glucoseuptakein kluyveromyceslactis:roleofthe hgt1 gene ... ·...

JOURNAL OF BACTERIOLOGY, Oct. 1996, p. 5860–5866 Vol. 178, No. 200021-9193/96/$04.0010Copyright q 1996, American Society for Microbiology

Glucose Uptake in Kluyveromyces lactis: Role of the HGT1 Genein Glucose Transport

PATRICK BILLARD,1 SANDRINE MENART,1 JOEL BLAISONNEAU,2 MONIQUE BOLOTIN-FUKUHARA,1

HIROSHI FUKUHARA,2 AND MICHELINE WESOLOWSKI-LOUVEL2,3*

Institut de Genetique et Microbiologie1 and Section de Biologie, Institut Curie,2 Centre Universitaire, 91405 Orsay Cedex,and Centre de Genetique Moleculaire et Cellulaire, Universite Claude Bernard, 69622 Villeurbanne Cedex,3 France

Received 17 June 1996/Accepted 12 August 1996

A gene for high-affinity glucose transport, HGT1, has been isolated from the lactose-assimilating yeastKluyveromyces lactis. Disruption strains showed much-reduced uptake of glucose at low concentrations andgrowth was particularly affected in low-glucose medium. The HGT1 nucleotide sequence implies that it encodesa typical transmembrane protein with 12 hydrophobic domains and with 26 to 31% amino acid identity withthe Hxtp family of glucose transport elements in Saccharomyces cerevisiae. Expression is constitutive (incontrast to RAG1, the major gene for low-affinity glucose uptake in K. lactis) and is controlled by several genesalso known to affect expression of RAG1. These include RAG5 (which codes for the single hexokinase of K.lactis), which is required for HGT1 transcription, and RAG4, which has a negative effect. The double mutantDhgt1Drag1 showed further reduced glucose uptake but still grew quite well on 2% glucose and was notcompletely impaired even on 0.1% glucose.

Eukaryotic and bacterial sugar transport proteins are relatedin protein sequence and cellular topology, forming the sugarpermease superfamily (1). Within this superfamily of proteinsare found transporters that act either by facilitated diffusion orby proton symport mechanisms (17). In the case of the yeastSaccharomyces cerevisiae, it is widely accepted that monosac-charides enter the cell by facilitated diffusion, and there are atleast nine candidate structural genes for glucose permeases:SNF3 (2, 3, 23); HXT1 (18); HXT2 (LGT2) (16, 34a); HXT3(15); HXT4 (LGT1) (25, 30); HXT5, HXT6, and HXT7 (27);and LGT3 (HXT11) (34a). (The existence of such a multigenefamily of glucose transporters in this yeast resembles the situ-ation the mammalian systems [12], in which six glucose carriershave already been identified.) Proof of their individual func-tions is difficult and is still being obtained (15, 27). They arealso classifiable as to their affinities for glucose and inducibili-ties; these aspects are also complex and being studied (15, 24,32, 33).In Kluyveromyces lactis, the situation may be simpler, with

only one low-affinity, inducible glucose transporter, encoded byRAG1 (11, 36), and a high-affinity uptake system which isconstitutively expressed (36). The expression of RAG1 is nec-essary for growth on high-glucose medium (5%) in the pres-ence of the respiratory inhibitor antimycin A (Rag1 pheno-type) (10); rag1 mutants are unable to grow on high-glucosemedium in the absence of respiration (Rag2 phenotype). Mu-tations of various genes involved in glycolysis also result in theRag2 phenotype (37). Among such mutants, we have identifiedrag4, rag5, and rag8, in which the induction of RAG1 transcrip-tion was impaired during growth on high-glucose medium (5).One of these trans-acting genes, RAG5, is the structural genefor the hexokinase, which is the only glucose-phosphorylatingenzyme in K. lactis (26).We describe here a new K. lactis gene, HGT1 (for high-

* Corresponding author. Mailing address: Centre de GenetiqueMoleculaire et Cellulaire, Universite Claude Bernard, Batiment 405,Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.Phone: 33 72 43 16 97. Fax: 33 72 43 11 81. Electronic mail address:[email protected].

