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J. Biol. Chem., Vol. 279, Issue 9, 8116-8125, February 27, 2004

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A Hexose Transporter Homologue Controls Glucose Repression in the Methylotrophic Yeast Hansenula polymorpha^*

Oleh V. Stasyk, Olena G. Stasyk, Janet Komduur¶, Marten Veenhuis¶, James M. Cregg||, and Andrei A. Sibirny**

From the Institute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov Street 14/16, Lviv 79005, Ukraine, the ^¶University of Groningen, Biological Centre, P. O. Box 14, 9750 AA Haren, The Netherlands, the ^||Keck Graduate Institute of Applied Life Sciences, 535 Watson Dr., Claremont, California 91711, and the ^**Rzeszów University, Cegelniana Street 12, 35-310 Rzeszów, Poland

Received for publication, October 6, 2003 , and in revised form, December 1, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Peroxisome biogenesis and synthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha are under the strict control of glucose repression. We identified an H. polymorpha glucose catabolite repression gene (HpGCR1) that encodes a hexose transporter homologue. Deficiency in GCR1 leads to a pleiotropic phenotype that includes the constitutive presence of peroxisomes and peroxisomal enzymes in glucose-grown cells. Glucose transport and repression defects in a UV-induced gcr1-2 mutant were found to result from a missense point mutation that substitutes a serine residue (Ser⁸⁵) with a phenylalanine in the second predicted transmembrane segment of the Gcr1 protein. In addition to glucose, mannose and trehalose fail to repress the peroxisomal enzyme, alcohol oxidase in gcr1-2 cells. A mutant deleted for the GCR1 gene was additionally deficient in fructose repression. Ethanol, sucrose, and maltose continue to repress peroxisomes and peroxisomal enzymes normally and therefore, appear to have GCR1-independent repression mechanisms in H. polymorpha. Among proteins of the hexose transporter family of baker's yeast, Saccharomyces cerevisiae, the amino acid sequence of the H. polymorpha Gcr1 protein shares the highest similarity with a core region of Snf3p, a putative high affinity glucose sensor. Certain features of the phenotype exhibited by gcr1 mutants suggest a regulatory role for Gcr1p in a repression pathway, along with involvement in hexose transport.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
If provided with a mixture of carbon substrates, yeast preferentially utilizes the one that supports the fastest growth rate. This is achieved by several coordinated regulatory mechanisms of metabolic adaptation. They include: (i) the induction of enzymes involved in the metabolism of a preferred substrate and (ii) repression and/or inactivation of enzymes involved in the metabolism of less preferred carbon sources. Carbon source-triggered repression (or catabolite repression) generally affects expression of the corresponding target genes at the transcriptional level. Among co-repressor substrates in Saccharomyces cerevisiae, glucose is best known (for review see Refs. 1 and 2). The main targets of glucose repression in S. cerevisiae are enzymes of gluconeogenesis and the glyoxylate cycle, mitochondrial enzymes involved in oxidative phosphorylation, and enzymes involved in transport and metabolism of alternative carbon substrates, such as galactose, sucrose, and maltose. Despite extensive studies and a growing number of genes known to be involved in glucose repression in this and other species, its actual mechanism, especially in the early stages of sensing and signal transduction, is not fully understood.

In methylotrophic yeasts, unique peroxisomal and cytosolic enzymes of methanol metabolism are under strict control of repression by various carbon substrates, e.g. glucose and ethanol (3, 4). Glucose and ethanol also trigger inactivation of peroxisomal enzymes by a process that involves the autophagic degradation of whole peroxisomes by vacuolar proteases (5–7). Previously, in the methylotrophic yeasts Pichia methanolica (formerly P. pinus) and Candida boidinii, we and others showed that glucose and ethanol repression and degradative inactivation of peroxisomal enzymes are controlled by separate carbon source-specific sets of regulatory genes (4, 8, 9). One complementation group of P. methanolica mutants defective in glucose repression was found to be deficient in phosphofructokinase activity. We proposed that this enzyme has a second signaling function in repression that is distinct from its catalytic activity (8).

