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Centro de Recursos Microbiológicos (CREM), Department of Life Sciences, Faculty of Sciences and Technology, New University of Lisbon, 2829-516 Caparica, Portugal
Correspondence
Paula Gonçalves
pmz{at}fct.unl.pt
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Jeffries, 2006; van Maris et al., 2006). Saccharomyces cerevisiae has been considered to be the organism with the highest potential for the production of ethanol from biomass hydrolysates, mainly because of its efficiency and robustness. However, S. cerevisiae is naturally unable to use pentose sugars as carbon and energy sources, which constitutes a major drawback, since hemicellulose hydrolysates contain high amounts of xylose and arabinose. Therefore, considerable research has focused on the metabolic engineering of S. cerevisiae to co-ferment the hexoses and pentoses derived from lignocellulosic feedstocks. Recombinant xylose-fermenting S. cerevisiae strains have been obtained by combining the expression of heterologous enzymes, either from the yeast Pichia stipitis (xylose reductase and xylitol dehydrogenase) or from the filamentous fungus Piromyces sp. (xylose isomerase), with the modulation of native enzymes that lead to positive effects on fermentation efficiency (Jeffries, 2006; van Maris et al., 2006; Hahn-Hägerdal et al., 2007).
In all the recombinant yeast strains currently available, xylose uptake occurs solely via the hexose facilitators (Hxt proteins and Gal2) that also accept xylose as a substrate, albeit with very low affinity (Km >100 mM; Hamacher et al., 2002). For this reason, xylose uptake is slow and dependent on the concentration of glucose present in the environment. Notably, an engineered S. cerevisiae strain selected for efficient xylose fermentation exhibits significantly improved xylose transport kinetics (Kuyper et al., 2005). Heterologous expression of a high-affinity xylose transporter is therefore expected to represent an asset for xylose-fermenting S. cerevisiae strains in which other limiting factors have already been optimized (Gárdonyi et al., 2003a; Karhumaa et al., 2006).
An attempt to isolate xylose transporters from P. stipitis by functional complementation yielded the low-affinity Sut1–3 transporters (Weierstall et al., 1999), but the isolation of a high-affinity xylose transporter from the same yeast remains elusive, despite the recent sequencing of its genome (Jeffries et al., 2007). Recently, we reported the cloning and characterization of two glucose/xylose transporter genes from Candida intermedia, designated GXF1 and GXS1 (Leandro et al., 2006). GXF1 encodes a glucose/xylose facilitator, whereas GXS1 encodes a glucose/xylose proton symporter, with Km values of 50 and 
0.4 mM, respectively. While Gxf1 functions very well in the heterologous context, S. cerevisiae expressing the symporter protein displays very weak transport activity. The different performances raise questions regarding the identification of factors hampering functional expression of the symporter, but not of the facilitator, in the host environment.
Deficient targeting of heterologous transporters to the plasma membrane has often been observed in S. cerevisiae (Wieczorke et al., 2003). Moreover, an apparent relationship between low- and high-affinity sugar transport systems has been detected in different yeasts (Spencer-Martins & van Uden, 1985; Cason et al., 1986; Lucas & van Uden, 1986; Verma et al., 1987; Ramos et al., 1988; Walsh et al., 1994), and these studies suggest that expression of transporter proteins is stringently regulated. In S. cerevisiae, a complex picture has emerged after the discovery of a surprisingly extensive set of HXT genes involved in hexose transport. Regulatory mechanisms governing the activity of the most important members of this family include both transcriptional regulation and specific targeting of transporters for degradation, dictated mainly by the sugar concentration in the growth medium (Gancedo, 1998; Rolland et al., 2002; Kresnowati et al., 2006).
