Arsenic trioxide uptake by hexose permeases in Saccharomyces cerevisiae

Arsenic trioxide is a toxic metalloid and carcinogen that is also used as an anticancer drug, and for this reason it is important to identify the routes of arsenite uptake by cells. In this study the ability of hexose transporters to facilitate arsenic trioxide uptake in Saccharomyces cerevisiae was examined. In the absence of glucose, strains with disruption of the arsenite efflux gene ACR3 accumulated high levels of (73)As(OH)(3). The addition of glucose inhibited uptake by approximately 80%. Disruption of FPS1, the aquaglyceroporin gene, reduced glucose-independent uptake by only about 25%, and the residual uptake was nearly completely inhibited by hexoses, including glucose, galactose, mannose, and fructose but not pentoses or disaccharides. A strain lacking FPS1, ACR3, and all genes for hexose permeases except for HXT3, HXT6, HXT7, and GAL2 exhibited hexose-inhibitable (73)As(OH)(3) uptake, whereas a strain lacking all 18 hexose transport-related genes (HXT1 to HXT17 and GAL2), FPS1 and ACR3, exhibited <10% of wild type (73)As(OH)(3) transport. When HXT1, HXT3, HXT4, HXT5, HXT7, or HXT9 was individually expressed in that strain, hexose-inhibitable (73)As(OH)(3) uptake was restored. In addition, the transport of [(14)C]glucose was inhibited by As(OH)(3). These results clearly demonstrate that hexose permeases catalyze the majority of the transport of the trivalent metalloid arsenic trioxide.     

The HXT1 gene product of Saccharomyces cerevisiae is a new member of the family of hexose transporters.

Two novel genes affecting hexose transport in the yeast Saccharomyces cerevisiae have been identified. The gene HXT1 (hexose transport), isolated from plasmid pSC7, was sequenced and found to encode a hydrophobic protein which is highly homologous to the large family of sugar transporter proteins from eucaryotes and procaryotes. Multicopy expression of the HXT1 gene restored high-affinity glucose transport to the snf3 mutant, which is deficient in a significant proportion of high-affinity glucose transport. HXT1 was unable to complement the snf3 growth defect in low copy number. The HXT1 protein was found to contain 12 putative membrane-spanning domains with a central hydrophilic domain and hydrophilic N- and C-terminal domains. The HXT1 protein is 69% identical to GAL2 and 66% identical to HXT2, and all three proteins were found to have a putative leucine zipper motif at a consensus location in membrane-spanning domain 2. Disruption of the HXT1 gene resulted in loss of a portion of high-affinity glucose and mannose transport, and wild-type levels of transport required both the HXT1 and SNF3 genes. Unexpectedly, expression of beta-galactosidase activity by using a fusion of the lacZ gene to the HXT1 promoter in a multicopy plasmid was maximal during lag and early exponential phases of growth, decreasing approximately 100-fold upon further entry into exponential growth. Deletion analysis of pSC7 revealed the presence of another gene (called ORF2) capable of suppressing the snf3 null mutant phenotype by restoring high-affinity glucose transport and increased low-affinity transport.     

Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on qlycolytic 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 (≥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.     

A novel signal transduction pathway in Saccharomyces cerevisiae defined by Snf3-regulated expression of HXT6.

We show that cells deleted for SNF3, HXT1, HXT2, HXT3, HXT4, HXT6, and HXT7 do not take up glucose and cannot grow on media containing glucose as a sole carbon source. The expression of Hxt1, Hxt2, Hxt3, Hxt6, or Gal2 in these cells resulted in glucose transport and allowed growth on glucose media. In contrast, the expression of Snf3 failed to confer glucose uptake or growth on glucose. HXT6 is highly expressed on raffinose, low glucose, or nonfermentable carbon sources but is repressed in the presence of high concentrations of glucose. The maintenance of HXT6 glucose repression is strictly dependent on Snf3 and not on intracellular glucose. In snf3 delta cells expression of HXT6 is constitutive even when the entire repertoire of HXT genes is present and glucose uptake is abundant. In addition, glucose repression of HXT6 does not require glucose uptake by HXT1, HXT2, HXT3 or HXT4. We show that a signal transduction pathway defined by the Snf3-dependent hexose regulation of HXT6 is distinct from but also overlaps with general glucose regulation pathways in Saccharomyces cerevisiae. Finally, glucose repression of ADH2 and SUC2 is intact in snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta hxt6 delta hxt7 delta gal2 cells, suggesting that the sensing and signaling mechanism for general glucose repression is independent from glucose uptake.     

