Glucose starvation-induced turnover of the yeast glucose transporter Hxt1

Background

The budding yeast Saccharomyces cerevisiae possesses multiple glucose transporters with different affinities for glucose that enable it to respond to a wide range of glucose concentrations. The steady-state levels of glucose transporters are regulated in response to changes in the availability of glucose. This study investigates the glucose regulation of the low affinity, high capacity glucose transporter Hxt1.

Methods and results

Western blotting and confocal microscopy were performed to evaluate glucose regulation of the stability of Hxt1. Our results show that glucose starvation induces endocytosis and degradation of Hxt1 and that this event requires End3, a protein required for endocytosis, and the Doa4 deubiquitination enzyme. Mutational analysis of the lysine residues in the Hxt1 N-terminal domain demonstrates that the two lysine residues, K12 and K39, serve as the putative ubiquitin-acceptor sites by the Rsp5 ubiquitin ligase. We also demonstrate that inactivation of PKA (cAMP-dependent protein kinase A) is needed for Hxt1 turnover, implicating the role of the Ras/cAMP-PKA glucose signaling pathway in the stability of Hxt1.

Conclusion and general significance

Hxt1, most useful when glucose is abundant, is internalized and degraded when glucose becomes depleted. Of note, the stability of Hxt1 is regulated by PKA, known as a positive regulator for glucose induction of HXT1 gene expression, demonstrating a dual role of PKA in regulation of Hxt1.     

Characterisation of glucose transport in Saccharomyces cerevisiae with plasma membrane vesicles (countertransport) and intact cells (initial uptake) with single Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 or Gal2 transporters

The yeast glucose transporters Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 and Gal2, individually expressed in an hxt1-7 null mutant strain, demonstrate the phenomenon of countertransport. Thus, these transporters, which are the most important glucose transporters in Saccharomyces cerevisiae, are facilitated diffusion transporters. Apparent K(m)-values from high to low affinity, determined from countertransport and initial-uptake experiments, respectively, are: Hxt6 0.9+/-0.2 and 1.4+/-0.1 mM, Hxt7 1.3+/-0.3 and 1.9+/-0.1 mM, Gal2 1.5 and 1.6+/-0.1 mM, Hxt2 2.9+/-0.3 and 4.6+/-0.3 mM, Hxt4 6.2+/-0.5 and 6.2+/-0.3 mM, Hxt3 28.6+/-6.8 and 34.2+/-3.2 mM, and Hxt1 107+/-49 and 129+/-9 mM. From both independent methods, countertransport and initial uptake, the same range of apparent K(m)-values was obtained for each transporter. In contrast to that in human erythrocytes, the facilitated diffusion transport mechanism of glucose in yeast was symmetric. Besides facilitated diffusion there existed in all single glucose transport mutants, except for the HXT1 strain, significant first-order behaviour.     

Starvation-induced degradation of yeast hexose transporter Hxt7p is dependent on endocytosis, autophagy and the terminal sequences of the permease

The yeast high-affinity glucose transporters Hxt6p and Hxt7p are rapidly degraded during nitrogen starvation in the presence of high concentrations of fermentable carbon sources. Our results suggest that degradation is mainly due to the stimulation of general protein turnover and not caused by a mechanism specifically triggered by glucose. Analysis of Hxt6p/7p stability and cellular distribution in end4, aut2 and apg1 mutants indicates that Hxt7p is internalized by endocytosis, and autophagy is involved in the final delivery of Hxt7p to the vacuole for proteolytic degradation. Internalization and degradation of Hxt7p were blocked after truncation of its N-terminal hydrophilic domain. Nevertheless, this fully functional and stabilized hexose transporter could not maintain fermentation capacity of the yeast cells under starvation conditions, indicating a regulatory constraint on glucose uptake.     

Determination of in vivo kinetics of the starvation-induced Hxt5 glucose transporter of Saccharomyces cerevisiae

We have investigated the role and the kinetic properties of the Hxt5 glucose transporter of Saccharomyces cerevisiae. The HXT5 gene was not expressed during growth of the yeast cells in rich medium with glucose or raffinose. However, it became strongly induced during nitrogen or carbon starvation. We have constructed yeast strains constitutively expressing only Hxt5, Hxt1 (low affinity) or Hxt7 (high affinity), but no other glucose transporters. Aerobic fed-batch cultures at quasi steady-state conditions, and aerobic and anaerobic chemostat cultures at steady-state conditions of these strains were used for estimation of the kinetic properties of the individual transporters under in vivo conditions, by investigating the dynamic responses of the strains to changes in extracellular glucose concentration. The K(m) value and the growth properties of the HXT5 single expression strain indicate that Hxt5 is a transporter with intermediate affinity.     