We now report on various aspects of the expression of the two aforementioned C. intermedia transporters in S. cerevisiae (transcription and translation efficiencies, subcellular localization and mRNA stability), with special attention given to the possible causes of the poor performance of the glucose/xylose proton symporter, and in particular addressing the co-expression of both transporters.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Yeast strains were routinely grown in YNB (yeast nitrogen base without amino acids; Difco) medium containing the indicated carbon source and the required supplements at 25 °C and 150 r.p.m. unless otherwise stated. Growth of the cultures was followed by monitoring OD640 with an Ultrospec 3100 pro spectrophotometer (GE Healthcare) (OD640=1 corresponds to 0.3 mg dry weight per ml). Sugar concentrations are given as percentages (w/v). Strains TMB 3201 and TMB 3001 were provided by Professor Bärbel Hahn-Hägerdal, University of Lund, Sweden, and strain C5 was provided by Professor Uwe Sauer, ETHZ, Switzerland.
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). Cultures were harvested at an OD640 of 0.7. Symport activity was assessed by the proton influx elicited by the addition of either glucose or xylose to an aqueous cell suspension adjusted to pH 5.
Isolation of yeast plasma membranes and Tricine SDS-PAGE.
C. intermedia PYCC 4715 cells cultivated in Verduyn medium (Verduyn et al., 1992) containing 0.5 % xylose, 2 % glucose or 4 % xylose, and S. cerevisiae strains MJY1, MJY2, MJY5 and MJY6 cultivated in Verduyn medium with 2 % glucose, were harvested during the exponential growth phase (OD640 1–2). Plasma and mitochondrial membranes were isolated as described previously (Leandro et al., 2006). Protein concentrations were determined using the BCA Protein Assay kit (Pierce). Aliquots of plasma membrane preparations containing 20 µg total protein were separated by Tricine SDS-PAGE (Schägger, 1994) in a 7.5 % polyacrylamide/0.23 % bisacrylamide gel.
Plasmid and strain construction.
A DNA fragment consisting of the GFP ORF with short flanking regions (45 bp each) homologous to the 3' end of the GXS1 ORF and to the beginning of the PGK1 terminator sequence was amplified by PCR using plasmid pT5II-GFP-GAP (Rodrigues de Sousa et al., 2004) as the template and primers GXS1-GFP_For (5'-ATGGACTCCAAAACTGAAGCTATTATGTCTGAAGAAGCTTCTGTTAGTAAAGGAGAAGAACTTTTC-3') and GFP-TER_Rev (5'-GAAAAGAAAAAAATTGATCTATCGATTTCAATTCAATTCAATTTACTATTTGTATAGTTCATCC-3'). The hxt-null S. cerevisiae TMB 3201 strain was simultaneously transformed with this PCR product and with plasmid pHXT7-GXS1 (Leandro et al., 2006), which had been linearized previously with XbaI. Ura+ transformants were first recovered in YNB maltose medium and subsequently plated onto medium containing glucose as the sole carbon and energy source. One of the transformants obtained, strain MJY6, was selected for further studies.
Plasmid pHXT7-GXS1–GFP was recovered from strain MJY6 and was used to transform ura– mutants (obtained through positive selection on medium containing 5-fluoroorotic acid) of S. cerevisiae strain TMB 3001 (Eliasson et al., 2000), resulting in strain MJY8.
The ura– mutants of S. cerevisiae strains TMB 3001 and C5 (Sonderegger & Sauer, 2003) were transformed with the high-copy-number plasmid pHXT7-GXS1 (Leandro et al., 2006), producing strains MJY7 and MJY9, respectively.
Plasmid pHXT7-GXS1 (Leandro et al., 2006) was digested with PstI and SacI to release the HXT7(promoter)-GXS1(ORF)-PGK(terminator) insert that was then subcloned into either µ plasmid YEplac181 or integrative plasmid YIplac204 (Gietz & Sugino, 1988), between the PstI and SacI sites, resulting in plasmids p(181)HXT7-GXS1 and p(204)HXT7-GXS1-i, respectively. Strain MJY12 was obtained by transformation of S. cerevisiae TMB 3201 with plasmid p(181)HXT7-GXS1. Strain MJY15 was obtained by transformation of strain MJY12 (TMB 3201+p(181)HXT7-GXS1) with plasmid pGXF1 (Leandro et al., 2006). Strain MJY13 was obtained by transformation of S. cerevisiae TMB 3201 with integrative plasmid p(204)HXT7-GXS1-i digested with EcoRV. Strain MJY16 was obtained by transformation of strain MJY13 (TMB 3201+p(204)HXT7-GXS1-i) with plasmid pGXF1 (Leandro et al., 2006).