The methylotrophic yeast Hansenula polymorpha (syn. Pichia angusta) is an important organism for biotechnological use, e.g. heterologous gene expression, and for basic research on peroxisome biogenesis and degradation (7, 10). There are several reports describing H. polymorpha mutants defective in glucose repression (11–13). Kramarenko et al. (11) demonstrated that glucose has to be phosphorylated in order to cause repression in H. polymorpha, and mutants impaired in activities for hexo- or glucokinases are insensitive to repression. However, the molecular nature of other mutations that impair glucose repression in H. polymorpha has not been determined (12, 13).

In a previous report, we described the isolation and characterization of H. polymorpha mutants in a gene named GCR1¹ that are defective in glucose repression (14).² Glucose-grown cells of gcr1 mutants exhibit pleiotropic alterations in cellular metabolism, namely: (i) the constitutive synthesis of the peroxisomal enzymes, alcohol oxidase (AO) and catalase, and the constitutive presence of peroxisomes; (ii) a decrease in growth rate; and (iii) a decrease in levels of glycolytic intermediates but wild-type levels of activity for each of the primary glycolytic enzymes. In addition, and unlike in wild-type cells, cytosolic enzymes required for methanol metabolism (formaldehyde and formate dehydrogenases) and -glucosidase are not repressed in gcr1 mutants grown in medium containing glucose along with either methanol or maltose, respectively. When shifted from methanol to glucose medium, AO is not inactivated in gcr1 mutants. We suggested that a glucose transport defect might be responsible for the phenotypes displayed by our gcr1 mutants (14, 15). Here, we further characterize the phenotype of gcr1 mutants, describe the cloning and sequence analysis of the GCR1 gene and its product, Gcr1p, the construction of a GCR1 deletion strain, and the identification of a missense mutation in a UV-induced mutant, gcr1-2. Finally, we discuss the possible involvement of Gcr1p in glucose sensing of repression in H. polymorpha.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Strains, Media, and Microbial Techniques—H. polymorpha strains used are listed in Table I. Auxotrophic strains AS8 (leu10), kindly supplied by Dr. P. Sudbery (University of Sheffield), and leu1-1 (both derived from NCYC495), were used in this study as the wild-type strains as indicated. The cells were grown at 37 °C in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) or a minimal medium (0.17% w/v) yeast nitrogen base without amino acids (Difco, Detroit, MI) with 0.5% w/v ammonium sulfate as a nitrogen source. Concentration of carbon sources was 1% if not indicated otherwise. Amino acids were added to a final concentration of 50 µg/ml as required. For solid media, agar was added to 2% (w/v). Sporulation/mating media and techniques were essentially as described (16). Absorbance was determined at 600 nm and yeast cells density calculated as mg dry weight per ml using a calibration curve. Cultivation of Escherichia coli DH5a and standard recombinant DNA techniques were performed essentially as described (17).

To localize a putative mutation in the gcr1-2 mutant allele, a number of pOS22 fragments comprised of different portions of GCR1 ORF were analyzed for ability to functionally complement the corresponding gcr1-2 leu1-1 mutant. The fragments were isolated after digestion of pOS22 with selected restriction enzymes, and as PCR products, with pOS22 as a template and primers: SO53 (5'-AATCGAAGCTCCCTTGAC-3'), SO56 (5'-TTCTTCGTCACCATCGGATT-3'), SO72 (5'-AACACCATGCAAATGTCGAG-3'). The fragments were co-transformed along with plasmid pYT3 (molar concentration ratio of fragment versus plasmid was 1:10 in each case) by electroporation into gcr1-2 leu1-1 strain. Transformants were selected for leucine prototrophy on minimal YNB plates with sucrose. Colonies were further replicated on l-Glc plates, and high, 55 mM glucose plates (h-Glc), for AO colony assay. To isolate the mutated gcr1-2 gene and identify the mutation, the total genomic DNAs of gcr1-2 leu1-1, and original gcr1-2 leu10 mutant were extracted and used as templates in PCR reactions with TaqDNA polymerase (Invitrogen, Life Technologies, Inc.), primers SO58 (5'-CCCAAGCTTAAACGGAGTAAATCCT-3') and SO72. Resulting 2.75-kb fragments were sequenced with the same GCR1-specific primers as used for sequencing of the wild-type gene. To exclude potential PCR amplification mistakes, two independently amplified fragments were sequenced in both directions. In addition, the wild-type fragment isolated by analogous PCR from leu10 parental strain was sequenced and served as a control. Nucleotide sequences were aligned using MacVector software to identify the site of a putative mutation.

