Sites for interaction between Gal80p and Gal1p in Kluyveromyces lactis: structural model of galactokinase based on homology to the GHMP protein family.

The induction of transcription of the galactose genes in yeast involves the galactose-dependent binding of ScGal3p (in Saccharomyces cerevisiae) or KlGal1p (in Kluyveromyces lactis) to Gal80p. This binding abrogates Gal80's inhibitory effect on the activation domain of Gal4p, which can then activate transcription. Here, we describe the isolation and characterization of new interaction mutants of K.lactis GAL1 and GAL80 using a two-hybrid screen. We present the first structural model for Gal1p to be based on the published crystal structures of other proteins belonging to the GHMP (galactokinase, homoserine kinase, mevalonate kinase and phosphomevalonate kinase) kinase family and our own X-ray diffraction data of Gal1p crystals at 3A resolution. The locations of the various mutations in the modelled Gal1p structure identify domains involved in the interaction with Gal80p and provide a structural explanation for the phenotype of constitutive GAL1 mutations.

Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha.

We have investigated the methylotrophic yeast Hansenula polymorpha as a host for the co-integration and expression of multiple heterologous genes using an rDNA integration approach. The ribosomal DNA (rDNA) of H. polymorpha was found to consist of a single rDNA cluster of about 50-60 repeats of an 8-kb unit located on chromosome II. A 2.4-kb segment of H. polymorpha rDNA encompassing parts of the 25S, the complete 5S and the non-transcribed spacer region between 25S and 18S rDNA was isolated and inserted into conventional integrative H. polymorpha plasmids harboring the Saccharomyces- cerevisiae-derived URA3 gene for selection. These rDNA plasmids integrated homologously into the rDNA repeats of a H. polymorpha (odc1) host as several independent clusters. Anticipating that this mode of multiple-cluster integration could be used for the simultaneous integration of several distinct rDNA plasmids, the host strain was co-transformed with a mixture of up to three different plasmids, all bearing the same URA3 selection marker. Transformations indeed resulted in mitotically stable strains harboring one, two, or all three plasmids integrated into the rDNA. The overall copy number of the plasmids integrated did not exceed the number of rDNA repeats present in the untransformed host strain, irrespective of the number of different plasmids involved. Strains harboring different plasmids co-expressed the introduced genes, resulting in functional proteins. Thus, this approach provides a new and attractive tool for the rapid generation of recombinant strains that simultaneously co-produce several proteins in desired stoichiometric ratios.

Overproduction of BiP negatively affects the secretion of Aspergillus niger glucose oxidase by the yeast Hansenula polymorpha.

We have cloned the Hansenula polymorpha BIP gene from genomic DNA using a PCR-based strategy. H. polymorpha BIP encodes a protein of 665 amino acids, which shows very high homology to Saccharomvces cerevisiae KAR2p. KAR2p belongs to the Hsp70 family of molecular chaperones and resides in the endoplasmic reticulum (ER)-lumen. H. polymorpha BiP contains a putative N-terminal signal sequence of 30 amino acids together with the conserved -HDEL sequence, the typical ER retention signal, at the extreme C-terminus. We have analysed the effect of BIP overexpression, placing the gene under control of the strong alcohol oxidase promoter (P(MOX)) on the secretion of artificially produced Aspergillus niger glucose oxidase (GOX) by H. polymorpha. BiP overproduction did not lead to any growth defects of the cells; at the subcellular level, proliferation of ER-like vesicles was observed. However, artificially enhanced BiP levels strongly affected GOX secretion and led to accumulation of this protein in the ER-like vesicles. This was not simply due to the high BiP overproduction, because it was also observed under conditions of low P(MOX) induction during growth of cells on glycerol. Vacuolar carboxypeptidase Y was properly sorted to its target organelle in the BiP overproducing strains.

Expression of mouse anticreatine kinase (MAK33) monoclonal antibody in the yeast Hansenula polymorpha.