TABLE 1. Yeast strains used

Strain Genotype Reference or source

K. lactis2359/152 MATa metA1-1 Rag1 34MW270-7B MATa metA1-1 uraA1-1

leu2 Rag1M. Wesolowski-Louvel Collection

MW270-7B/Dh1 MATa metA1-1 uraA1-1leu2 hgt1::lacZ LEU2aRag1

This work

MW270-7B/Dh2 MATa metA1-1 uraA1-1leu2 hgt1::LEU2bRag1

This work

MW270-7B/Dr MATa metA1-1 uraA1-1leu2 rag1::URA3cRag2

This work

MW270-7B/Dh2 Dr MATa metA1-1 uraA1-1leu2 hgt1::LEU2brag1::URA3c Rag2

This work

2360/7 MATa lysA1-1 rag1-1Rag2

A. Algeri

MW109-8C/FA49 MATa lysA1-1 rag4-5Rag2

M. Wesolowski-Louvel Collection

PM6-7A/VV41 MATa uraA1-1 adeT-600d rag5-1 Rag2

5

MW109-8C/FA42 MATa lysA1-1 rag8-2Rag2

5

MW239-13B MATa uraA1-1 rag4-5rag5-1

M. Wesolowski-Louvel Collection

S. cerevisiaeMCY1409 MATa snf3-D4::HIS3

his3-D200 ura3-52lys2-801 ade2-101SUC2

M. Carlson

W303-1A MATa SUC2 ade2-1can1-100 his3-11,15trp1-1 leu2-3,112ura3-1

31

a Insertion mutation; transplacement of chromosomal HGT1 by hgt1::lacZLEU2 (see Materials and Methods).b Substitution mutation; internal deletion of chromosomal HGT1 replaced by

LEU2 marker (see Materials and Methods).c Substitution mutation; internal deletion of chromosomal RAG1 replaced by

URA3 marker (36).d Previously ade1 (35).

5860

on January 14, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

http://jb.asm.org/

glucose transporter), which may code for the major, or eventhe sole, high-affinity glucose carrier in this yeast. Like RAG1,the expression of HGT1 is under the control of several RAGgenes: RAG5 positively controls transcription, and RAG4 has anegative effect. Dhgt1Drag1 double mutants are very markedlyimpaired in glucose uptake compared with the wild type.

MATERIALS AND METHODS

Strains and plasmids. Table 1 lists the yeast strains used in this study. Agenomic library of K. lactis was constructed on a multicopy vector, KEp6 (38),from the DNA of the standard Rag1 strain 2359/152 (Table 1).The centromeric vector YCp50 (28) and the ARS1-containing vector pAB107

(a gift from F. Sherman, University of Rochester, Rochester, N.Y.) were used forintroducing the HGT1 gene into S. cerevisiae. SNF3-containing plasmid pBL8(20), kindly provided by M. Carlson (Columbia University, New York, N.Y.), wasused to generate an SNF3 probe.Culture conditions and genetic methods. Yeasts were grown at 288C either in

a complete medium containing 1% yeast extract–1% Bacto Peptone (Difco,Detroit, Mich.) supplemented with 2% glucose or in a minimal medium con-taining 0.7% Yeast Nitrogen Base (Difco) without amino acids, with auxotrophicsupplements as required, and with a specified carbon source.Genetic methods have been described previously (10, 29, 34).Yeast transformation. Replicative transformation of K. lactis was performed