Construction of a GCR1 Deletion Strain—A vector capable of deleting most of the HpGCR1 ORF was constructed in two steps. In the first step, a 0.5-kb fragment containing sequences just 5' of the methionine initiator ATG of GCR1 was amplified by PCR using plasmid pOS22 as a template with Vent DNA polymerase (NEB, Beverly, MA). The primers for this PCR, SO58 and SO57 (5'-ATCCTGCAGGTTTGTAATAGTTGATTG-3') included restriction sites for HindIII and PstI, respectively. The 5'-flanking fragment was inserted into HindIII/PstI-digested plasmid pYT1 (18), carrying ScLEU2 gene as a selectable marker, to create pOS28. For the second step, a 1.2-kb SalI-SalI fragment of pOS22 composed of sequences from the 3' terminus of GCR1, beginning at nucleotide 1480 of the ORF and adjusted 3'-flanking region of 1.1-kb was inserted in the correct orientation into SalI-digested pOS28 to create pOS29. The latter plasmid was digested with HindIII and XbaI, releasing a 3.9-kb fragment comprised of ScLEU2 flanked by GCR1 5' and 3' sequences and transformed into the leu1-1 met6 strain by electroporation. This double auxotrophic hybrid was isolated from the spore progeny of a diploid strain resulting from crossing of leu1-1 and met6. Transformants were selected and analyzed for Gcr^- phenotype as described in the main text. To confirm deletion of the GCR1 gene, genomic DNAs were isolated from several Gcr^- transformants and used as templates in PCR reactions with two sets of oligonucleotide primers. One set was composed of primer SO56, complementary to sequences in the 3'-flanking region of the GCR1 ORF, present in both, the wild type and a deletion allele. Second primer, SO72, hybridized to sequences in the 5' region of the wild-type GCR1 ORF that were absent in the gcr1 allele. The other set of primers contained the same 3'-flanking sequence primer SO56 and a second primer complementary to a sequence in ScLEU2, SO90 (5'-TAAGAAGATCGTCGTTTTGCC-3'). The first set of primers produced a 1.3-kb long fragment with only the wild-type genomic DNA as template, while the second set generated a 1.9-kb long fragment with genomic DNAs of each of the putative candidate gcr1-deletion transformants as template, but not with wild-type DNA. One transformant, gcr1::ScLEU2 leu1-1 met6, was utilized throughout this study as a gcr1 deletion strain.

Construction of Strains with Fluorescently Labeled Peroxisomes— pOS18, an E. coli-H. polymorpha shuttle vector capable of expressing a peroxisome-targeted red shifted form of the green fluorescent protein (EGFP-PTS1) under control of the H. polymorpha AO promoter (P_MOX) was constructed. As a first step, plasmid pOGP-2 was constructed by introducing an adapter fragment encoding the last nine amino acids and stop codon of Pichia pastoris Pex8p (including the PTS1 sequence, C-terminal AKL) into a pEGFP-C3 vector (Clontech Laboratories, Inc., Palo Alto, CA) as described (23). It resulted in a chimeric gene encoding EGFP with the last nine amino acids of Pex8p fused in-frame to its C terminus (EGFP-PTS1). As the next step, the EGFP-PTS1 0.8-kb fragment was amplified from pOGP2 by PCR with primers SO40 (5'-GTGAAGCTTATGGTGAGCAAGGGCGAG-3') and SO2 (5'-AGCTTACCGGTTTATAACTTTGCGGTTGATTGGGCG-3') that introduced HindIII flanking sites immediately 5' and 3' of the EGFP fusion gene. The fragment was inserted into the unique HindIII site of pET1 (18) downstream of P_MOX in required orientation to produce pOS18. The latter was linearized in the unique StuI site in the P_MOX sequence and transformed into leu1-1 wild type and gcr1-2 leu1-1 strains. Isolated prototrophic transformants were grown on methanol plates to induce P_MOX, and fluorescence was examined directly in yeast colonies with fluorescent microscope. To identify stable integrants, individual fluorescent clones were examined for mitotic stability by repeated shifting from selective methanol minimal to non-selective YPD medium. Two strains, WT (GFP-PTS1), a wild type, and gcr1-2 (GFP-PTS1) (Table I.) were further utilized in this study. To isolate a gcr1 GFP-PTS1-expressing strain, ade11 (GFP-PTS1) wild-type strain was isolated first in the same way as described above, by transforming leu1-1 ade11 with pOS18. The resulting strain was crossed with a gcr1 met6-null mutant, and recombinant prototrophic gcr1 (GFP-PTS1) was isolated from spore progeny as a fluorescent clone on glucose medium also unable to grow on l-Glc plates.

