Comparison of the MTT1- and MAL31-like maltose transporter genes in lager yeast strains.

Maltose transporter genes were isolated from four lager yeast strains and sequenced. All four strains contain at least two different types of maltose transporter genes, MTT1 and MAL31. In addition, 'long' 2.7 kb, and 'short' 2.4 kb, versions of each type exist. The size difference is caused by the insertion of two repeats of 147 bp into the promoter regions of the long versions of the genes. As a consequence of the insertion, two Mal63-binding sites move 294 bp away from the transcription initiation site. The 2.4- and 2.7-kb versions are further highly similar. Only the 2.4-kb versions and not the 2.7-kb versions of MTT1 could restore the rapid growth of lager yeast strain A15 on maltotriose in the presence of antimycin A. These results suggest that insertion of the two repeats into the promoter region of the 'long versions' of MTT1 genes led to a diminished expression of these genes. None of the tested long and short versions of the MAL31 genes were able to restore this growth. As the promoter regions of the MTT1 and MAL31 genes are identical, small differences in the protein sequence may be responsible for the different properties of these genes.

Proteome analysis of aerobically and anaerobically grown Saccharomyces cerevisiae cells.

The yeast Saccharomyces cerevisiae is able to grow under aerobic as well as anaerobic conditions. We and others previously found that transcription levels of approximately 500 genes differed more than two-fold when cells from anaerobic and aerobic conditions were compared. Here, we addressed the effect of anaerobic growth at the post-transcriptional level by comparing the proteomes of cells isolated from steady-state glucose-limited anaerobic and aerobic cultures. Following two-dimensional gel electrophoresis and mass spectrometry we identified 110 protein spots, corresponding to 75 unique proteins, of which the levels differed more than two-fold between aerobically and anaerobically-grown cells. For 21 of the 110 spots, the intensities decreased more than two-fold whereas the corresponding mRNA levels increased or did not change significantly under anaerobic conditions. The intensities of the other 89 spots changed in the same direction as the mRNA levels of the corresponding genes, although to different extents. For some genes of glycolysis a small increase in mRNA levels, 1.5-2 fold, corresponded to a 5-10 fold increase in protein levels. Extrapolation of our results suggests that transcriptional regulation is the major but not exclusive mechanism for adaptation of S. cerevisiae to anaerobic growth conditions.

The Saccharomyces cerevisiae Wss1 protein is only present in mother cells.

The Saccharomyces cerevisiae WSS1 (Weak Suppressor of Smt3) gene has initially been identified as a multicopy suppressor of a mutation in SMT3 encoding the small ubiquitin-like modifier. Later, multiple functions related to DNA replication and repair have been found for WSS1. Here, we report the subcellular location of the Wss1 protein. Fluorescence microscopy of strains expressing a Wss1p-green fluorescent protein (GFP) fusion shows that the protein is present in a single sharp spot near the nuclear membrane, distinct from the spindle pole bodies and nucleolus. In dividing cells, the spot is exclusively present in the mother cell, suggesting a mother cell-specific function of WSS1.

Disruption of genes encoding pyruvate dehydrogenase kinases leads to retarded growth on acetate and ethanol in Saccharomyces cerevisiae.

Two open reading frames, YIL042c (PKP1) and YGL059w, with 25% sequence similarity to human pyruvate dehydrogenase kinases, were shown to have protein kinase activity. Using GFP fusions, it was demonstrated that the proteins localize in discrete submitochondrial regions. Strains with a null mutation in these loci grew poorly on acetate and ethanol as carbon sources. Doubling times increased from ca. 4 h in the wild-type to > 6 h for the mutants. Growth rates of the mutants could be restored to wild-type levels by simultaneous disruption of the PDA1 gene, encoding the E1alpha subunit of the pyruvate dehydrogenase complex. This observation and the pyruvate dehydrogenase activities measured in the mutant strains and the wild-type grown on glucose or acetate suggest that the slow growth phenotype on C2 carbon sources is caused by a futile cycle in which phosphoenolpyruvate is converted back to acetyl coenzyme A.

Why does Kluyveromyces lactis not grow under anaerobic conditions? Comparison of essential anaerobic genes of Saccharomyces cerevisiae with the Kluyveromyces lactis genome.