The methylotrophic yeast Hansenula polymorpha HM1-39 (ura 3 and leu 2) was used as a host strain for the expression of the Fab fragment of the MAK33 monoclonal antibody. The MAK33 antibody reacts specifically with creatine kinase-M. The cDNA of kappa and gamma chains were inserted between the FMD or MOX promoter and the MOX terminator within the expression plasmids. In addition, the secretion signal sequence of the mating factor-alpha (prepro segment) and a fragment from glucoamylase with its secretion signal peptide, were also inserted in the expression plasmids for efficient secretion and production of the MAK33 monoclonal antibody. The co-expression of kappa and gamma chains was achieved by double transformation with kappa and then with gamma chain-expressing plasmids. The cells of H. polymorpha HM1-39 showed high mitotic stability and both uracil+ and leucine+ phenotypic stability after double transformation. Northern analysis showed a high rate of transcription of either kappa or gamma chain mRNA but not both, when the cells were grown in an induction medium. Protein analysis of double-transformed cells showed the monomers of the MAK33 antibody (kappa and gamma chains) were not assembled into a heterodimeric functional form. The expressed proteins of light and heavy chains represent about 11-12% of total cell protein and are found more inside than outside the cell. The expressed monomers show antigen-binding affinity in the Ouchterlony diffusion test; and the binding activity exhibited by cell-free extract was more than that of the cell culture supernatant.

Mutations in the SAM domain of STE50 differentially influence the MAPK-mediated pathways for mating, filamentous growth and osmotolerance in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the MAPKKK Ste11p is involved in three mitogen-activated protein kinase (MAPK) pathways required for mating, filamentous growth and the SHO1-dependent response to hyperosmolarity. All three pathways are also dependent on Ste50p. Ste50p and Ste11p interact constitutively via their N-terminal regions, which include putative SAM domains. Here we show that the interaction of Ste50p and Ste11p is differentially required for modulation of Ste11p function during mating, filamentous growth and the SHO1-dependent response to hyperosmolarity. Two derivatives of Ste50p with mutations in the SAM domain were isolated and characterised. The mutant Ste50 proteins showed reduced binding to Ste11p and a tendency to form homodimers in two-hybrid and in vitro binding assays. Interestingly, these two Ste50p-SAM mutants were associated with increased activation of the mating and filamentous-growth pathways, but a reduction in the SHO1-dependent growth response to hyperosmolarity, relative to the wild-type Ste50p. Moreover, when exposed to hyperosmolarity, these Ste50p-SAM mutants activate genes in the mating (FUS1) and filamentous-growth (FLO11) pathways to higher levels than does the wild type. Thus the Ste50p-Ste11p interaction may differentially modulate the flow of information through the various MAPK-mediated pathways.

The HTR1 gene is a dominant negative mutant allele of MTH1 and blocks Snf3- and Rgt2-dependent glucose signaling in yeast.

Saccharomyces cerevisiae HTR1 mutants are severely impaired in the uptake of glucose. We have cloned dominant HTR1 mutant alleles and show that they encode mutant forms of the Mth1 protein. Mth1 is shown to be involved in carbon source-dependent regulation of its own, invertase and hexose transporter gene expression. The mutant forms block the transduction of the Snf3- and Rgt2-mediated glucose signals upstream of the Rgt1 transcriptional regulator.

Galactose induction in yeast involves association of Gal80p with Gal1p or Gal3p.

Gal1p carries out two functions in the galactose pathway of yeast. It activates Gal4p by interacting with Gal80p--a function that can also served by Gal3p--and it catalyzes the formation of galactose-1-phosphate. Recently, we and others have presented biochemical evidence for complex formation between Gal1p and Gal80p. Here, we extend these data and present genetic evidence for an interaction between Gal1p and Gal80p in vivo, using a two-hybrid assay. Interaction between Gal1p and Gal80p depends on the presence of galactose, but not on the catalytic activity of Gal1p. A new class of Kluyveromyces lactis mutants was isolated, designated Klgal1-m, which have lost the derepressing activity but retain galactokinase activity, indicating that the two Gal1p activities are functionally independent. The KlGal1-m proteins are defective in their ability to interact with Gal80p in a two-hybrid assay. The locations of gall-m mutations identify putative interaction sites in Gal1p and Gal80p. A dominant mutation, KlGAL1-d, leads to a high level of constitutive expression of genes of the galactose pathway. The behavior of chimeric proteins consisting of Gal3p and KlGal1p sequences indicates that both the N-terminal and C-terminal halves of KlGal1p are involved in specific interaction with KlGal80p.