Biochemical Methods—Preparation of crude cell free extracts was performed as described previously (23). AO activity was measured in cell-free extracts as described (24), and expressed as micromoles of product/min/mg of protein, or in permeabilized whole cells (8), and expressed in micromoles of product/min/g dry weight. Protein concentration was determined by the method of Lowry (25). Extracellular glucose concentration was measured with a glucose oxidase-based enzymatic kit Diagluc (UBT Ltd., Lviv, Ukraine), and methanol concentration in culture media with the alcohol oxidase-based enzymatic kit Alcotest as described (26).

Glucose Uptake Assays—For glucose transport assays, cells were grown on 1% glucose YNB medium until mid-logarithmic phase and harvested at a cell density of 1–1.5 mg dry weight/ml. Cells were washed twice by centrifugation in distilled water at 3,000 x g. Sugar transport was measured at 20 °C, starting with the addition of 0.1 ml of the uniformly labeled [¹⁴C]glucose or [¹⁴C]fructose in a final volume of 0.2 ml, in 0.1 M potassium phosphate buffer (pH 6.0). Cell concentration was 50 mg/ml. Ten seconds later, transport was stopped by adding 8 ml of ice-cold 0.8 M glucose in the same buffer essentially as described (27). Samples were immediately filtered under vacuum and washed twice with 10 ml of ice-cold glucose solution. The treatment of control samples differed in that the cold glucose was added first to the cells and labeled sugar. Thereafter, the reaction was kept at 0 °C. Samples were transferred to scintillation vials with 2 ml of scintillation liquid. A portion of the reaction mixture served as a reference to determine the total radioactivity. The radioactivity was measured with a liquid scintillation counter (Rac-Beta 1219, LKB). The final glucose concentration ranged from 0.5 to 50 mM. The glucose consumption rate (V_Glc) was expressed as grams per hour per gram of dry weight.

Electron and Fluorescence Microscopy—Cells were fixed and prepared for electron microscopy and immunocytochemistry as described previously (28). Immunolabeling was performed on ultrathin sections of Unicryl-embedded cells using specific antibodies against H. polymorpha AO and goat anti-rabbit antibodies conjugated to 15 nm gold particles (Amersham Biosciences) according to the instructions of the manufacturer. Fluorescence microscopy was performed essentially as described (23).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cloning and Sequence Analysis of the GCR1 Gene—The GCR1 gene was isolated by functional complementation of a gcr1-2 leu1-1 mutant (see "Experimental Procedures") with an H. polymorpha genomic DNA library (18). To clone the gene, we made use of the severe growth defect of the mutant at low extracellular glucose concentrations (14, 15). Library transformants were selected simultaneously for leucine prototrophy (Leu⁺) and for restored ability to grow on low glucose (5 mM) agar medium (l-Glc⁺). Four transformants displaying a Leu⁺ l-Glc⁺ phenotype were further examined for AO activity in colonies grown on high (55 mM) glucose plates. All displayed a wild-type phenotype, i.e. full repression of AO synthesis by glucose (Aog^-). To isolate the GCR1 gene, total DNA was extracted from the transformants, and, after transformation of the total genomic DNA preparations into E. coli and amplification, four plasmids were recovered. All four were able to transform the gcr1-2 leu1-1 strain to Leu⁺, l-Glc⁺, and Aog^- phenotypes at high efficiency, suggesting that the plasmids most likely each harbored the complementing GCR1 gene. Restriction mapping of these four plasmids revealed identical 2.0-kb PstI and 1.2-kb SalI fragments in the genomic DNA inserts in each. Both restriction fragments were found to originate from within an 3.3-kb long region of genomic DNA present in one of the plasmids, named pOS22. Subsequent sequence analysis of this fragment revealed a single open reading frame (ORF) of 1,623 bp, the putative GCR1 gene, predicted to encode a polypeptide of 541 amino acids (Fig. 1A). This ORF was subsequently shown to be GCR1 (see below).

