoding beta-galactosidase and lactose permease, respectively, are responsible for the ability of the milk yeast Kluyveromyces lactis to utilise lactose. They are divergently transcribed and separated by an unusually large intergenic region of 2.6 kbp. Mapping of the upstream border of the beta-galactosidase gene (LAC4) promoter by introduction of mutations at the chromosomal locus showed that LAC4 and LAC12 share the same upstream activation sites (UAS). The UASs represent binding sites for the trans-activator LAC9, a K. lactis homologue of GAL4, conforming to the consensus sequence 5'-CGG(N5)A/T(N5)CCG-3'. Two binding sites are located in front of each of the genes at almost symmetrical positions. beta-galactosidase activity measurements as well as quantitation of LAC4 and LAC12 mRNA levels demonstrated that all four sites are required for full induction. LAC4 proximal and a LAC12 proximal sites cooperate in activating transcription of both genes. These sites are more than 1.7 kbp apart and the distal site is located more than 2.3 kbp upstream of the respective start of transcription. Thus, the distance between interacting sites is larger than in any of the well characterised yeast promoters. The contribution to gene activation differs for individual binding sites and correlates with the relative affinity of LAC9 for these sites in vitro suggesting that LAC9 binding is a rate limiting step for LAC promoter function.