A novel feature of DNA recognition: a mutant Gcn4p bZip peptide with dual DNA binding specificities dependent of half-site spacing.

Homodimeric DNA-binding proteins with relaxed half-site spacing requirements for their DNA targets have been described. As an example, the yeast transcriptional activator Gcn4p binds in vitro equally well to the AP1 site (5'A4T3G2A1C0T1'C2'A3'T4'3') and the ATF/CREB site (5'A4T3G2A1C0G0'T1'C2'A3'T4'3'), which have identical but differently spaced half-site blocks. We describe a novel feature for the bZip class of DNA-binding proteins. The N-14 mutant of a Gcn4p-derived bZip peptide shows a diametrically opposed base-pair recognition specificity depending on the half-site spacing of its DNA target: on pseudo-palindromic, AP1 site-like binding sites, guanine is required in position 2 for proper binding; in contrast, on palindromic, ATF/CREB site-like targets, position 2 must be cytosine to prevent a loss of binding. Modeling studies suggest that the different base-pair requirements on differently spaced DNA targets are due to minimal alterations of the distances between the relevant atoms of the N-14 side-chain and the corresponding target groups on the DNA. Although the N-14 peptide does not have a natural counterpart, its behavior hints at the possibility that dual binding modi dependent on half-site spacing may occur also for natural homodimeric DNA-binding proteins.

Cloning and characterization of three genes (SUT1-3) encoding glucose transporters of the yeast Pichia stipitis.

We have identified and characterized three genes, SUT1, SUT2 and SUT3, that encode glucose transporters of the yeast Pichia stipitis. When expressed in a Saccharomyces cerevisiae hxt null mutant strain that is unable to take up monosaccharides, all three proteins restored growth on glucose. Sequencing of the genes revealed open reading frames coding for 553 amino acids in the case of SUT1, and for 550 amino acids in the case of SUT2 and of SUT3. The derived protein sequences are closely related to one another, and show distinct sequence similarities to the S. cerevisiae hexose transporter family and to monosaccharide transporters of other organisms. The Sut2 and Sut3 proteins are nearly identical and differ only in one amino acid. Determination of substrate specificities and kinetic parameters of the individual Sut proteins expressed in a S. cerevisiae hxt1-7 mutant revealed Sut1, Sut2 and Sut3 as glucose transporters with K(m) values in the millimolar range. The proteins were also able to transport xylose and other monosaccharides, but with a considerably lower affinity. In P. stipitis, transcription of SUT1 was strongly induced by glucose and was independent of the oxygen supply. In contrast, SUT2 and SUT3 were only expressed under aerobic conditions, but independent of the carbon source. Cells disrupted for the SUT1 gene did not show any obvious growth phenotype, however low-affinity glucose uptake was lost. Further investigations suggest that the Sut proteins constitute a subfamily of glucose transporters in P. stipitis, and that other and probably unrelated proteins exist additionally mediating high-affinity glucose and xylose uptake.

Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae.

The hexose transporter family of Saccharomyces cerevisiae comprises 18 proteins (Hxt1-17, Gal2). Here, we demonstrate that all these proteins, except Hxt12, and additionally three members of the maltose transporter family (Agt1, Ydl247, Yjr160) are able to transport hexoses. In a yeast strain deleted for HXT1-17, GAL2, AGT1, YDL247w and YJR160c, glucose consumption and transport activity were completely abolished. However, as additional deletion of the glucose sensor gene SNF3 partially restored growth on hexoses, our data indicate the existence of even more proteins able to transport hexoses in yeast.

Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach.