Although some yeast species, e.g. Saccharomyces cerevisiae, can grow under anaerobic conditions, Kluyveromyces lactis cannot. In a systematic study, we have determined which S. cerevisiae genes are required for growth without oxygen. This has been done by using the yeast deletion library. Both aerobically essential and nonessential genes have been tested for their necessity for anaerobic growth. Upon comparison of the K. lactis genome with the genes found to be anaerobically important in S. cerevisiae, which yielded 20 genes that are missing in K. lactis, we hypothesize that lack of import of sterols might be one of the more important reasons that K. lactis cannot grow in the absence of oxygen.

Yeast 14-3-3 proteins.

14-3-3 proteins form a family of highly conserved proteins which are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. They interact with more than 200 different, mostly phosphorylated proteins. The molecular consequences of 14-3-3 binding are diverse: this binding may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization, to the interaction with other proteins or to shielding of binding sites. The binding partners, and hence the 14-3-3 proteins, are involved in almost every cellular process and 14-3-3 proteins have been linked to several diseases, such as cancer, Alzheimer's disease, the neurological Miller-Dieker and spinocerebellar ataxia type 1 diseases and bovine spongiform encephalopathy (BSE). The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe both have two genes encoding 14-3-3 proteins, BMH1 and BMH2 and rad24 and rad25, respectively. In these yeasts, 14-3-3 proteins are essential in most laboratory strains. As in higher eukaryotes, yeast 14-3-3 proteins bind to numerous proteins involved in a variety of cellular processes. Recent genome-wide studies on yeast strains with impaired 14-3-3 function support the participation of 14-3-3 proteins in numerous yeast cellular processes. Given the high evolutionary conservation of the 14-3-3 proteins, the experimental accessibility and relative simplicity of yeasts make them excellent model organisms for elucidating the function of the 14-3-3 protein family.

The KlPGS1 gene encoding phosphatidylglycerolphosphate synthase in Kluyveromyces lactis is essential and assigned to chromosome I.

The phosphatidylglycerolphosphate synthase (CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase, EC 2.7.8.5) is an essential enzyme in biosynthesis of cardiolipin. In this work we report the isolation, heterological cloning, molecular characterization and physical mapping of the Saccharomyces cerevisiae PEL1/PGS1 homologue from Kluyveromyces lactis. The pel1 mutant strain of S. cerevisiae was used to isolate this homologue by screening a K. lactis genomic library. The novel cloned gene was named KlPGS1. Its coding region was found to consist of 1623 bp. The corresponding protein exhibits 55% amino acid identity to its S. cerevisiae counterpart. The presence of the mitochondrial presequence indicates its mitochondrial localization. Sporulation and ascus dissection of diploids heterozygous for single-copy disruption of KlPGS1 revealed that the KlPGS1 gene, is essential in K. lactis. Using a DIG-dUTP-labeled DNA probe-originated from the KlPGS1 gene and Southern hybridization of contour-clamped homogeneous electric field (CHEF)-separated K. lactis chromosomal DNA, the KlPGS1 gene was assigned to chromosome I. The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the Accession No. AY176328.

Efficient gene targeting in Kluyveromyces lactis.

Integration of a DNA fragment in a host genome requires the action of a double-strand break (DSB) repair mechanism. Homologous recombination (HR) is initiated by binding of Rad52p to DNA ends and results in targeted integration. Binding of the Ku heterodimer (Ku70p/Ku80p) results in random integration via non-homologous end joining (NHEJ). In contrast to Saccharomyces cerevisiae, the budding yeast Kluyveromyces lactis shows variable, but in general low, gene targeting efficiency. To study and to improve gene targeting efficiency, K. lactis has been used as a model. The KlRAD51, KlRAD52 and KlKU80 genes have been isolated and deletion mutants for these genes have been constructed. Efficiency of gene targeting was determined at the KlADE2 locus using targeting constructs with different lengths of homologous flanking sequences. In wild-type K. lactis, the gene targeting efficiency ranged from 0% with 50 to 88% with 600 bp flanks. The Klku80 mutant, however, showed >97% gene targeting efficiency independently of the size of the homologous flanks. These results demonstrate that deletion of the NHEJ mechanism results in a higher gene targeting efficiency. Furthermore, increased gene targeting efficiency was achieved by the transformation of wild-type K. lactis with the KlADE2 deletion construct in the presence of excess small DNA fragments. Using this method, PCR-generated deletion constructs containing only 50 bp of homologous flanking sequences resulted in efficient targeted gene replacement.