by electroporation (21) using an electropulsator, model TRX GHT 1287 (Jouan,Toulouse, France). For integrative transformation of K. lactis, the proceduredescribed by Dohmen et al. (8) was followed. S. cerevisiae was transformed by alithium acetate method (14). Complementation of the snf3 mutation of S. cer-evisiae was defined as restoration of growth on 2% raffinose minimal medium inthe presence of antimycin A (1 mg/ml) (23).Cloning of HGT1. The entire HGT1 gene of K. lactis was cloned by screening

the genomic library of K. lactis, using as a probe the 630-bp ClaI internalfragment of the partial HGT1 gene, which had been cloned as part of the 2C5plasmid (Fig. 1). Ten positive clones (out of 2 3 104 E. coli transformant clonesplated) were obtained; these defined three classes of plasmids with overlappinginserts of 7.0 and 8.5 kbp.Preparation of yeast RNA, Northern (RNA) blot analysis, and DNA manipu-

lation. Standard procedures for nucleic acid manipulation were essentially thosecompiled by Maniatis et al. (19). Total RNA was extracted from cells grown toan A600 of about 2 to 3. RNA was fractionated by electrophoresis on a 1.2%agarose-formaldehyde gel. Scanning of the Northern blots was performed byusing a Phosphorimager 425 (Molecular Dynamics). For sequencing, DNA frag-ments were cloned into pBluescript vectors (Stratagene, La Jolla, Calif.) andprocessed by standard techniques using both the M13 universal and reverseprimers (Pharmacia-France, Les Ulis, France) and T7 DNA polymerase (Phar-macia).Measurement of glucose uptake. The method used to measure glucose uptake

was essentially based on that established by Walsh et al. (32) with slight modi-

fications. Cells were grown to an A600 of 2.0 in 2% glucose complete medium,harvested by centrifugation, washed with 100 mM potassium phosphate buffer(pH 6.5), and suspended in the same buffer to a cellular density of 60 to 100 mg(wet weight) ml21. Eighty microliters of cell suspension was mixed with 20 ml ofD-[U-14C]glucose at final concentrations ranging from 0.2 to 160 mM.Uptake activity at each concentration was determined at 258C after 5 s of

incubation. The reaction was quenched by addition of chilled (08C) 100 mM

FIG. 1. Restriction maps of recombinant plasmids carrying the HGT1 gene. In the 2C5 plasmid, the coding sequence (black box) is interrupted by the mini-Muelement MudIIZKI, leading to a fusion of the 59 region of HGT1 with the lacZ gene. The hatched box corresponds to pBR322 DNA. The pJM202 restriction maprepresents the shortest DNA clone containing the entire HGT1 gene among the clones detected by hybridization with probe 1 of the 2C5 plasmid. The diagram at thebottom represents the strategy used for the disruption of theHGT1 locus (see Materials and Methods). The hatched box corresponds to the LEU2 fragment. The arrowsindicate the direction of transcription. Abbreviations: B, BamHI; Bg, BglII; C, ClaI; E, EcoRI; H, HindIII; P, PstI; S, SalI; EV, EcoRV; Hp, HpaI. Restriction sites inbrackets are those which have been eliminated by ligation.

FIG. 2. Eadie-Hofstee plot showing glucose uptake in the wild-typeMW270-7B strain and in the Drag1, Dhgt1, and Drag1Dhgt1 null mutants. TheDhgt1 mutant used in the present experiment was MW270-7B/Dh2 strain(hgt1::LEU2 substitution mutation); similar results were obtained with thehgt1::lacZ mutant (not shown). Glucose uptake was measured as described inMaterials and Methods. Uptake rate (V) is expressed as nanomoles of glucoseper milligram (dry cell weight) per minute; glucose concentration (S) is millimo-lar.