View larger version (36K): [in this window] [in a new window]   FIG. 1. Nucleotide and amino acid sequences of the H. polymorpha GCR1 gene and its product. A, twelve predicted membrane-spanning segments (TM 1–12) are numbered and underlined. A putative uORF is underlined, a potential TATA box is shown in bold, and potential binding sites for a Mig1p-like repressor protein are shown in italics in the GCR1 5'-region. B, hydrophobicity profile derived from the predicted amino acid sequence of Gcr1p according to the method of Eisenberg et al. (37) with a window size of 17 amino acids. Hydropathy values are on the y-axis, and the residue numbers are on the x-axis.

View larger version (77K): [in this window] [in a new window]   FIG. 2. Alignment of selected amino acid sequence regions of H. polymorpha Gcr1p, S. cerevisiae Snf3p and Rgt2p, K. lactis Rag4p, and N. crassa Rco3p. A, transmembrane domain regions. The serine residue (S85) altered in the gcr1-2 mutant to phenylalanine by a missense mutation is shown in bold and indicated by an asterisk. Predicted membrane spanning segments designated as TM 1–12 are shown with an overline. B, C-terminal regions. Conserved amino acid residues are shown with black shaded areas indicating identical residues, and light gray areas indicating similar residues.

Cloning of the gcr1-2 Mutant Allele—The phenotype of the gcr1-2 mutant was similar but not identical to that of a gcr1 strain. We determined the molecular nature of the gcr1-2 mutation by isolating the mutated gene from genomic DNA of gcr1-2 by PCR, and sequencing (see "Experimental Procedures" for details). A point mutation was identified that caused a transition from C to T at position 254 of the GCR1 ORF. In addition, only those fragments of pOS22 that contained the N-terminal part of the wild-type GCR1 gene including nucleotide 254, were able to rescue our gcr1-2 mutation when integrated into the mutant genome, thus confirming the 5' location of the site of the mutation in the gene (not shown). The mutation resulted in the substitution of a semi-conserved serine residue (Ser⁸⁵) with a phenylalanine (Fig. 2A). At this position, only one of three amino acid residues is found in hexose transporters: alanine, glycine, or, in a majority of proteins, serine.

Effect of Carbon Substrates on AO Repression and Peroxisome Biogenesis in gcr1 Mutants—Defects in the GCR1 gene lead to synthesis of the peroxisomal enzyme AO in glucose-grown cells (Table II and Ref. 14). The level of AO induction in glucose-grown mutant cells was comparable or higher relative to wild-type cells induced by methanol under analogous conditions (1.7 units/mg). Remarkably, the AO-repression defect was more pronounced in the missense gcr1-2 mutant relative to the gcr1 mutant (Table II). The defects in repression were associated with the presence of AO-containing peroxisomes in mid-exponential glucose-grown cells of gcr1 mutants (Fig. 3 and Ref. 14).

View larger version (149K): [in this window] [in a new window]   FIG. 3. Peroxisome biogenesis in a gcr1 mutant. Electron microscopic images of cells of wild-type strain grown in batch culture with (A) methanol and (B) glucose; C, immunogold detection of AO in glucose-grown gcr1 mutant cell section; D, gcr1 mutant grown in glucose medium. P, peroxisome; V, vacuole; N, nucleus; M, mitochondrion. Bar, 1 micron.

View larger version (59K): [in this window] [in a new window]   FIG. 4. Fluorescence microscopy images of wild-type, gcr1-2, and gcr1 strains expressing peroxisome-targeted EGFP-PTS1 fusion protein on different carbon substrates. Left panels (A–E), mid-exponential cells were analyzed for the presence of fluorescent peroxisomes with an excitation light wavelength of 490 nm. Right panels (A1–E1), the same field of cells in visible light. The same fluorescence intensity setting was used for all images.