Regulation of transcription by Saccharomyces cerevisiae 14-3-3 proteins.

14-3-3 proteins form a family of highly conserved eukaryotic proteins involved in a wide variety of cellular processes, including signalling, apoptosis, cell-cycle control and transcriptional regulation. More than 150 binding partners have been found for these proteins. The yeast Saccharomyces cerevisiae has two genes encoding 14-3-3 proteins, BMH1 and BMH2. A bmh1 bmh2 double mutant is unviable in most laboratory strains. Previously, we constructed a temperature-sensitive bmh2 mutant and showed that mutations in RTG3 and SIN4, both encoding transcriptional regulators, can suppress the temperature-sensitive phenotype of this mutant, suggesting an inhibitory role of the 14-3-3 proteins in Rtg3-dependent transcription [van Heusden and Steensma (2001) Yeast 18, 1479-1491]. In the present paper, we report a genome-wide transcription analysis of a temperature-sensitive bmh2 mutant. Steady-state mRNA levels of 60 open reading frames were increased more than 2.0-fold in the bmh2 mutant, whereas those of 78 open reading frames were decreased more than 2.0-fold. In agreement with our genetic experiments, six genes known to be regulated by Rtg3 showed elevated mRNA levels in the mutant. In addition, several genes with other cellular functions, including those involved in gluconeogenesis, ergosterol biosynthesis and stress response, had altered mRNA levels in the mutant. Our data show that the yeast 14-3-3 proteins negatively regulate Rtg3-dependent transcription, stimulate the transcription of genes involved in ergosterol metabolism and in stress response and are involved in transcription regulation of multiple other genes.

Isolation of an acetyl-CoA synthetase gene (ZbACS2) from Zygosaccharomyces bailii.

A gene homologous to Saccharomyces cerevisiae ACS genes, coding for acetyl-CoA synthetase, has been cloned from the yeast Zygosaccharomyces bailii ISA 1307, by using reverse genetic approaches. A probe obtained by PCR amplification from Z. bailii DNA, using primers derived from two conserved regions of yeast ACS proteins, RIGAIHSVVF (ScAcs1p; 210-219) and RVDDVVNVSG (ScAcs1p; 574-583), was used for screening a Z. bailii genomic library. Nine clones with partially overlapping inserts were isolated. The sequenced DNA fragment contains a complete ORF of 2027 bp (ZbACS2) and the deduced polypeptide shares significant homologies with the products of ACS2 genes from S. cerevisiae and Kluyveromyces lactis (81% and 82% identity and 84% and 89% similarity, respectively). Phylogenetic analysis shows that the sequence of Zbacs2 is more closely related to the sequences from Acs2 than to those from Acs1 proteins. Moreover, this analysis revealed that the gene duplication producing Acs1 and Acs2 proteins has occurred in the common ancestor of S. cerevisiae, K. lactis, Candida albicans, C. glabrata and Debaryomyces hansenii lineages. Additionally, the cloned gene allowed growth of S. cerevisiae Scacs2 null mutant, in medium containing glucose as the only carbon and energy source, indicating that it encodes a functional acetyl-CoA synthetase. Also, S. cerevisiae cells expressing ZbACS2 have a shorter lag time, in medium containing glucose (2%, w/v) plus acetic acid (0.1-0.35%, v/v). No differences in cell response to acetic acid stress were detected both by specific growth and death rates. The mode of regulation of ZbACS2 appears to be different from ScACS2 and KlACS2, being subject to repression by a glucose pulse in acetic acid-grown cells.

The spoilage yeast Zygosaccharomyces bailii forms mitotic spores: a screening method for haploidization.

Zygosaccharomyces bailii ISA 1307 and the type strain of this spoilage yeast show a diploid DNA content. Together with a rather peculiar life cycle in which mitotic but no meiotic spores appear to be formed, the diploid DNA content explains the observed difficulties in obtaining auxotrophic mutants. Mitotic chromosome loss induced by benomyl and selection on canavanine media resulted in three haploid strains of Z. bailii. This new set of Z. bailii strains allows the easy isolation of recessive mutants and is suitable for further molecular genetic studies.

Self-association of the spindle pole body-related intermediate filament protein Fin1p and its phosphorylation-dependent interaction with 14-3-3 proteins in yeast.