VOL. 178, 1996 HIGH-AFFINITY GLUCOSE TRANSPORT IN K. LACTIS 5861


.org/D

ownloaded from

http://jb.asm.org/

potassium phosphate buffer (pH 6.5) containing 500 mM nonlabelled glucose,and the cells were rapidly collected on glass-fiber filters and washed twice with 10ml of the same buffer. As controls, 0-s incubations were performed for eachdifferent substrate concentration tested. For these incubations the cell suspen-sion was added to 10 ml of chilled 100 mM potassium phosphate buffer (pH 6.5)containing the labelled glucose. D-[U-14C]glucose was purchased from Dupont/New England Nuclear (Les Ulis, France).Disruption of HGT1 and RAG1 genes. Two types of hgt1 mutants were con-

structed.(i) In the 2C5 plasmid, the HGT1 gene sequence was interrupted by fusion

with the lacZ gene carried by the mini-Mu MudIIZK1 transposon (Fig. 1) (21a).

This transposon also carried the S11 fragment, which promotes replication in K.lactis (4), and the auxotrophic marker LEU2 from S. cerevisiae, which comple-ments the leu2 mutation of K. lactis. After removal of the S11 fragment, theconstruction could be used directly for integrative transformation in the form ofa NotI cassette. The plasmid was linearized by BglII and SalI cuts (Fig. 1). Theresulting 17-kbp fragment was used to replace the HGT1 wild-type chromosomallocus with the hgt1-lacZ fusion by transformation of the strain MW270-7B toleucine prototrophy. The resulting strain is referred as MW270-7B/Dh1 (Table1).(ii) Once the entire HGT1 gene was cloned, a true null mutant was con-

structed. The 2.4-kbp EcoRI-HindIII fragment of the pJM202 plasmid (Fig. 1)

FIG. 3. Nucleotide and deduced amino acid sequences of the HGT1 gene. Numbers at the left correspond to the nucleotide position relative to the A of the putativeATG translational start codon; numbers at the right correspond to the codon positions relative to that ATG. In the 59 untranslated region, the MIG1-target consensussequence is in italic boldface letters and the 8- and 10-bp direct repeats are underlined with dotted lines and double lines, respectively. In the coding portion, highlyhydrophobic domains are in boldface letters and potential N-glycosylation sites are underlined.

5862 BILLARD ET AL. J. BACTERIOL.


.org/D

ownloaded from

http://jb.asm.org/

was cloned into the pBluescript KS1 phagemid (Stratagene). The internal 680-bpHpaI-EcoRV fragment was then replaced by a 2.0-kbp HpaI fragment fromYEp13 vector, containing the LEU2 gene of S. cerevisiae (Fig. 1). The 3.4-kbpfragment generated by PstI digestion of the resulting plasmid was used to trans-form the two strains MW270-7B and MW270-7B/Dr to leucine prototrophy. Thetransformants thus obtained, MW270-7B/Dh2 and MW270-7B/Dh2 Dr (Table 1),both contain an hgt1 null allele.The RAG1 gene was disrupted by using the strategy described previously (38).Nucleotide sequence accession number. The HGT1 nucleotide sequence has

been assigned GenBank accession number U22525.

RESULTS AND DISCUSSION

HGT1 gene product is necessary for high-affinity glucoseuptake. The HGT1 gene was obtained as a lacZ fusion (plas-mid 2C5, Fig. 1) in the course of screening for strongly ex-pressed K. lactis genes (21a). The screening process used apromoter probe system based upon translational gene fusionsbetween genomic DNA fragments of K. lactis and the lacZgene of Escherichia coli. A partial nucleotide sequence of thefused gene, corresponding to the 287 N-terminal amino acidsfused to the lacZ gene, established that its putative product wasa hydrophobic transmembrane protein, sharing approximately25 to 30% identity with Snf3p, Hxt1p, and Hxt2p glucose per-meases of S. cerevisiae (3, 18, 16) and with the low-affinityglucose carrier Rag1p of K. lactis (11, 36).