View larger version (59K): [in this window] [in a new window]   FIG. 5. Peroxisome degradation in gcr1 mutants. A, Western blot analysis of AO degradation. Cells pregrown in YPS medium were induced in methanol medium for 19 h and shifted to fresh glucose medium. Aliquots were taken at time points 0, 2, 4, and 6 h to measure AO activity and for Western blotting for AO. Equal amounts of protein were loaded per lane. The initial AO activity (time point 0) for the wild-type strain was 2.77 units/mg, gcr1-2; 1.5 units/mg and gcr1, 0.78 units/mg. The residual AO specific activity in time point 6 was for the wild-type strain, 1.5%; gcr1-2, 46%; and gcr1, 35% of the corresponding initial activity. B, electron microscopic image of gcr1 cell 2 h after transfer from methanol medium to fresh glucose medium. Additional membrane enwrapping autophagic peroxisome (specific for macropexophagy) is indicated by arrow. P, peroxisome; V, vacuole; N, nucleus; M, mitochondrion. C, combined fluorescent microscopic images of the wild type and gcr1 cells with GFP-PTS1 labeled peroxisomes and FM64-labeled vacuoles. Time points indicated on the top correspond to methanol-induced cells (0), and those after 2 and 4 h of glucose adaptation.

Glucose Uptake and Consumption in H. polymorpha gcr1-2— The glucose repression defect in the gcr1-2 mutant was accompanied by retarded growth on glucose (14). Since glucose phosphorylation activity was normal in gcr1-2 cells, we suggested that a defect in glucose transport might be the primary cause of mutant catabolite repression deficiency (14, 15). In further investigations of this phenotype, we have determined that the rate of glucose consumption by the gcr1-2 mutant relative to the wild-type strain was decreased at all extracellular sugar concentrations (Fig. 6). The relative difference between the two strains was most pronounced at low extracellular glucose concentrations (e.g. at 5 mM the rate was 3.5-fold slower in the mutant relative to wild type), but this effect diminished with increasing glucose concentrations (e.g. at 55 mM the rate was only 1.3-fold slower than wild type) (Fig. 6). Using [¹⁴C]glucose, we observed that the kinetics of glucose uptake in the gcr1-2 mutant closely matched that of glucose consumption (Fig. 6). In contrast, fructose was transported and consumed by the mutant at wild-type rates (not shown).

View larger version (15K): [in this window] [in a new window]   FIG. 6. Glucose uptake and utilization as a function of extracellular glucose concentration. Glucose uptake (open symbols, dashed lines) and consumption from the medium (solid symbols and lines) were measured in wild-type (circles) and gcr1-2 (triangles) strains as described under "Experimental Procedures." The glucose consumption rate (VGlc) is given in grams per hour per gram of cells (dry weight).

Effect of Extracellular Glucose Concentration on AO Repression in gcr1 Mutants—The deficiency of the gcr1-2 mutant in AO repression is not a function of extracellular glucose concentration and, consequently, glucose uptake. In cells incubated in high-glucose (55 mM) medium, where the transport defect in the gcr1-2 mutant is less pronounced, AO activity was the highest (Table II). In low-glucose (5 mM) medium, where glucose uptake in the gcr1-2 mutant is severely impaired, AO activity was lower. A similar pattern of AO activity levels relative to glucose concentration was also displayed by our gcr1 strain. In the wild-type strain, any of these glucose concentrations was sufficient to completely repress AO synthesis (Table II).

Growth of the gcr1-2 Mutant in Glucose/Methanol Mixtures—Consistent with the classical catabolite repression paradigm (29), a wild-type strain of H. polymorpha utilizes glucose first when incubated in a glucose/methanol mixture, while enzymes of methanol utilization remain repressed. In the gcr1-2 mutant, we observed that the utilization of these carbon sources was reversed, i.e. glucose consumption initiates following exhaustion of methanol from the medium (Fig. 7). Furthermore, methanol, when administrated as a pulse to mid-exponential glucose-grown gcr1-2 cells, transiently blocked growth and glucose utilization of the strain. After a lag-period of several hours, gcr1-2 cells resumed growth, utilizing methanol as carbon substrate (not shown). Addition of methanol to glucose-grown wild-type cells did not affect the growth and substrate utilization pattern. Consistent with our previous data, fructose remained the preferred substrate in the gcr1-2 mutant grown in fructose/methanol mixtures (not shown).