The Fin1 protein of the yeast Saccharomyces cerevisiae forms filaments between the spindle pole bodies of dividing cells. In the two-hybrid system it binds to 14-3-3 proteins, which are highly conserved proteins involved in many cellular processes and which are capable of binding to more than 120 different proteins. Here, we describe the interaction of the Fin1 protein with the 14-3-3 proteins Bmh1p and Bmh2p in more detail. Purified Fin1p interacts with recombinant yeast 14-3-3 proteins. This interaction is strongly reduced after dephosphorylation of Fin1p. Surface plasmon resonance analysis showed that Fin1p has a higher affinity for Bmh2p than for Bmh1p (K(D) 289 versus 585 nm). Sequences in both the central and C-terminal part of Fin1p are required for the interaction with Bmh2p in the two-hybrid system. In yeast strains lacking 14-3-3 proteins Fin1 filament formation was observed, indicating that the 14-3-3 proteins are not required for this process. Fin1 also interacts with itself in the two-hybrid system. For this interaction sequences at the C terminus, containing one of two putative coiled-coil regions, are sufficient. Fin1p-Fin1p interactions were demonstrated in vivo by fluorescent resonance energy transfer between cyan fluorescent protein-labeled Fin1p and yellow fluorescent protein-labeled Fin1p.

A microarray-assisted screen for potential Hap1 and Rox1 target genes in Saccharomyces cerevisiae.

Saccharomyces cerevisiae adapts to altered oxygen availability by differentially expressing a number of genes. Under aerobic conditions oxygen control of gene expression is exerted through the activator Hap1 and the repressor Rox1. The Hap1 transcription factor senses cellular heme status and increases expression of aerobic genes in response to oxygen. The repression of hypoxic genes under normoxic conditions results from Hap1-mediated activation of ROX1 transcription. To allow the identification of additional Hap1 and Rox1 target genes, genome-wide expression was analysed in aerobically, chemostat-cultivated hap1 and rox1 null mutants. The microarray results show that deletion of HAP1 causes a lower transcript level of 51 genes. Transcription of 40 genes was increased in rox1 mutant cells compared to wild-type cells. Combining these results with our previously described transcriptome data of aerobically and anaerobically grown cells and with computational analysis of the promoters identified 24 genes that are potentially regulated by Hap1, and 38 genes satisfied the criteria of being direct targets of Rox1. In addition, this work provides further evidence that Rox1 controls transcription of anaerobic genes through repression under normoxic conditions.

The Saccharomyces cerevisiae Fin1 protein forms cell cycle-specific filaments between spindle pole bodies.

The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of approximately 10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells.

Inactivation of the Kluyveromyces lactis KlPDA1 gene leads to loss of pyruvate dehydrogenase activity, impairs growth on glucose and triggers aerobic alcoholic fermentation.

The KlPDA1 gene, encoding the E1alpha subunit of the mitochondrial pyruvate-dehydrogenase (PDH) complex was isolated from a Kluyveromyces lactis genomic library by screening with a 1.1 kb internal fragment of the Saccharomyces cerevisiae PDA1 gene. The predicted amino acid sequence encoded by KlPDA1 showed 87% similarity and 79% identity to its S. cerevisiae counterpart. Disruption of KIPDA1 resulted in complete absence of PDH activity in cell extracts. The maximum specific growth rate on glucose of null mutants was 3.5-fold lower than that of the wild-type, whereas growth on ethanol was unaffected. Wild-type K. lactis CBS 2359 exhibits a Crabtree-negative phenotype, i.e. no ethanol was produced in aerobic batch cultures grown on glucose. In contrast, substantial amounts of ethanol and acetaldehyde were produced in aerobic cultures of an isogenic Klpda1 null mutant. A wild-type specific growth rate was restored after introduction of an intact KlPDA1 gene but not, as previously found for S. cerevisiae pda1 mutants, by cultivation in the presence of leucine. The occurrence of aerobic fermentation and slow growth of the Klpda1 null mutant indicate that, although present, the enzymes of the PDH bypass (pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase) could not efficiently replace the PDH complex during batch cultivation on glucose. Only at relatively low growth rates (D = 0.10 h(-1)) in aerobic, glucose-limited chemostat cultures, could the PDH bypass completely replace the PDH complex, thus allowing fully respiratory growth. This resulted in a lower biomass yield [g biomass (g glucose)-1] than in the wild-type due to a higher consumption of ATP in the PDH bypass compared to the formation of acetyl-CoA via the PDH complex.