Glucose sensing and signalling properties in Saccharomyces cerevisiae require the presence of at least two members of the glucose transporter family.

The kinetics of glucose transport in a number of different mutants of Saccharomyces cerevisiae with multiple deletions in the glucose transporter gene family were determined. The deletions led to differences in maximal rate and affinity for glucose uptake by the cells, dependent on the growth conditions. At the same time, there were changes in glucose repression, as determined by expression of invertase activity. Only in the strain with genes HXT1-4 and SNF3 deleted but carrying HXT6/7 were glucose uptake kinetics and invertase activity independent of the presence or concentration of glucose in the growth medium. Some degree of glucose sensitivity was recovered if the SNF3 or HXT2 gene was present in the multiple-deletion background. It is hypothesized that during growth on glucose, both modulation of the kinetics of glucose uptake and derepression of invertase activity require the presence of more than one active gene of the glucose transporter family.     

Combined phylogenetic and neighbourhood analysis of the hexose transporters and glucose sensors in yeasts

The sugar porter family in yeasts encompasses a wide variety of transporters including the hexose transporters and glucose sensors. We analysed a total of 75 members from both groups in nine hemiascomycetous species, with complete and well-annotated genomes: Saccharomyces cerevisiae, Candida glabrata, Zygosaccharomyces rouxii, Kluyveromyces thermotolerans, Saccharomyces kluyverii, Kluyveromyces lactis, Eremothecium gossypii, Debaryomyces hansenii and Yarrowia lipolytica . We present a model for the evolution of the hexose transporters and glucose sensors, supported by two types of complementary evidences: phylogeny and neighbourhood analysis. Five lineages of evolution were identified and discussed according to different mechanisms of gene evolution: lineage A for HXT1, HXT3 , HXT4, HXT5 , HXT6 and HXT7 ; lineage B for HXT2 and HXT10 ; lineage C for HXT8 ; lineage D for HXT14 ; and lineage E for SNF3 and RGT2 .     

Dynamic response of the expression of hxt1, hxt5 and hxt7 transport proteins in Saccharomyces cerevisiae to perturbations in the extracellular glucose concentration

Glucose transport in Saccharomyces cerevisiae relies on a multi-factorial uptake system. The modulation of its efficiency depends on the differential expression of various sets of hexose transport-related proteins whose glucose affinity differs considerably. The expression of three different glucose transport proteins (HXT1, HXT5 and HXT6/7 with low-, intermediate- and high-affinity, respectively) was monitored as a result of modified extracellular glucose concentrations. Cultivation at glucose-limited (continuous) conditions was instantly replaced by a batch (and thus, non-limited) mode. Further, to mimic concentration gradients in large-scale production bioreactors, multiple and rapid transient glucose pulses were applied to chemostat cultivation. Antibodies against the HXT-proteins were used to monitor the proteins' expression levels prior to and after perturbing the external glucose concentrations. HXT5 and HXT6/7 were either expressed during the starvation-like steady-state phases in the chemostat cultivations, whereas HXT1 could not be detected at all. HXT1, however, is subsequently expressed during the excess of glucose in the batch mode, while the HXT5 and HXT6/7 transporters were at least found to decline. These findings coincide well with the transporters' affinity profiles. As a result of repeated and rapid transient glucose pulses during continuous fermentation, especially HXT6/7 pointed out to alter the protein expression pattern.     

Physiological characterization of putative high-affinity glucose transport protein Hxt2 of Saccharomyces cerevisiae by use of anti-synthetic peptide antibodies.