Northern blotting.
Cultures were harvested at OD640 0.7 and frozen in liquid nitrogen prior to RNA isolation. Total RNA isolation and gel electrophoresis were performed as described elsewhere (Griffioen et al., 1996). The gels were blotted onto Hybond-N nylon membranes (Amersham Biosciences). A 163 bp fragment of the GXF1 gene and a 300 bp fragment of the GXS1 gene were used as gene-specific probes, as described previously (Leandro et al., 2006). A 733 bp fragment of the PDA1 (pyruvate dehydrogenase) gene, amplified from genomic DNA of S. cerevisiae TMB 3201 with primers PDAP1 (5'-ATGGCTTGTGACGCCTTGTA-3') and PDAP2 (5'-GACCAGCAATTGGATCGTTC-3'), was used as a PDA1-specific probe. A 192 bp fragment of the ACT1 (actin) gene, amplified from genomic DNA of S. cerevisiae TMB 3201 with primers ActFor (5'-AGTGAACGTGGTTACTCTTTC-3') and ActRev (5'-GGCTCTGAATCTTTCGTTAC-3'), was used as an ACT1-specific probe. Probes were labelled with either [
-32P]ATP or [-32P]CTP (Amersham Biosciences) using the Prime-a-Gene Labelling system (Promega). Hybridization and washing conditions were as described elsewhere (Griffioen et al., 1996).
Inhibition of transcription with 1,10-phenanthroline.
For inhibition of transcription the method described by Parker et al. (1991) was used. Yeast was cultivated at 25 °C in 1 l YNB medium containing 1 % (w/v) ethanol up to OD640 0.8. A 200 ml volume was kept on ice to prepare cell suspensions for the determination of symport activity. The remaining 800 ml was divided into two aliquots, each subsequently concentrated 10x in the same medium. Either 1,10-phenanthroline alone, to a final concentration of 100 µg ml–1, or 1,10-phenanthroline plus glucose (2 %, final concentration) was added to the aliquots. Samples (4 ml) were collected immediately following addition of inhibitor (time 0) and after incubation for 5, 10, 20, 40 and 60 min at 25 °C. The cell pellets were rapidly frozen in liquid nitrogen and stored at –80 °C. Total RNA was isolated from the frozen cells using TRIzol reagent (Invitrogen).
Microscopy.
Live GFP-transformed cells were spotted onto microscope slides, and epifluorescence images were acquired using an Olympus BX50 microscope equipped with a U-ULH 100 W mercury high-pressure bulb and a U-MWIB2 filter set for GFP (Olympus). Images were obtained with a digital camera (Olympus C3030-ZOOM).
Miscellaneous.
Molecular biology techniques were performed using standard protocols (Sambrook et al., 1989). Yeast transformations were performed using the lithium acetate method (Gietz & Woods, 2002). Restriction enzymes and ligase were purchased from Roche. Escherichia coli strain MOSBlue (Amersham Biosciences) was used as the host for DNA manipulations. Primers were obtained from MWG Biotech.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), a strain devoid of any other glucose/xylose transporters (Hamacher et al., 2002), its activity was very low both when expressed from a low-copy-number or from a high-copy-number vector. This was consistently observed for the several independent transformants examined. Expression was driven by a strong constitutive promoter, either a truncated version of the HXT7 promoter (Hamacher et al., 2002) or the PGK1 promoter (Kingsman et al., 1990), which should result in high constitutive transcription levels. Nevertheless, to evaluate whether the weak Gxs1 transporter activity in the S. cerevisiae transformants was due to low mRNA levels, we determined both symport activity and mRNA levels in the original C. intermedia PYCC 4715 strain from which the gene was isolated and in S. cerevisiae MJY2 (Leandro et al., 2006) expressing Gxs1p from a multicopy vector. Although mRNA levels are similar in the S. cerevisiae transformant carrying the multicopy plasmid and in C. intermedia (Fig. 1), xylose symport activity was considerably higher in the latter (Fig. 2). In contrast, it should be noted that expression of Gxf1p, the glucose/xylose facilitator also isolated from C. intermedia, resulted in similar mRNA levels in both yeasts but much higher glucose/xylose transport rates in S. cerevisiae (Leandro et al., 2006). We therefore concluded that the poor symport activity was not due to lack of GXS1 mRNA in S. cerevisiae transformants under the conditions examined. This finding led us to examine the role of post-transcriptional events in the expression of Gxs1p.
