Kinetic characterization of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression.

In Saccharomyces cerevisiae, there are a large number of genes (HXT1-HXT17/SNF3/RGT2) encoding putative hexose transporters which, together with a galactose permease gene (GAL2), belong to a superfamily of monosaccharide facilitator genes. We have performed a systematic analysis of the HXT1-7 and GAL2 genes and their function in hexose transport. Glucose uptake was below the detection level in the hxt1-7 null strain growing on maltose. Determination of the kinetic parameters of individual hexose transporter-related proteins (Hxtp) expressed in the hxt null background revealed Hxt1p and Hxt3p as low-affinity transporters (Km(glucose) = 50-100 mM), Hxt2p and Hxt4p as moderately low in affinity (Km(glucose) about 10 mM), and Hxt6p, Hxt7p as well as Gal2p as high-affinity transporters (Km(glucosse) = 1-2 mM). However, Hxt2p kinetics in cells grown on low glucose concentrations showed a high-affinity (Km = 1.5 mM) and a low-affinity component (Km = 60 mM). Furthermore, we investigated the involvement of glucose transport in glucose signalling. Glucose repression of MAL2, SUC2 and GAL1 was not dependent on a specific transporter but, instead, the strength of the repression signal was dependent on the level of expression, the properties of the individual transporters and the kind of sugar transported. The strength of the glucose repression signal correlated with the glucose consumption rates in the different strains, indicating that glucose transport limits the provision of a triggering signal rather then being directly involved in the triggering mechanism.

Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on glycolytic flux.

In Saccharomyces cerevisiae, hexose uptake is mediated by HXT proteins which belong to a superfamily of monosaccharide facilitators. We have identified three more genes that encode hexose transporters (HXT5, 6, 7). Genes HXT6 and HXT7 are almost identical and located in tandem 3' adjacent to HXT3 on chromosome IV. We have constructed a set of congenic strains expressing none or any one of the seven known HXT genes and followed growth and flux rates for glucose utilization. The hxt null strain does not grow on glucose, fructose or mannose, and both glucose uptake and flux rate were below the detection level. Expression of either HXT1, 2, 3, 4, 6 or 7 is basically sufficient for aerobic growth on these sugars. In most of the constructs, glucose was the preferred substrate compared to fructose or mannose. There is a considerable variation in flux and growth rates with 1% glucose, dependent on the expression of the individual HXT genes. Expression of either HXT2, 6 or 7 in the null background is sufficient for growth on 0.1% glucose, while growth of strains with only HXT1, 3 or 4 requires higher (> or = 1%) glucose concentrations. These results demonstrate that individual HXT proteins can function independently as hexose transporters, and that most of the metabolically relevant HXT transporters from S. cerevisiae have been identified.

The TYE7 gene of Saccharomyces cerevisiae encodes a putative bHLH-LZ transcription factor required for Ty1-mediated gene expression.

In Saccharomyces cerevisiae, expression of a gene adjacent to the retrotransposon Ty1 is often mediated by Ty-internal sequences. We have identified novel mutants, tye7, which are affected in Ty1-mediated expression of ADH2 through a Ty1 sequence distal to the 5' long terminal repeat sequence. The TYE7 gene has been isolated and characterized. It encodes a 33 kDa protein whose N-terminal third is extremely rich in serine residues (28%). Within its C-terminal sequence, a remarkable similarity to Myc and Max proteins can be found. Thus, TYE7 is a potential member of the basic region/helix-loop-helix/leucine-zipper protein family. TYE7 function is not essential for growth. It may primarily function as a transcriptional activator in Ty1-mediated gene expression, as has been confirmed by the activation of reporter gene expression by a LexA-TYE7 hybrid protein. ADH2 activation by defined Ty1 derivatives revealed that TYE7 acts positively through the more distal Ty1 enhancer element (region D), and negatively in a region between A (the 5' proximal enhancer element) and D.

Glucose uptake and metabolism in grr1/cat80 mutants of Saccharomyces cerevisiae.