Effect of glucose on xylose utilization in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> harboring the xylose reductase gene

We have constructed recombinant Saccharomyces cerevisiae JH1 harboring a xylose reductase gene (xyl1) isolated from Pichia stipitis. However, JH1 still utilizes glucose more easily than xylose. Therefore, in this study, we characterized the effect of a glucose supplement on xylose utilization, the expression level of xylose reductase as a recombinant gene in JH1, and the expression levels of two hexose transporters (Hxt4 and Hxt7) due to co-fermentation of different concentrations of glucose and xylose. Co-fermentation using 20 g/l of glucose increased xylose consumption up to 11.7 g/l, which was 7.9-fold that of xylose fermentation without a glucose supplement. In addition, we found xyl1 mRNA levels dramatically increased as cells grew under co-fermentation conditions with supplementary glucose; this result is consistent with a significant decrease in the xylose concentration 48 h after cultivation. In addition, the expression levels of Hxt4 and Hxt7 were strongly activated by the presence of glucose and xylose; in particular, Hxt7 showed a 2.9-fold increased expression relative to that of recombinant S. cerevisiae JHM with only a backbone vector, pYES2. The results of this study suggest that xylose utilization would be improved by activation of hexose transporters induced by glucose (rather than xylose) reductase expression.     

Comprehensive chimeric analysis of amino acid residues critical for high affinity glucose transport by Hxt2 of Saccharomyces cerevisiae

Chimeras of Hxt2 and Hxt1, high affinity and low affinity glucose transporters, respectively, of Saccharomyces cerevisiae, were previously constructed by random replacement of each of the 12 transmembrane segments (TMs) of Hxt2 with the corresponding region of Hxt1. Characterization of these chimeras revealed that at least TMs 1, 5, 7, and 8 of Hxt2 are required for high affinity transport activity. To determine which amino acid residues in these TMs are important for high affinity glucose transport, we systematically shuffled all of the 20 residues in these regions that differ between Hxt2 and Hxt1. Analysis of 60 independent mutant strains identified as expressing high affinity and high capacity glucose transport activity by selection on glucose-limited agar plates revealed that Leu-201 in TM5 of Hxt2 is most important for such activity and that either Cys-195 or Phe-198 is also required for maximal activity.     

Glucose sensing and signaling by two glucose receptors in the yeast Saccharomyces cerevisiae.

How eukaryotic cells sense availability of glucose, their preferred carbon and energy source, is an important, unsolved problem. Bakers' yeast (Saccharomyces cerevisiae) uses two glucose transporter homologs, Snf3 and Rgt2, as glucose sensors that generate a signal for induction of expression of genes encoding hexose transporters (HXT genes). We present evidence that these proteins generate an intracellular glucose signal without transporting glucose. The Snf3 and Rgt2 glucose sensors contain unusually long C-terminal tails that are predicted to be in the cytoplasm. These tails appear to be the signaling domains of Snf3 and Rgt2 because they are necessary for glucose signaling by Snf3 and Rgt2, and transplantation of the C-terminal tail of Snf3 onto the Hxt1 and Hxt2 glucose transporters converts them into glucose sensors that can generate a signal for glucose-induced HXT gene expression. These results support the idea that yeast senses glucose using two modified glucose transporters that serve as glucose receptors.     

Identification of a key residue determining substrate affinity in the human glucose transporter GLUT1

Asn³³¹ in transmembrane segment 7 of the yeast Saccharomyces cerevisiae transporter Hxt2 has been identified as a single key residue for high-affinity glucose transport by comprehensive chimera approach. The glucose transporter GLUT1 of mammals belongs to the same major facilitator superfamily as Hxt2 and may therefore show a similar mechanism of substrate recognition. The functional role of Ile²⁸⁷ in human GLUT1, which corresponds to Asn³³¹ in Hxt2, was studied by its replacement with each of the other 19 amino acids. The mutant transporters were individually expressed in a recently developed yeast expression system for GLUT1. Replacement of Ile²⁸⁷ generated transporters with various affinities for glucose that correlated well with those of the corresponding mutants of the yeast transporter. Residues exhibiting high affinity for glucose were medium-sized, non-aromatic, uncharged and irrelevant to hydrogen-bond capability, suggesting an important role of van der Waals interaction. Sensitivity to phloretin, a specific inhibitor for the presumed exofacial glucose binding site, was decreased in two mutants, whereas that to cytochalasin B, a specific inhibitor for the presumed endofacial glucose binding site, was unchanged in the mutants. These results suggest that Ile²⁸⁷ is a key residue for maintaining high glucose affinity in GLUT1 as revealed in Hxt2 and is located at or near the exofacial glucose binding site.     

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.     