Characterization and quantification of the Hxt2 (hexose transport) protein of Saccharomyces cerevisiae indicate that it is one of a set of differentially expressed high-affinity glucose transporters. The protein product of the HXT2 gene was specifically detected by antibodies raised against a synthetic peptide encompassing the 13 carboxyl-terminal amino acids predicted by the HXT2 gene sequence. Hxt2 migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad band or closely spaced doublet with an average M(r) of 47,000. Hxt2 cofractionated with the plasma membrane ATPase, Pma1, indicating that it is a plasma membrane protein. Hxt2 was not solubilized by high pH or urea but was solublized by detergents, which is characteristic of an integral membrane protein. Expression of the Hxt2 protein was measured under two different conditions that produce expression of high-affinity glucose transport: a medium shift from a high (2.0%) to a low (0.05%) glucose concentration (referred to below as high and low glucose) and growth from high to low glucose. Hxt2 as measured by immunoblotting increased 20-fold upon a shift from high-glucose to low-glucose medium, and the high-affinity glucose transport expressed had a strong HXT2-dependent component. Surprisingly, Hxt2 was not detectable when S. cerevisiae growing in high glucose approached glucose exhaustion, and the high-affinity glucose transport expressed under these conditions did not have an HXT2-dependent component. The role of Hxt2 in growth during aerobic batch culture in low-glucose medium was examined. An hxt2 null mutant grew and consumed glucose significantly more slowly than the wild type, and this phenotype correlated directly with appearance of the Hxt2 protein.     

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.     

A new hexose transporter from Cryptococcus neoformans: molecular cloning and structural and functional characterization

We carried out a screen for Cryptococcus neoformans genes involved in resistance to copper ion toxicity and identified a new hexose transporter (Hxt) gene, HXT1. Hxt1 consists of 520 amino acids and functions to transport hexoses such as glucose. Although Hxt1 conferred copper resistance to Saccharomyces cerevisiae, disruption of the HXT1 gene showed that Hxt1 is not necessary for copper resistance. In virulence tests, an hxt1 mutant strain showed 12% less phenoloxidase activity than the wild-type strain, and no difference in the ability to form melanin was identified. In addition, the hxt1 mutant strain showed virulence similar to that of the wild-type strain in experiments with Caenorhabditis elegans. However, the hxt1 mutant strain generated larger capsules than were generated by the wild-type strain. Thus, Hxt1 appears to be involved in capsule formation.     

High-Copy Suppression of Glucose Transport Defects by Hxt4 and Regulatory Elements in the Promoters of the Hxt Genes in Saccharomyces Cerevisiae

HXT4, a new member of the hexose transporter (HXT) family in Saccharomyces cerevisiae was identified by its ability to suppress the snf3 mutation in multicopy. Multicopy HXT4 increases both high and low affinity glucose transport in snf3 strains and increases low and high affinity transport in wild-type strains. Characterization of HXT4 led to the discovery of a new class of multicopy suppressors of glucose transport defects: regulatory elements in the promoters of the HXT genes. We have designated these sequences DDSEs (DNA sequence dependent suppressing element). Multicopy HXT4 and DDSEs in the HXT1 HXT2, HXT3 and HXT4 promoters were found to restore growth to snf3 and grr1 strains on low glucose media. The DDSE in the HXT4 promoter was refined to a 340-bp sequence 450 bp upstream of the HXT4 translational start. This region was found to contain an 183-amino acid open reading frame. Extensive analysis indicates that the DNA sequence itself and not the encoded protein is responsible for suppression. The promoters of SNF3 and of other glycolytic genes examined did not suppress snf3 in multicopy. Suppression of snf3 by DDSE is dependent on the presence of either HXT2 or HXT3.     

Molecular basis of fructose utilization by the wine yeast Saccharomyces cerevisiae: a mutated HXT3 allele enhances fructose fermentation

Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Delta strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.     

Growth and Glucose Repression Are Controlled by Glucose Transport in Saccharomyces cerevisiae Cells Containing Only One Glucose Transporter

A set of Saccharomyces cerevisiae strains with variable expression of only the high-affinity Hxt7 glucose transporter was constructed by partial deletion of the HXT7 promoter in vitro and integration of the gene at various copy numbers into the genome of an hxt1-7 gal2 deletion strain. The glucose transport capacity increased in strains with higher levels of HXT7 expression. The consequences for various physiological properties of varying the glucose transport capacity were examined. The control coefficient of glucose transport with respect to growth rate was 0.54. At high extracellular glucose concentrations, both invertase activity and the rate of oxidative glucose metabolism increased manyfold with decreasing glucose transport capacity, which is indicative of release from glucose repression. These results suggest that the intracellular glucose concentration produces the signal for glucose repression.     