Glucose repression in the yeast Saccharomyces cerevisiae designates a global regulatory system controlling the expression of various sets of genes required for the utilization of alternate carbon sources. In a screen, designed for the selection of mutants with reduced glycolytic flux we obtained isolates which were shown by complementation of the cloned wild-type gene to be allelic to the glucose repression mutants grr1/cat80/cot2 previously described. We demonstrate that the grr1 lesion lead to a concentration-dependent decrease in glycolytic flux on glucose. It is very likely that this is caused by a significant decrease in the expression of various genes encoding hexose transporters (HXT1,3) leading to a reduced glucose-uptake rate. In contrast, expression of the maltose permease gene (MAL11) and maltose utilization is normal. There is indirect evidence that grr1 affects the uptake of amino acids, and others have shown that the sugar-induced transport of divalent cations is impaired. These effects are not glucose-specific. We suggest that Grr1, a putative cytoplasmic protein, has a central function in the sensing of nutritional conditions for a variety of unrelated substances, and that relief from glucose repression may be a corollary of this defect in sensing.

Reverse transcriptase activity of an intron encoded polypeptide.

A number of group II introns from eukaryotic organelles and prokaryotes contain open reading frames for polypeptides with homology to retroviral reverse transcriptases (RTs). We have used the yeast transposon (Ty) system to express ORFs for RTs from eukaryotic organelles. This includes the mitochondrial coxI intron i1 from the fungus Podospora anserina, the plastid petD intron from the alga Scenedesmus obliquus and the mitochondrial RTL gene from the alga Chlamydomonas reinhardtii. The ORFs were fused with the TYA ORF from the yeast retrotransposon Ty to produce virus-like particles in the recipient strains with detectable amounts of the RT-like polypeptides. Analysis of the heterologous gene products revealed biochemical evidence that the P. anserina intron encodes an RNA-directed DNA polymerase with properties typically found for RTs of viral or retrotransposable origin. In vitro assays showed that the intron encoded RT is sensitive to RT inhibitors such as N-ethylmaleimide and dideoxythymidine triphosphate but is insensitive against the DNA polymerase inhibitor aphidicolin. The direct biochemical evidence provided here supports the idea that intron encoded RTs are involved in intron transposition events.

Isolation of the TYE2 gene reveals its identity to SWI3 encoding a general transcription factor in Saccharomyces cerevisiae.

The TYE2 gene was identified by recessive mutations which result in a significant reduction of Ty-mediated ADH2 expression. We cloned the TYE2 gene and analyzed its sequence. A large open reading frame of 825 codons was found encoding a rather hydrophilic, 93-kilodalton protein which contains a highly acidic region at its N-terminus. By sequence comparison we found that TYE2 is identical to gene SWI3 which has recently been shown to encode a nuclear protein which may function as a global transcription activation factor. The TYE2/SWI3 protein is necessary for the initiation of Ty1 transcription at its major initiation site in the delta element. Furthermore TYE2 function seems to be important for the expression of a variety of Ty-unrelated functions such as ADH1 expression, sporulation, growth on maltose, galactose, raffinose, and on non-fermentable carbon sources.

Glucose uptake and catabolite repression in dominant HTR1 mutants of Saccharomyces cerevisiae.

Growth and carbon metabolism in triosephosphate isomerase (delta tpi1) mutants of Saccharomyces cerevisiae are severely inhibited by glucose. By using this feature, we selected for secondary site revertants on glucose. We defined five complementation groups, some of which have previously been identified as glucose repression mutants. The predominant mutant type, HTR1 (hexose transport regulation), is dominant and causes various glucose-specific metabolic and regulatory defects in TPI1 wild-type cells. HTR1 mutants are deficient in high-affinity glucose uptake and have reduced low-affinity transport. Transcription of various known glucose transporter genes (HXT1, HXT3, and HXT4) was defective in HTR1 mutants, leading us to suggest that HTR mutations affect a negative factor of HXT gene expression. By contrast, transcript levels for SNF3, which encodes a component of high-affinity glucose uptake, were unaffected. We presume that HTR1 mutations affect a negative factor of HXT gene expression. Multicopy expression of HXT genes or parts of their regulatory sequences suppresses the metabolic defects of HTR1 mutants but not their derepressed phenotype at high glucose concentrations. This suggests that the glucose repression defect is not a direct result of the metabolically relevant defect in glucose transport. Alternatively, some unidentified regulatory components of the glucose transport system may be involved in the generation or transmission of signals for glucose repression.

Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent xylose reductase-encoding gene (XYL1) from the xylose-assimilating yeast Pichia stipitis.

The XYL1 gene of the yeast Pichia stipitis has been isolated from a genomic library using a specific cDNA probe, and its nucleotide (nt) sequence has been determined. In the 5' noncoding region of the P. stipitis XYL1 gene a TATAAA element (known to be necessary for transcription initiation in most yeast genes) is found at nt -81, and two CCAAT recognition motifs (often referred to as the CCAAT box) are present at nt -146 and -106. The XYL1 encodes a polypeptide of 35,927 Da that constitutes a NADH/NADPH-dependent xylose reductase (XR). The enzyme is part of the xylose-xylulose pathway that is absent or only weakly expressed in Saccharomyces cerevisiae. Extensive homology is found to the N terminus of the XR of Pachysolen tannophilus and Candida shehatae. None of the known cofactor binding domains found in many NAD-dependent dehydrogenases are present in the protein. Transformants of S. cerevisiae containing XYL1 of P. stipitis synthesize an active XR. Fusion of XYL1 with the prokaryotic tac promoter leads to a gene that can be expressed in S. cerevisiae and Escherichia coli.

Characterization of trans-acting mutations affecting Ty and Ty-mediated transcription in Saccharomyces cerevisiae.

By recessive mutations, we have identified five genes, TYE1-TYE5, that are required for Ty-mediated expression of ADH2. These tye mutations not only suppress transcription of ADH2 when associated with a Ty element but are also defective in transcription of all Ty1 and Ty2 elements. Moreover, some of these mutations cause growth defects on non-fermentable carbon sources as well as sporulation defects. tye mutations also strongly suppress ADH2 expression when controlled by a polyA/T insertion mutation. Genetic analysis revealed that genes TYE3 and TYE4 are allelic to the previously identified genes SNF2 and SNF5 which code for transcription factors. These findings suggest that TYE gene products influence transcription of many genes rather than specifically Ty and Ty-mediated transcription. We have also found that null alleles of certain STE genes (ste7, ste11 and ste12), known to affect cell-type specific gene expression and expression of some Ty-adjacent genes, have a clear effect on Ty-controlled ADH2 expression depending on the carbon source. On the basis of ADH2 transcript levels in glucose-grown cells, all three ste alleles cause of five-fold reduction of ADH2 expression/transcription. In ethanol-grown cells, ste11 and ste12 mutations caused an almost complete loss of Ty-mediated ADH2 activation while ste7 has only a rather moderate effect. Surprisingly, ste11 and ste12 mutations lead to a significant increase in total Ty transcript levels. This would indicate that the STE12 protein, which is known to bind specifically to Ty1 sequences and thereby serve as an activator of a Ty-adjacent gene, can negatively modulate Ty transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical basis of mitochondrial acetaldehyde dismutation in Saccharomyces cerevisiae.

As reported previously, Saccharomyces cerevisiae cells deficient in all four known genes coding for alcohol dehydrogenases (ADH1 through ADH4) produce considerable amounts of ethanol during aerobic growth on glucose. It has been suggested that ethanol production in such adh0 cells is a corollary of acetaldehyde dismutation in mitochondria. This could be substantiated further by showing that mitochondrial ethanol formation requires functional electron transport, while the proton gradient or oxidative phosphorylation does not interfere with reduction of acetaldehyde in isolated mitochondria. This acetaldehyde-reducing activity is different from classical alcohol dehydrogenases in that it is associated with the inner mitochondrial membrane and also is unable to carry out ethanol oxidation. The putative cofactor is NADH + H+ generated by a soluble, matrix-located aldehyde dehydrogenase upon acetaldehyde oxidation to acetate. This enzyme has been purified from mitochondria of glucose-grown cells. It is clearly different from the known mitochondrial aldehyde dehydrogenase, which is absent in glucose-grown cells. Both acetaldehyde-reducing and acetaldehyde-oxidizing activities are also present in the mitochondrial fraction of fermentation-proficient (ADH+) cells. Mitochondrial acetaldehyde dismutation may have some significance in the removal of surplus acetaldehyde and in the formation of acetate in mitochondria during aerobic glucose fermentation.

Characterization of products of TY1-mediated reverse transcription in Saccharomyces cerevisiae.