Identification by comprehensive chimeric analysis of a key residue responsible for high affinity glucose transport by yeast HXT2

Hxt2 and Hxt1 are, respectively, high affinity and low affinity facilitative glucose transporter paralogs of Saccharomyces cerevisiae. We have previously investigated which amino acid residues of Hxt2 are important for high affinity transport activity. Studies with all the possible combinations of 12 transmembrane segments (TMs) of Hxt2 and Hxt1 revealed that TMs 1, 5, 7, and 8 of Hxt2 are necessary for high affinity transport. Systematic shuffling of the 20 amino acid residues that differ between Hxt2 and Hxt1 in these TMs subsequently identified 5 residues as important for such activity: Leu(59) and Leu(61) (TM1), Leu(201) (TM5), Asn(331) (TM7), and Phe(366) (TM8). We have now studied the relative importance of these 5 residues by individually replacing them with each of the other 19 residues. Replacement of Asn(331) yielded transporters with various affinities, with those of the Ile(331), Val(331), and Cys(331) mutants being higher than that of the wild type. Replacement of the Hxt2 residues at the other four sites yielded transporters with affinities similar to that of the wild type but with various capacities. A working homology model of the chimeric transporters containing Asn(331) or its 19 replacement residues indicated that those residues at this site that yield high affinity transporters (Ile(331), Val(331), Cys(331)) face the central cavity and are within van der Waals distances of Phe(208) (TM5), Leu(357) (TM8), and Tyr(427) (TM10). Interactions via these residues of the four TMs, which compose a half of the central pore, may thus play a pivotal role in formation of a core structure for high affinity transport.     

Degradation of the hexose transporter Hxt5p in Saccharomyces cerevisiae

BACKGROUND INFORMATION: Hxt5p is a member of a multigene family of hexose transporter proteins which translocate glucose across the plasma membrane of the yeast Saccharomyces cerevisiae. In contrast with other major hexose transporters of this family, Hxt5p expression is regulated by the growth rate of the cells and not by the external glucose concentration. Furthermore, Hxt5p is the only glucose transporter expressed during stationary phase. These observations suggest a different role for Hxt5p in S. cerevisiae. Therefore we studied the metabolism and localization of Hxt5p in more detail. RESULTS AND CONCLUSIONS: Inhibition of HXT5 expression in stationary-phase cells by the addition of glucose, which increases the growth rate, led to a decrease in the amount of Hxt5 protein within a few hours. Addition of glucose to stationary-phase cells resulted in a transient phosphorylation of Hxt5p on serine residues, but no ubiquitination was detected. The decrease in Hxt5p levels is caused by internalization of the protein, as observed by immunofluorescence microscopy. In stationary-phase cells, Hxt5p was localized predominantly at the cell periphery and upon addition of glucose to the cells the protein translocated to the cell interior. Electron microscopy demonstrated that the internalized Hxt5p-HA (haemagglutinin) protein was localized to small vesicles, multivesicular bodies and the vacuole. These results suggest that internalization and degradation of Hxt5p in the vacuole occur in an ubiquitination-independent manner via the endocytic pathway.     

Analysis of Saccharomyces cerevisiae hexose carrier expression during wine fermentation: both low- and high-affinity Hxt transporters are expressed

The transport of glucose and fructose into yeast cells is a critical step in the utilization of sugars during wine fermentation. Hexose uptake can be carried out by various Hxt carriers, each possessing distinct regulatory and transport-kinetic properties capable of influencing yeast fermentation capacity. We investigated the expression pattern of the hexose transporters Hxt1 to 7 at the promoter and protein levels in Saccharomyces cerevisiae during wine fermentation. The Hxt1p carrier was expressed only at the beginning of fermentation, and had no role during stationary phase. The Hxt3p carrier was the only one to be expressed throughout fermentation, displaying maximal expression at growth arrest and slowly decreasing in abundance over the course of the stationary phase. The high-affinity carriers Hxt6p and Hxt7p displayed similar expression profiles, with expression induced at entry into stationary phase and persisting throughout the phase. The expression of these two carriers occurred despite the presence of high amounts of hexoses, and the proteins were stably expressed when the cells were starved for nitrogen. The Hxt2p transporter was only transiently expressed during lag phase, which suggests a role for the protein in growth initiation. Characterization of glucose transport kinetics indicated the presence of a shift in the low-affinity component that is consistent with a predominant expression of Hxt1p during growth phase and of Hxt3p during stationary phase. In addition, a high-affinity uptake component consistent with functional expression of Hxt6p/Hxt7p was identified during stationary phase.     

Role of hexose transport in control of glycolytic flux in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.     

Role of Hexose Transport in Control of Glycolytic Flux in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.     