To determine whether HGT1 affected glucose transport,chromosomal HGT1 was replaced by hgt1::lacZ LEU2 (seeMaterials and Methods); this was confirmed by Southern anal-ysis (data not shown). Genetic cross of one of these Leu1

transplacents with a leu2 strain gave 2:2 segregation of leu2 in22 tetrads dissected, indicating that the LEU2 integration tookplace at a single locus in the genome.The kinetics of glucose uptake as a function of glucose

concentration were examined (Fig. 2). The parental wild-typestrain MW270-7B displayed the same pattern of glucose up-take as was observed with other wild-type strains previouslytested (36), i.e., there were components of both low affinity(Km of about 40 mM, steep slope) and high affinity (Km ofabout 1 mM, shallow slope). (The low-affinity component waspresent only in glucose-grown cells [data not shown].) Thehgt1::lacZ mutant (Fig. 2) showed only the low-affinity compo-nent, suggesting that the HGT1 gene product would be themajor or even the sole high-affinity glucose carrier, or at leasta positive regulator of high-affinity transport.Isolation of the whole HGT1 gene and deduced amino acid

sequence of its product. Plasmids carrying the entire HGT1DNA gene sequence were isolated from a K. lactis genomiclibrary by colony hybridization as described in Materials andMethods. One of these plasmids (pJM202) with a 7.0-kbpinsert is shown in Fig. 1. The nucleotide sequence of a 2.4-kbpEcoRI-HindIII DNA fragment revealed a single open readingframe of 552 codons (Fig. 3) similar in size and predictedstructure (including 12 hydrophobic regions postulated to betransmembrane segments) to the transporter family. Hgt1pwould share 26 to 28% identity with the glucose carriers of theHxtp family (Hxt1, -2, -3, -4, -5, -6, and -7p), including Rag1pof K. lactis itself, the glucose transporter GlcP from the cya-nobacterium Synechocystis sp. strain PCC 6803 (39), and thequinate permeases Qa-y and Qut-d from Neurospora crassaand Aspergillus nidulans, respectively (9, 13). In addition, 31%identity was found with Snf3p of S. cerevisiae (which seems tohave regulatory roles in high-affinity glucose transport systembut may not itself be a transporter [1, 6, 23].) Snf3p (884 aminoacids long) possesses a large carboxy-terminal tail of 300 aminoacids which was not found in Hgt1p. Two 8-bp and three 10-bpperfect direct repeats between positions 225 and 2482 werefound in the 59-untranslated region of the gene (Fig. 3). Nu-cleotide sequence analysis also revealed the presence of themotif 59-CCCCAGACTAA-39 at position 2294 to 2304,which is almost identical (one base difference) to the consensusbinding site for the yeast protein Mig1p, WWWWTSYGGGG(22).In spite of the sequence similarity, HGT1 could not restore

the growth of a snf3 null mutant (strain MCY 1409) (Table 1)on raffinose-antimycin A media (23) either on centromericvector YCp50 or on multicopy vector pAB107 (see Materialsand Methods). Even in multiple copies, HGT1 was unable torestore the Rag1 phenotype to a rag1 mutant of K. lactis.(Indeed, when we cloned the RAG1 gene by complementationof rag1 using a multicopy genomic library of K. lactis, only theRAG1 gene was isolated [36].) This latter result shows thatHGT1 and RAG1 have distinct functions in glucose transport.Expression of HGT1. As mentioned previously, high-affinity

glucose uptake in K. lactis is constitutive (36). This is also trueof expression of the HGT1 gene (Fig. 4A). Interestingly, twoconstitutive mRNAs were found. The most abundant, of 2.0kb, had mRNA/actin ratios of 1.5 and 2 for glycerol/lactate andglucose, respectively, and the other, of 2.6 kb, had respectivemRNA/actin ratios of 0.5 and 0.4. The two RNA species weresimultaneously lost in the hgt1::LEU2mutant (Fig. 4A, lane 4).In the hgt1::lacZ mutant (lane 3), a larger transcript was ob-