Transposition of the yeast transposable element, Ty, has been shown to require a reverse transcription process. By analysing the extrachromosomal Ty-specific nucleic acid molecules associated with overproduced Ty virus-like particles (Ty-VLPs), we identified several reverse transcribed cDNA strands. Most of them resemble the characteristic intermediates of the reverse transcription process described for authentic retroviruses: a (-) strong-stop DNA strand covalently bound to an RNA primer, two elongated (-) strands with one or two long terminal repeat (LTR) sequences and a (+) strong-stop DNA. Surprisingly, complete (+) strands and full-length linear duplex Ty DNA could not be detected. The structural features of two additional (+) strands may indicate some differences between the mechanisms of (+) strand synthesis in Ty and other retrotransposons or retroviruses.

Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene, XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant.

A P. stipitis cDNA library in lambda gt11 was screened using antisera against P. stipitis xylose reductase and xylitol dehydrogenase, respectively. The resulting cDNA clones served as probes for screening a P. stipitis genomic library. The genomic XYL2 gene was isolated and the nucleotide sequence of the 1089 bp structural gene, and of adjacent non-coding regions, was determined. The XYL2 open-reading frame codes for a protein of 363 amino acids with a predicted molecular mass of 38.5 kDa. The XYL2 gene is actively expressed in S. cerevisiae transformants. S. cerevisiae cells transformed with a plasmid, pRD1, containing both the xylose reductase gene (XYL1) and the xylitol dehydrogenase gene (XYL2), were able to grow on xylose as a sole carbon source. In contrast to aerobic glucose metabolism, S. cerevisiae XYL1-XYL2 transformants utilize xylose almost entirely oxidatively.

Ethanol formation in adh0 mutants reveals the existence of a novel acetaldehyde-reducing activity in Saccharomyces cerevisiae.

A strain of Saccharomyces cerevisiae has been constructed which is deficient in the four alcohol dehydrogenase (ADH) isozymes known at present. This strain (adh0), being irreversibly mutated in the genes ADH1, ADH3, and ADH4 and carrying a point mutation in the gene ADH2 coding for the glucose-repressible isozyme ADHII, still produces up to one third of the theoretical maximum yield of ethanol in a homofermentative conversion of glucose to ethanol. Analysis of the glucose metabolism of adh0 cells shows that the lack of all known ADH isozymes results in the formation of glycerol as a major fermentation product, accompanied by a significant production of acetaldehyde and acetate. Treatment of glucose-growing adh0 cells with the respiratory-chain inhibitor antimycin A leads to an immediate cessation of ethanol production, demonstrating that ethanol production in adh0 cells is dependent on mitochondrial electron transport. Reduction of acetaldehyde to ethanol in isolated mitochondria could also be demonstrated. This reduction is apparently linked to the oxidation of acetaldehyde to acetate. Preliminary data suggest that this novel type of ethanol formation in S. cerevisiae is associated with the inner mitochondrial membrane.

Overexpression, purification and properties of alcohol dehydrogenase IV from Saccharomyces cerevisiae.

We have purified ADHIV, a novel alcohol dehydrogenase (ADH) isozyme in the yeast Saccharomyces cerevisiae, after increasing the normally low amount of ADHIV protein in laboratory strains. This was done by overexpression of the structural gene (ADH4) on a 2micro-based multicopy vector. Characterization of the purified enzyme revealed a dimeric structure as well as a different substrate specificity and pH profile as compared to other alcohol dehydrogenase isozymes. On the other hand, we could demonstrate that ADHIV is activated by zinc ions, like the other yeast alcohol dehydrogenase isozymes, and not by ferrous ions, like a structurally similar alcohol dehydrogenase from the bacterium Zymomonas mobilis.

Processing of TY1 proteins and formation of Ty1 virus-like particles in Saccharomyces cerevisiae.