In a systematic approach to the study of Saccharomyces cerevisiae genes of unknown function, 150 deletion mutants were constructed (1 double, 149 single mutants) and phenotypically analysed. Twenty percent of all genes examined were essential. The viable deletion mutants were subjected to 20 different test systems, ranging from high throughput to highly specific test systems. Phenotypes were obtained for two-thirds of the mutants tested. During the course of this investigation, mutants for 26 of the genes were described by others. For 18 of these the reported data were in accordance with our results. Surprisingly, for seven genes, additional, unexpected phenotypes were found in our tests. This suggests that the type of analysis presented here provides a more complete description of gene function.

Two novel gene expression systems based on the yeasts Schwanniomyces occidentalis and Pichia stipitis.

Two non-Saccharomyces yeasts have been developed as hosts for heterologous gene expression. The celD gene from Clostridium thermocellum, encoding a heat-stable cellulase, served as the test sequence. The first system is based on the amylolytic species Schwanniomyces occidentalis, the second on the xylolytic species Pichia stipitis. The systems comprise auxotrophic host strains (trp5 in the case of S. occidentalis; trp5-10, his3 in the case of P. stipitis) and suitable transformation vectors. Vector components consist of an S. occidentalis-derived autonomously replicating sequence (SwARS) and the Saccharomyces cerevisiae-derived TRP5 sequence for plasmid propagation and selection in the yeast hosts, an ori and an ampicillin-resistance sequence for propagation and selection in a bacterial host. A range of vectors has been engineered employing different promoter elements for heterologous gene expression control in both species. Homologous elements derived from highly expressed genes of the respective hosts appeared to be of superior quality: in the case of S. occidentalis that of the GAM1 gene, in the case of P. stipitis that of the XYL1 gene. Further elements tested are the S. cerevisiae-derived ADH1 and PDC1 promoter sequences.

Ste50p is involved in regulating filamentous growth in the yeast Saccharomyces cerevisiae and associates with Ste11p.

STE50 is required to sustain pheromone-induced signal transduction in S. cerevisiae. Here we report that Ste50p is involved in regulating pseudohyphal development. Both of these processes are also dependent on Ste11p. Deletion of STE50 leads to defects in filamentous growth, which can be suppressed by overproduction of Ste11p. Overexpression of STE11 also suppresses the mating defects of ste50 mutants. We have analysed the physical association between Ste50p and Ste11p in extracts of cells harvested under various conditions. A Ste11p-Ste50p complex can be isolated from extracts of cells in which the pheromone response has been activated, as well as from normally growing cells. Formation of the Ste50p-Ste11p complex does not require G(alpha), G(beta), Ste20p or Ste5p. Oligomerisation of Ste11p is shown to be independent of activation of the pheromone response pathway, and occurs in the absence of Ste50p. We conclude that Ste50p is necessary for Ste11p activity in at least two differentiation programmes: mating and filamentous growth.

Cloning and characterization of the gene encoding a repressible acid phosphatase (PHO1) from the methylotrophic yeast Hansenula polymorpha.

A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with a calculated M(r) of 49400. The encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression.

The activation specificities of wild-type and mutant Gcn4p in vivo can be different from the DNA binding specificities of the corresponding bZip peptides in vitro.

Single amino acid substitutions which previously have been shown to alter the DNA binding specificity of a Gcn4p bZip peptide in vitro were transformed to full length Gcn4p, and activation of a test promoter carrying various palindromic and pseudo-palindromic binding sites was measured. All mutations were found to have different phenotypes, and the first change-of-specificity mutants for Gcn4p in vivo are described. The comparison of plasmids encoding no protein or a particular Gcn4p mutant with broadened activation specificity in gcn4 and gcn4 acr1 genetic backgrounds revealed three new DNA targets of the yeast Acr1p repressor. Surprisingly, we found the activation specificities Gcn4p and the mutants tested in vivo to be generally different from DNA binding specificities of the corresponding bZip peptides in vitro. Especially, the proteins respond differently, in vitro and in vivo, on changes in half site spacing of the DNA binding sites. We present data which largely exclude that the differences between in vivo and in vitro-derived results are due to differences in protein structure, or to the presence of competing protein factors in the yeast cell. We conclude that the differences between in vitro and in vivo-derived results are caused by differences in the degree of flexibility of the target DNA sequences in vitro and in vivo.