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). Both sequences of the GXS1 and GXF1 genes were screened for the utilization of this codon, but neither gene encoded leucine as CUG. Since the two C. intermedia genes are very similar in codon usage despite the difference in transport activities when expressed in S. cerevisiae, codon bias was also excluded as a possible source of poor Gxs1 activity.
Protein levels of C. intermedia transporters in S. cerevisiae
It has been often observed that plasma membrane proteins expressed in an heterologous context may be deficiently targeted or present serious problems for the secretion pathway of the host cell, in particular when overexpressed (Wieczorke et al., 2003; Flegelova et al., 2006). To examine this question, we constructed a Gxs1–GFP fusion for use as a reporter for the subcellular localization of heterologously expressed Gxs1p. In most living cells of strain MJY6, which expresses the fusion protein from the same multicopy expression system present in strain MJY2 (expressing GXS1 only; Leandro et al., 2006), fluorescence could clearly be observed at the periphery of the cell, as would be expected for a protein targeted to the plasma membrane (Fig. 3). Moreover, strain MJY6 grows on glucose, thereby confirming that the Gxs1–GFP fusion protein is a functional glucose transporter located in the plasma membrane. To gain insight into the relative abundance of heterologous Gxs1p and the Gxs1–GFP fusion in the plasma membrane of S. cerevisiae, plasma membranes were isolated from strains MJY2 and MJY6 and run on SDS-PAGE optimized for the separation of membrane proteins. A comparative analysis of the band patterns showed that the clearly noticeable Gxs1p band present in plasma membranes isolated from C. intermedia cells grown in 0.5 % xylose (inducing conditions; Gárdonyi et al., 2003b; Leandro et al., 2006) also appears in the plasma membrane fraction of strain MJY2 as a highly prominent band (Fig. 4). In the membrane of strain MJY6 a strong band is visible corresponding to a molecular mass of approximately 67 kDa, which is the expected molecular mass of the fusion protein Gxs1–GFP (an increase of 27 kDa due to the GFP moiety). This demonstrates that both native Gxs1p and the Gxs1–GFP fusion protein are abundantly produced and correctly directed to the plasma membrane. Indeed, Gxs1p appears to be the second most abundant protein in the plasma membrane, following the plasma membrane ATPase which corresponds to the very prominent band at 95 kDa (Fig. 4).
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). The transformants expressing Gxs1–GFP exhibited a clear retention of fluorescence in intracellular structures, suggesting that this mutation affects trafficking of Gxs1–GFP to the plasma membrane (results not shown). We concluded, therefore, that neither an incorrect subcellular localization nor insufficient production of the protein accounts for the observed deficit in Gxs1p- mediated glucose/xylose symport activity in S. cerevisiae TMB 3201.