We have analysed functional properties of putative proteins encoded by the yeast transposable element, Ty1, by overexpression of TY genes. High-level expression was achieved by appropriate fusion of a Ty sequence, TY9C, to the yeast ADH1 promoter and transformation of yeast cells with this construction. As shown recently by others (Garfinkel et al. 1985; Mellor et al. 1985c) TY overexpression leads to an increase in particle-bound reverse transcriptase activity and to an intracellular accumulation of virus-like particles (Ty-VLPs). We have used a number of deletions in the second open reading frame (TYB) to identify functional domains required for processing and assembly of Ty proteins. Deletions in the TYB region with homology to acid proteases result in overproduction of an unprocessed form of the TYA protein (pro-TYA) which represents the major protein of Ty-VLPs. One particular mutant construction, TY9C-delta 36, led to the accumulation of a particle-bound, 160 kDa protein which cross-reacted with a mouse antiserum raised against purified pro-TYA protein. This supports the hypothesis that TYB is expressed as a TYA/TYB fusion protein which is processed by a TYB-encoded protease activity. Ty-VLPs are formed in the absence of protein processing and even when the TYB gene is not expressed. Thus, we assume that the assembly of Ty particles occurs prior to processing of Ty proteins.

Gene conversion and reciprocal exchange in a Ty-mediated translocation in yeast.

A haploid yeast mutant carrying a reciprocal translocation was analyzed. Cloning and comparison of sequences involved in the translocation event in wildtype and mutant revealed that the crossover between nonhomologous chromosomes has occurred within Ty sequences. By DNA sequence analysis it could be demonstrated that the reciprocal recombination event is accompanied by a short segment of non-reciprocal exchange (gene conversion) in the immediate vicinity of the crossover. Analysis of the translocation mutant and revertant isolates also indicated that the regulatory effect of Ty elements on adjacent genes can be modified by discrete changes within a Ty element.

Carbon source dependence of transposable element-associated gene activation in Saccharomyces cerevisiae.

Seven cis-dominant mutations leading to the overproduction of the glucose-repressible alcohol dehydrogenase isozyme ADHII (structural gene, ADH2) in Saccharomyces cerevisiae have previously been shown to be due to insertion of a transposable element, Ty, in the 5' regulatory region of the ADH2 gene. We showed that although mating-competent cells (a, alpha, a/a, or alpha/alpha cells) overproduced both ADHII enzyme and ADH2 mRNA, mating-incompetent cells (a/alpha or ste-cells) produced much less ADHII enzyme and ADH2 mRNA. This mating type effect on ADH2 expression was greatest in the presence of a normally derepressing carbon source, glycerol, and much less apparent in the presence of a repressing carbon source, glucose. In addition, Ty insertion led to an aberrant carbon source response in mating-incompetent cells--the normally glucose-repressible ADHII becomes glycerol repressible. The mating type effect and aberrant carbon source response in mating-incompetent cells was specific for Ty-associated mutations in the 5' flanking region of the ADH2 gene in that a non-Ty mutation in the same region did not show these effects. Finally, Ty1 RNA levels also showed a/alpha, suppression, which was apparent only during growth on a nonfermentable carbon source such as glycerol. This suggests that Ty-mediated gene expression is subject to regulation by both mating competence and carbon catabolites.

The synthesis of yeast pyruvate decarboxylase is regulated by large variations in the messenger RNA level.

The yeast PDC1 gene coding for the fermentative enzyme pyruvate decarboxylase was isolated. This DNA sequence was used to identify the corresponding messenger RNA by hybridization. It could be shown that the synthesis of pyruvate decarboxylase is efficiently regulated by variations in the amount of PDC1 mRNA. Very low levels of PDC1 mRNA were found in cells growing in a medium containing ethanol. Glucose addition to these cells leads to a rapid accumulation of PDC1 mRNA. The PDC1 mRNA levels found in different mutants and in cells growing in media containing carbon sources other than glucose or ethanol suggest that the amount of PDC1 mRNA in yeast cells is affected by a number of different factors.

δ Sequences mediate DNA rearrangements in Saccharomyces cerevisiae.

A solo δ sequence flanking the 5' end of the ADHII structural gene, ADR2, can promote a number of DNA rearrangements some of which were investigated in detail. In a selective system haploid mutants were screened in which a solo S sequence flanking ADR2 had been joined to a Ty element. Three different types of events can create such a structure: Reintegration of a Ty sequence at the δ-ADR2 site, inversion of ADR2 and flanking material, and transposition of ADR2 along with 3' flanking material. The involvement of reciprocal or non-reciprocal exchange mechanisms in creating such events are discussed.