Identification of a Key Residue Determining Substrate Affinity in the Yeast Glucose Transporter Hxt7: A TWO-DIMENSIONAL COMPREHENSIVE STUDY*

We previously identified Asn³³¹ in transmembrane segment 7 (TM7) as a key residue determining substrate affinity in Hxt2, a moderately high-affinity facilitative glucose transporter of Saccharomyces cerevisiae. To gain further insight into the structural basis of substrate recognition by yeast glucose transporters, we have now studied Hxt7, whose affinity for glucose is the highest among the major hexose transporters. The functional role of Asp³⁴⁰ in Hxt7, the residue corresponding to Asn³³¹ of Hxt2, was examined by replacing it with each of the other 19 amino acids. Such replacement of Asp³⁴⁰ generated transporters with various affinities for glucose, with the affinity of the Cys³⁴⁰ mutant surpassing that of the wild-type Hxt7. To examine the structural role of Asp³⁴⁰ in the substrate translocation pathway, we performed cysteine-scanning mutagenesis of the 21 residues in TM7 of a functional Cys-less Hxt7 mutant in conjunction with exposure to the hydrophilic sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS). The transport activity of the D340C mutant of Cys-less Hxt7, in which Asp³⁴⁰ is replaced with Cys, was completely inhibited by pCMBS, indicating that Asp³⁴⁰ is located in a water-accessible position. This D340C mutant showed a sensitivity to pCMBS that was ∼70 times that of the wild-type Hxt7, and it was protected from pCMBS inhibition by the substrates d-glucose and 2-deoxy-d-glucose but not by l-glucose. These results indicate that Asp³⁴⁰ is situated at or close to a substrate recognition site and is a key residue determining high-affinity glucose transport by Hxt7, supporting the notion that yeast glucose transporters share a common mechanism for substrate recognition.     

Regulation of Hxt3 and Hxt7 Turnover Converges on the Vid30 Complex and Requires Inactivation of the Ras/cAMP/PKA Pathway in Saccharomyces cerevisiae

Eukaryotic cells adjust their intracellular protein complement as a mechanism to adapt to changing environmental signals. In Saccharomyces cerevisiae the hexose transporters Hxt3 and Hxt7 are expressed and function on the plasma membrane in high and low glucose abundance, respectively. By contrast, Hxt3 is endocytosed and degraded in the vacuole when cells are starved of glucose and Hxt7 in response to rapamycin treatment or when nitrogen is limiting. Yeast uses several signaling pathways, including the TORC1 and Ras/cAMP/Protein Kinase A (PKA) pathways, to adapt to nutrient changes in the environment. The multi-protein Vid30 complex (Vid30c), an E3 ubiquitin ligase required for the degradation of FBPase, assists in this adaptation process in a mechanism that is poorly understood. Here we show the endocytosis and the subsequent degradation of both Hxt3 and Hxt7, in response to different nutrient signals, is dependent on components of the Vid30c. Additionally, we define the signaling events required for the turnover of Hxt3 and Hxt7 by showing that Hxt3 turnover requires Ras2 and PKA inactivation, whereas Hxt7 turnover requires TORC1 and Ras2 inactivation. Further investigation led us to identify Rim15, a kinase that is inhibited by both the TORC1 and Ras/cAMP/PKA pathways, as a key downstream effector in signaling both turnover events. Finally, we show that the turnover of both Hxt3 and Hxt7 is dependent on the essential E3 ubiquitin ligase, Rsp5, indicating that the role of the Vid30c might be indirect of Hxt ubiquitylation.     

Crucial effects of amino acid side chain length in transmembrane segment 5 on substrate affinity in yeast glucose transporter Hxt7

We previously identified Asp(340) in transmembrane segment 7 (TM7) as a key determinant of substrate affinity in Hxt7, a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae. To gain further insight into the structural basis of substrate recognition by Hxt7, we performed cysteine-scanning mutagenesis of 21 residues in TM5 of a Cys-less form of Hxt7. Four residues were sensitive to Cys replacement, among which Gln(209) was found to be essential for high-affinity glucose transport activity. The 17 remaining sites were examined further for the accessibility of cysteine to the hydrophilic sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS). Among the Cys mutants, T213C was the only one whose transport activity was completely inhibited by 0.5 mM pCMBS. Moreover, this mutant was protected from pCMBS inhibition by the substrate d-glucose and by 2-deoxy-D-glucose but not by L-glucose, indicating that Thr(213) is situated at or close to a substrate recognition site. The functional role of Thr(213) was further examined with its replacement with each of the other 19 amino acids in wild-type Hxt7. Such replacement generated seven functional transporters with various affinities for glucose. Only three mutants, those with Val, Cys, and Ser at position 213, exhibited high-affinity glucose transport activity. All of these residues possess a side chain length similar to that of Thr, indicating that side chain length at this position is a key determinant of substrate affinity. A working homology model of Hxt7 indicated that Gln(209) and Thr(213) face the central cavity and that Thr(213) is located within van der Waals distance of Asp(340) (TM7).