View larger version (18K): [in this window] [in a new window]   FIG. 7. Growth and carbon substrate consumption in glucose and methanol medium mixture. Kinetics of growth (squares), glucose (circles), and methanol (triangles) consumption in wild-type (solid symbols) and gcr1-2 (open symbols) strains grown in batch culture. Initial glucose and methanol concentrations were 55 mM and 1% v/v respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We demonstrated that deficiency in GCR1 does not block autophagic peroxisome degradation upon adaptation of methanol-grown cells to glucose. Nevertheless, gcr1 mutants are capable of preferentially utilizing methanol in the presence of glucose. This fact suggests that glucose metabolism is required for pexophagy to proceed. The gcr1 mutants may serve as a unique model to study molecular mechanisms of peroxisome homeostasis. An intriguing question is how the fate of pre-existing and newly formed peroxisomes is regulated in the mutants under pexophagy-triggering conditions.

A gcr1 mutant retains the capability to grow well on elevated concentrations of glucose, suggesting that other sugar transporter(s) facilitate the uptake of glucose in the absence of Gcr1p. Two kinetically distinct glucose transport systems have been described in H. polymorpha: a high and a low-affinity system (40). Our data suggest that Gcr1p could be primarily involved in high-affinity glucose transport, as growth of gcr1 mutants at glucose concentrations of less than 5 mM is severely hampered. However, to determine whether Gcr1p is, in fact, a functional glucose transporter, further studies are required.

For S. cerevisiae hexose transport mutants, glucose uptake capacity determines the strength of the repression signal (41, 42). However, in our H. polymorpha gcr1-2 mutant, the defect in AO repression did not correlate with a concentration-dependent glucose transport capacity, as AO levels were higher in cells fed with higher concentrations of glucose. Thus, it is possible that Gcr1p somehow functions directly in AO repression, rather than just as a sugar carrier. The phenomenon of methanol inhibition of glucose utilization in the gcr1-2 mutant, also suggests a regulatory function for Gcr1p. We hypothesize that the glucose transport deficiency in the gcr1-2 mutant alone is not sufficient to cause this regulatory effect. Alternatively, this phenomenon may be a common physiological feature of H. polymorpha mutants deficient in glucose repression (13), and may result from altered carbon fluxes through glycolysis and gluconeogenesis pathways.

All closely related potential orthologues of Gcr1p are integral proteins of the plasma membrane and are involved in glucose sensing and/or glucose repression. This sensing/repression function and sequence similarity separates these transporters into their own subgroup within the hexose transporter family (30). The closest GCR1 homologues in S. cerevisiae, ScSNF3, and ScRGT2, are thought to encode high and low affinity glucose sensors, respectively, that are non-functional as sugar carriers. Both gene products are involved primarily in the regulation of glucose transport via differential induction of other hexose transporters in response to changes in extracellular glucose concentrations, but are not thought to act directly in the repression pathway (33, 34). Two other close Gcr1p homologues, K. lactis Rag4p and N. crassa Rco3p, seem to be directly involved in a signaling mechanism for repression, in addition to sugar transport (35, 36). For instance, NcRco3p regulates catabolite repression of conidiation genes, the synthesis of several repressible enzymes, as well as glucose transport in this fungus (36). Like ScSNF3 and ScRGT2, NcRCO3 is expressed at a low level (36). However, there are no conclusive data, as to whether it is a functional transporter or a sensor protein.

HpGcr1p, in contrast with the other putative sensor homologues, ScSnf3p, ScRgt2p, NcRco3p and KlRag4p, lacks an elongated protein-specific C-terminal region (43). For ScSnf3p, this region is functionally essential for glucose signaling (33). A unique short amino acid sequence at the Gcr1p C terminus exhibits similarity to the putative C-terminal "glucose-sensing" domains of ScSnf3p, ScRgt2p, and KlRag4p (Fig. 2B). Although, this similarity is too low to conclude that Gcr1p plays a role in glucose sensing from sequence comparison alone, the concept that Gcr1p is a sensor protein that affects glucose transport via a signaling mechanism similar to ScSnf3p and ScRgt2p is a reasonable working hypothesis.