Cell growth restoration and high level protein expression by the promoter of hexose transporter, HXT7, from Saccharomyces cerevisiae

The promoters of high-affinity hexose transporter, HXT6 and HXT7, are sufficient for complementary expression of invertase to restore the growth of Saccharomyces cerevisiae in raffinose medium. The HXT7 promoter produced higher invertase activity at 139- and 30-fold of the basal activity in strains GN 3C.2 and W303-1, respectively. In addition, the HXT7 promoter expressed 3- to 9-fold more of enhanced green fluorescent protein than that of the constitutive ADH1 in three different S. cerevisiae strains, even during short-term incubation in glucose medium. Therefore, HXT7 promoter could be used for heterologous protein expression in S. cerevisiae.     

A SWEET surprise: Anaerobic fungal sugar transporters and chimeras enhance sugar uptake in yeast

In the yeast Saccharomyces cerevisiae, microbial fuels and chemicals production on lignocellulosic hydrolysates is constrained by poor sugar transport. For biotechnological applications, it is desirable to source transporters with novel or enhanced function from nonconventional organisms in complement to engineering known transporters. Here, we identified and functionally screened genes from three strains of early-branching anaerobic fungi (Neocallimastigomycota) that encode sugar transporters from the recently discovered Sugars Will Eventually be Exported Transporter (SWEET) superfamily in Saccharomyces cerevisiae. A novel fungal SWEET, NcSWEET1, was identified that localized to the plasma membrane and complemented growth in a hexose transporter-deficient yeast strain. Single cross-over chimeras were constructed from a leading NcSWEET1 expression-enabling domain paired with all other candidate SWEETs to broadly scan the sequence and functional space for enhanced variants. This led to the identification of a chimera, NcSW1/PfSW2:TM5-7, that enhanced the growth rate significantly on glucose, fructose, and mannose. Additional chimeras with varied cross-over junctions identified residues in TM1 that affect substrate selectivity. Furthermore, we demonstrate that NcSWEET1 and the enhanced NcSW1/PfSW2:TM5-7 variant facilitated novel co-consumption of glucose and xylose in S. cerevisiae. NcSWEET1 utilized 40.1% of both sugars, exceeding the 17.3% utilization demonstrated by the control HXT7(F79S) strain. Our results suggest that SWEETs from anaerobic fungi are beneficial tools for enhancing glucose and xylose co-utilization and offers a promising step towards biotechnological application of SWEETs in S. cerevisiae.     

Characterization of glucose transport mutants of Saccharomyces cerevisiae during a nutritional upshift reveals a correlation between metabolite levels and glycolytic flux

Saccharomyces cerevisiae shows a marked preference for glucose and fructose, revealed by the repression of genes whose products are involved in processing other carbon sources. This response seems to be driven by sugar phosphorylation in the first steps of glycolysis rather than by the external sugar concentration. To gain a further insight into the role of the internal sugar signalling mechanisms, were measured the levels of upper intracellular glycolytic metabolites and adenine nucleotides in three mutant strains, HXT1, HXT7 and TM6*, with progressively reduced uptake capacities in comparison with the wild type. Reducing the rate of sugar consumption caused an accumulation of hexose phosphates upstream of the phosphofructokinase (PFK) and a reduction of fructose-1,6-bisphosphate levels. Mathematical modelling showed that these effects may be explained by changes in the kinetics of PFK and phosphoglucose isomerase. Moreover, the model indicated a modified sensitivity of the pyruvate dehydrogenase and the trichloroacetic acid cycle enzymes towards the NAD/NADH in the TM6* strain. The activation of the SNF1 sugar signalling pathway, previously observed in the TM6* strain, does not correlate with a reduction of the ATP : AMP ratio as reported in mammals. The mechanisms that may control the glycolytic rate at reduced sugar transport rates are discussed.