Gxs1 expression in the presence of glucose/xylose facilitators
In the course of our attempts to improve functional expression of Gxs1p in S. cerevisiae, different strain backgrounds were tested, among which were two strains that grow relatively well on xylose, TMB 3001 (Eliasson et al., 2000) and C5 (Sonderegger & Sauer, 2003). Both strains differ from the host used to construct MJY2 and MJY6 in that they carry all native S. cerevisiae hexose transporters (Hxt proteins). Strains TMB 3001 and C5 were transformed with plasmid pHXT7-GXS1, yielding strains MJY7 and MJY9. Surprisingly, we failed to detect symport activity in glucose-grown cells of both strains. We reasoned that the absence of symport activity might be related to the presence in the host strains of the hexose transporters operating through facilitated diffusion. This could be the result of a specific interaction between Gxs1p and one or more of the Hxt proteins, or could be the consequence of an increased glucose uptake in these strains. In the latter case this effect would be expected to be independent of the particular functional transporter present. To investigate this possibility, we compared symport activity in two hxt-null strains (Leandro et al., 2006): MJY12, carrying only the GXS1 symporter gene; and MJY15, carrying both the GXS1 (symporter) and the GXF1 (facilitator) C. intermedia genes. In MJY15, unlike in MJY12, we failed to detect symport activity (Fig. 6a). This is in line with the view that the presence of an efficient facilitated glucose transport system may cause a decrease in Gxs1 activity. We subsequently analysed, by Northern blotting, whether this decrease took place at the mRNA level. In these experiments (Fig. 5) we examined both GXF1 and GXS1 mRNA levels. GXF1 mRNA levels were found to be unaltered upon co-expression with other transporters (Fig. 5a) in the MJY1, MJY5 and MJ15 strains. However, GXS1 mRNA was clearly abundant in MJY2, MJY3 and MJY12, where the gene encodes the only glucose/xylose transport system present, and below the detection limit in MJY5, MJY15 and MJY16 co-expressing Gxf1p, and in MJY7 and MJY9, where the native HXT transporter genes are present (Fig. 5b). A similar effect was observed for the expression of the Gxs1–GFP fusion protein: in MJY6, where it is the sole transporter present, the respective mRNA is abundant, whereas it is undetectable in strain MJY8, which differs from the former only by the presence of the Hxt proteins (Fig. 5b).
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). In contrast, the amounts of Gxf1 protein in the plasma membrane of glucose-grown cells were similar in strains MJY1 and MJY5, as expected from the observations concerning GXF1 mRNA in these strains (Figs 4 and 5).
A simple explanation of all our observations would be a sharp drop in the copy number of the plasmid carrying the GXS1 gene, caused by the fact that Gxs1 activity becomes dispensable for the cell when passive glucose transporters are present in high-glucose medium. Indeed, in strain MJY5 we detected that although the plasmid copy number did not decrease, as judged by a Southern blot experiment designed to quantify plasmid-borne copies of the LEU2 gene, many plasmids seemed to have lost the GXS1 gene (results not shown). However, this does not explain the decrease in GXS1 mRNA levels in strain MJY7 (GXS1+HXTs). This strain exhibited low GXS1 mRNA levels (Fig. 5) and virtually no symport activity (Fig. 6b, c) when cultivated in minimal medium with glucose. However, when MJY7 was cultivated in ethanol medium, symport activity was found to be present (Fig. 6b, c). In ethanol-grown cells of MJY7, proton movements elicited by the addition of glucose to an aqueous cell suspension suggest that both passive and active transport systems were operating simultaneously (Fig. 6c): the initial, transient alkalinization caused by the proton influx associated with symport activity was soon counteracted by the acidification due to proton efflux resulting from the activation of the plasma membrane ATPase. This could mean that glucose uptake rates increased considerably, in comparison with the situation in which the low-capacity Gxs1p was the only transporter present and no acidification was observed (results not shown). These results favour the hypothesis that the symporter co-exists with the Hxt facilitators at the plasma membrane of strain MJY7 in ethanol-grown but not in glucose-grown cells. This was confirmed by Northern blotting (see Fig. 5 and time zero in Fig. 7) and by quantitative real-time RT-PCR (not shown), both of which indicated that GXS1 mRNA is present in ethanol-grown cells of strain MJY7, while it is almost undetectable in glucose-grown cells of the same strain. In view of these data, there seem to be two prerequisites for the reduction in the amount of GXS1 mRNA: the presence of a high-capacity glucose facilitator in the plasma membrane, to bring about vigorous glucose uptake, and the presence of glucose in the growth medium.
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; Kresnowati et al., 2006).