Catabolite inactivation of the high-affinity hexose transporters Hxt6 and Hxt7 of Saccharomyces cerevisiae occurs in the vacuole after internalization by endocytosis.

After addition of high concentrations of glucose, rates of high-affinity glucose uptake in Saccharomyces cerevisiae decrease rapidly. We found that the high-affinity hexose transporters Hxt6 and Hxt7 are subject to glucose-induced proteolytic degradation (catabolite inactivation). Degradation occurs in the vacuole, as Hxt6/7 were stabilized in proteinase A-deficient mutant cells. Degradation was independent of the proteasome. The half-life of Hxt6 and Hxt7 strongly increased in end4, ren1 and act1 mutant strains, indicating that the proteins are delivered to the vacuole by endocytosis. Moreover, both proteins were also stabilized in mutants defective in ubiquitination. However, the initial signal that triggers catabolite inactivation is not relayed via the glucose sensors Snf3 and Rgt2.

A region of the cellobiohydrolase I promoter from the filamentous fungus Trichoderma reesei mediates glucose repression in Saccharomyces cerevisiae, dependent on mitochondrial activity.

The upstream activating region that controls cellulose-induced expression of the glucose-repressible cellobiohydrolase I gene (UARcb1) of the filamentous fungus Trichoderma reesei is shown to mediate transcription and glucose repression of a reporter gene in Saccharomyces cerevisiae, a unicellular microorganism that lacks the genes required for the utilization of cellulose. Glucose-controlled transcription mediated by UARcb1 requires the products of the genes SNF1 and SSN6, a protein kinase and a repressor, respectively, that regulate glucose-repressible yeast genes. Previously, it has been shown that mitochondrial function is implicated in cellobiohydrolase I gene expression in T. reesei and this sensitivity to the metabolic state of the mitochondria was shown to be transcriptionally controlled by the 5'-flanking sequence of the cbh1 gene [Abrahão-Neto et al. (1995) Biochemistry 34, 10456-10462]. Remarkably, transcription of the reporter gene controlled by UARcb1 in S. cerevisiae also showed a requirement for active mitochondria, suggesting that a common mechanism involving mitochondrial activity controls glucose-repressible genes in both microorganisms.

Production of recombinant proteins by methylotrophic yeasts.

The methylotrophic yeasts Hansenula polymorpha, Pichia pastoris and Candida boidinii have been developed as production systems for recombinant proteins. The favourable and most advantageous characteristics of these species have resulted in an increasing number off biotechnological applications. As a consequence, these species--especially H. polymorpha and P. pastoris--are rapidly becoming the systems of choice for heterologous gene expression in yeast. Recent advances in the development of these yeasts as hosts for the production of heterologous proteins have provided a catalogue of new applications, methods and system components.

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.

Characterization of a glucose-repressed pyruvate kinase (Pyk2p) in Saccharomyces cerevisiae that is catalytically insensitive to fructose-1,6-bisphosphate.

We have characterized the gene YOR347c of Saccharomyces cerevisiae and shown that it encodes a second functional pyruvate kinase isoenzyme, Pyk2p. Overexpression of the YOR347c/PYK2 gene on a multicopy vector restored growth on glucose of a yeast pyruvate kinase 1 (pyk1) mutant strain and could completely substitute for the PYK1-encoded enzymatic activity. PYK2 gene expression is subject to glucose repression. A pyk2 deletion mutant had no obvious growth phenotypes under various conditions, but the growth defects of a pyk1 pyk2 double-deletion strain were even more pronounced than those of a pyk1 single-mutation strain. Pyk2p is active without fructose-1,6-bisphosphate. However, overexpression of PYK2 during growth on ethanol did not cause any of the deleterious effects expected from a futile cycling between pyruvate and phosphoenolpyruvate. The results indicate that the PYK2-encoded pyruvate kinase may be used under conditions of very low glycolytic flux.