The mutation in gcr1-2 is predicted to result in the substitution of a serine residue for a phenylalanine at position 85 in the amino acid sequence of Gcr1p. This amino acid change resides in the second predicted transmembrane segment (see Fig. 2A), which overlaps with a leucine zipper motif in many hexose transporters (30). The leucine zipper motif has been proposed to be essential for hetero- or homo-oligomerization of hexose transporters (30). It was also shown that the glucose-sensing function of ScSnf3p requires the presence of at least one of the HXT gene products, suggesting their possible interaction (44). Although this putative leucine zipper motif appears to be rather degenerate in Gcr1p and its closest homologues, Gcr1^S85Fp may be unable to form oligomeric structures or to correctly interact with other downstream components involved in signaling for repression. Alternatively, a mutation in the second TM may lead to cytoplasmic mislocalization of Gcr1p, as demonstrated for one of the ScSnf3p mutants, namely in snf3-72 (45). This question will be addressed in further studies. The second membrane-spanning segment of hexose transporters is not thought to be involved in formation of a channel pore (31).

Remarkably, the glucose repression defect in the gcr1-2 mutant is more profound than in the gcr1 strain. In addition, the gcr1-2 strain is not defective in fructose-mediated repression, while the gcr1 strain is. Similarly, certain mutants in ScSNF3 and NcRCO3 also exhibit phenotypes different from their respective null mutant strains. For example, an rco3¹ mutant was able to conidiate within the agar of solid medium, while the deletion mutant, rco3³, like the wild-type strain, could not. Other glucose-repressible phenotypes were the same between N. crassa rco3 mutants (36). These results can be explained if one assumes that the peculiar and distinct phenotypes of the missense mutants are the result of an abnormal function conferred on the protein, whereas null phenotypes result from the abolition of all functions of that protein.

Results with the gcr1 mutants suggest the existence of specific GCR1-independent repression pathway(s) in H. polymorpha for sucrose, maltose and ethanol. In both the missense gcr1-2 and gcr1 mutants, these substrates continue to repress AO synthesis and to support a wild-type growth rate on these substrates. In contrast, trehalose, mannose, fructose, and xylose seem to have GCR1-dependent repression and transport mechanisms. This observation suggests that, in H. polymorpha, hydrolysis of a disaccharide to hexose residues precedes the transport step for trehalose, but not maltose and sucrose, a conclusion that is consistent with other reports (40).

The sugar-specific phenotype of gcr1 mutants provides an opportunity for their exploitation as hosts for P_MOX-directed expression of heterologous proteins (7), as demonstrated in this report with EGFP-PTS1. Such an expression system would combine the advantages of the strong regulatable P_MOX with the utilization of convenient sugar substrates (i.e. sucrose and glucose, respectively) for growth and induction of gcr1-based production strains, while avoiding the use of toxic and flammable methanol. We have also successfully utilized the glucose repression-deficient mutants derived from gcr1, for AO production in glucose medium (46, 47).

In conclusion, we have identified and characterized a gene essential for glucose repression in H. polymorpha. This gene, GCR1, encodes a hexose transporter homologue. We have also demonstrated that deletion of this gene, or a point mutation that causes a single amino acid substitution relieves repression triggered by several but not all sugar substrates. Our data implicate Gcr1p in an early stage of the repression mechanism, functioning either in glucose transport or glucose signaling.

	FOOTNOTES

 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY465112 [GenBank] .

^* This research was supported by International Association of European Union Grant 01-0583. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported in part by FIRCA Grant TW00547 from the National Institutes of Health, Fogarty International Center.

To whom correspondence should be addressed: Institute of Cell Biology, Drahomanov St. 14/16, Lviv 79005, Ukraine. Tel.: 380-322-740363; Fax: 380-322-721648; E-mail: sibirny{at}biochem.lviv.ua.

¹ The abbreviations used are: GCR, glucose catabolite repression; AO, alcohol oxidase; TM, transmembrane domain; P_MOX, promoter of the AO gene; EGFP, enhanced green fluorescent protein; PTS, peroxisomal targeting signal; ORF, open reading frame.

² The authors chose to use the name of the gene as it appeared in their original reports (14, 15), despite the fact that it coincides with the name of a different S. cerevisiae GCR1 gene, involved in the translational activation of glycolytic proteins.

	ACKNOWLEDGMENTS

 
We thank Dr. A. R. Kulachkovsky for performing part of the transmission electron microscopy studies described in this article.

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