We sought to determine whether GXS1 mRNA stability was affected by glucose. This was accomplished in experiments in which de novo mRNA synthesis was stopped by the addition of 1,10-phenanthroline, a compound often used as a transcription inhibitor in the determination of the half-life of mRNAs (Parker et al., 1991). S. cerevisiae MJY7 (GXS1+HXTs) was first cultivated in ethanol medium. In two parallel experiments, 1,10-phenanthroline was subsequently added to the cells alone or simultaneously with glucose. Samples were collected at time zero and periodically after the addition of inhibitor, and total RNA was isolated. GXS1, as well as PDA1 and ACT1 (as loading controls), mRNA levels in the samples were analysed by Northern blotting. As expected, a decrease in GXS1 mRNA levels was observed in all experiments, due to inhibition of transcription, but it remained above the detection level until 1 h after the addition of 1,10-phenanthroline. However, when glucose was added simultaneously with 1,10-phenanthroline, GXS1 mRNA levels could no longer be detected after 40 min (Fig. 7), strongly suggesting that glucose accelerated GXS1 mRNA decay.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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); and (ii) S. cerevisiae transformants expressing Gxs1p as the sole transporter exhibit glucose and xylose proton symport activity and are able to grow on glucose, though they hardly grow on xylose.
It is of note that a xylose (high-affinity) transporter gene recently isolated from the fungus Trichoderma reesei (Hypocrea jecorina) is also unable to support growth of S. cerevisiae with xylose as the sole carbon and energy source. However, growth on xylose improved after prolonged culture on xylose medium supplemented with maltose as a consequence of mutations that occurred spontaneously in the host strain (Saloheimo et al., 2007). A similar observation has been described for the expression of the mammalian glucose transporters GLUT1 and GLUT4 in S. cerevisiae (Wieczorke et al., 2003). In this case, mutations in the host strain were required for correct targeting of the protein to the plasma membrane. The mechanism(s) by which host mutations improve functional expression of heterologous membrane proteins are still unclear, but one possibility is the activation of auxiliary factors. It has been suggested that such factors could help folding of permeases, form complexes with permeases to activate them, or have a regulatory role in the activity of certain transporters (Saloheimo et al., 2007).
Another possible explanation for the reduced activity of Gxs1p in S. cerevisiae could be related to the interactions between the protein and the surrounding lipids in the membrane. The lipid composition of the plasma membrane may vary considerably as a function of the growth conditions and the yeast (Hunter & Rose, 1971; Opekarová & Tanner, 2003). Should such differences occur between S. cerevisiae and C. intermedia, they could affect the biochemical properties of heterologously expressed integral membrane proteins from C. intermedia. For example, a change in the hydrophobic thickness of the lipid bilayer (defined by the lengths of the fatty acyl chains in the lipid components) would result in significant changes in the conformation of membrane proteins, which are likely to cause changes in activity (Opekarová & Tanner, 2003; Lee, 2004).
Based on available knowledge, it is not possible to outline general methods to improve functionality of heterologous membrane proteins in S. cerevisiae, since different proteins seem to require specific and distinct conditions to be correctly targeted to the plasma membrane and/or display their full activity (Wieczorke et al., 2003; Flegelova et al., 2006). This is illustrated by our observations of the deleterious effect on Gxs1p expression of a mutation selected for improving functional expression of the mammalian GLUT1 transporter in S. cerevisiae (results not shown). In the work described here, having excluded the possibility that Gxs1p is deficiently produced or incorrectly targeted, we postulate that poor activity could be due to a more subtle shortcoming of the environment in the heterologous host, such as the absence of an auxiliary protein or a different membrane composition.
In the course of our attempts to improve functional expression of Gxs1p in S. cerevisiae we found that co-expression of glucose/xylose facilitators, such as Hxts, with Gxs1p strongly reduced GXS1 mRNA levels and, consequently, symport activity. This occurred in glucose-grown but not in ethanol-grown cells, suggesting that the reduction in the levels of the GXS1 transcript relates to the enhancement of glucose influx and not to a specific interaction between transporters. In this respect, the decrease in GXS1 mRNA levels resembles many other glucose-triggered cellular responses that operate mainly at the transcription level (Gancedo, 1998; Rolland et al., 2002). However, the glucose-responsive decrease in GXS1 transcript levels was rather unexpected, because constitutive glucose repression-insensitive S. cerevisiae promoters drive the expression of the respective cDNA in all our strains. The possibility that glucose affects GXS1 mRNA stability, instead of the rate of transcription, was investigated, and we found evidence for a decreased lifetime of GXS1 mRNA in the presence of glucose. The mechanism affecting GXS1 expression might also explain the failure of attempts to co-express pairs of Hxt proteins with different kinetic properties, driven by constitutive promoters (E. Boles, personal communication). Many different genes in S. cerevisiae have been shown to be regulated at the level of mRNA stability in response to glucose, e.g. SUC2 (encoding invertase; Cereghino & Scheffler, 1996) and JEN1 (encoding a lactate transporter; Andrade et al., 2005). In addition, a recent whole-genome analysis of the glucose response in S. cerevisiae has also revealed that large sets of genes involved in the tricarboxylic acid cycle and storage carbohydrate metabolism are downregulated by glucose at the level of mRNA stability (Kresnowati et al., 2006).
The model proposed for mRNA turnover in yeast comprises three sequential steps: shortening of the poly(A) tail, decapping, and 5'–3' exonucleolytic degradation (Prieto et al., 2000). It has been proposed that glucose causes the 5' cap of the mRNA to become more accessible to decapping, and that competition occurs between the decapping machinery and the assembly of the translation initiation complex for the 5' cap region (Scheffler et al., 1998; de la Cruz et al., 2002). The outcome of this competition seems to depend on both the sequence and the length of the 5' untranslated region (UTR). The presence of a long poly(A) tail may also affect this competition in favour of translational initiation and mRNA stabilization (Prieto et al., 2000). Moreover, conserved 3' UTR motifs have been identified in glucose-downregulated genes that may be involved in specific degradation (Kresnowati et al., 2006).
In the case of GXS1 mRNA, it is unlikely that either 5' or 3' UTR signals mediate glucose-accelerated degradation, because the UTRs present in this mRNA (namely the 5' UTR from the HXT7 and PGK1 genes and the 3' UTR from the PGK1 gene) do not mediate glucose-accelerated mRNA degradation in either their natural contexts or other fusion constructs (Kingsman et al., 1990; Hauf et al., 2000; Hamacher et al., 2002). It follows that such a signal is most probably located in the coding region of the GXS1 gene. The possibility that destabilization of GXS1 mRNA in the presence of high glucose concentrations also occurs in C. intermedia to reinforce transcriptional repression, possibly by a mechanism similar to that operating in S. cerevisiae transformants, cannot be discarded. We have evidence supporting conservation of regulatory mechanisms in S. cerevisiae and C. intermedia, at least at the transcriptional level: the GXF1 (facilitator) gene is very efficiently expressed in S. cerevisiae from its own (C. intermedia) promoter and, in addition, it seems to be similarly regulated by the carbon source (Leandro et al., 2006), i.e. it is poorly expressed in the absence of glucose or xylose (data not shown).
In different yeasts displaying multiple transport systems for glucose or fructose, the expression of those sugar transporters is stringently regulated according to the needs of the cell. The expression profile is determined by the external sugar concentration, and is usually linked to the mechanistic and kinetic properties of the various transporters. In particular, active transport systems are often prevented from operating unless they are strictly required (Spencer-Martins & van Uden, 1985; Cason et al., 1986; Verma et al., 1987; Lucas & van Uden, 1988; Nobre et al., 1999; Gonçalves et al., 2000; Gárdonyi et al., 2003b). In S. cerevisiae, several regulatory mechanisms have been shown to prevent inappropriate expression of sugar transporters. Both transcriptional repression and the specific removal from the membrane and degradation of transporters can be involved (Krampe et al., 1998; Kresnowati et al., 2006). We did not find any evidence for the specific degradation of Gxs1p in the presence of high glucose concentrations, even with a functioning facilitated glucose transport system (results not shown). Our work suggests that regulation of mRNA stability might be an additional, ubiquitous mechanism used by yeast cells to enforce downregulation of high-affinity transporters, in particular those that may pose an unnecessary energetic burden on the cells by operating when sugar is plentiful and fermentation is the preferred metabolic route. A comparative study of the functional expression of the two C. intermedia transporters in S. cerevisiae constitutes an attractive model to investigate important bottlenecks in the heterologous expression of sugar transporters.
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ACKNOWLEDGEMENTS |
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Edited by: D. Burke
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Received 5 December 2007;
revised 14 February 2008;
accepted 25 February 2008.
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