FIG. 4. Northern blot analysis of HGT1 mRNA. Each slot was loaded withapproximately 15 mg of total RNA prepared and electrophoresed as described inMaterials and Methods. The probe used to detect the HGT1 mRNA was theinternal probe 1 (Fig. 1) radiolabelled by random priming by using the Oligola-belling Kit from Pharmacia. The actin gene from K. lactis (7) was used as aninternal reference. (A) The wild-type strain 2359/152 was grown either on 2%glycerol (lane 1) or on 2% glucose (lane 2) complete medium (the same resultwas observed with the parental wild-type strain MW270-7B [not shown]); the twohgt1 mutants MW270-7B/Dh1 (lane 3) and MW270-7B/Dh2 (lane 4) were grownon 2% glucose. (B) Northern blots of rag mutants grown on 2% glucose.



.org/D

ownloaded from

http://jb.asm.org/

served, perhaps corresponding to the fusion product of the 59half of HGT1 with lacZ.The low-affinity carrier gene RAG1 is regulated at the tran-

scriptional level by several RAG genes (5). Therefore, we in-vestigated whether the expression of HGT1 was also affectedby these rag mutations (37) in glucose-grown cultures (Fig.4B). While the level of the 2.6-kb mRNA did not significantlyvary in all of the rag mutants tested, the level of the 2.0-kbspecies was severely decreased in rag5 (hexokinase) mutants(26). On the other hand, this transcript was increased in rag4mutants, indicating a negative control of HGT1 expression byRAG4. A rag4 rag5 double mutant showed a high level ofHGT1 transcript, indicating that rag4 is epistatic on rag5 in thetranscriptional regulation of HGT1. In contrast, mutations ofthe RAG8 gene (coding for a casein kinase I homolog [unpub-lished data]) required for RAG1 transcription did not modifythe transcription of HGT1. HGT1 transcription was normal inthe rag1 mutant (Fig. 4B), and RAG1 expression was unaf-fected in the hgt1 null mutant (data not shown). These resultsindicate that the two likely permease genes are similarly af-fected by the hexokinase gene but are differently regulated byother genes.Southern blots. Low-stringency Southern hybridization (Fig.

5A) with an internal radioactive probe of HGT1 (630-bp ClaIfragment) revealed only a single band in K. lactis correspond-ing to HGT1. This finding indicates that HGT1 is present inonly a single copy in the K. lactis genome and reinforces theidea that the two detected mRNAs originate from HGT1.No signal was observed with S. cerevisiae from the HGT1

probe (Fig. 5A); an SNF3 probe, under identical low-strin-gency conditions, gave a single band of expected size with S.cerevisiae DNA and no signal with K. lactis DNA (Fig. 5B),

even after prolonged film exposure. Thus, no close HGT1homolog exists in S. cerevisiae, and no close homolog for SNF3exists in K. lactis.Hybridization of RAG1 or HXT4 probes under the same

conditions of stringency gave multiple bands with genomicDNA from S. cerevisiae corresponding to the multiple HXTgenes, and a single signal (the RAG1 gene) was detected withK. lactis DNA (24a).rag1 and rag1 hgt1 mutants. Starting with the wild-type

strain MW270-7B (Table 1) and using various in vitro con-structions (see Materials and Methods), an isogenic set ofsingle and double null mutants for RAG1 and HGT1 genes wasconstructed (Table 1). As referred to above, single null mu-tants hgt1::lacZ (MW270-7B/Dh1) and hgt1::LEU2 (MW270-7B/Dh2) showed an apparent absence of high-affinity uptake.Analogously, as was already known (36), Drag1 (MW270-7B/Dr) lacked low-affinity transport (Fig. 2). Under these condi-tions, the double null mutant Drag1Dhgt1 (MW270-7B/Dh2 Dr)showed a highly reduced level of glucose uptake, whether thecells were grown on 2% glucose (Fig. 2) or on glycerol medium(not shown). Nevertheless, some low-affinity, low-capacity glu-cose transport was still detected (Fig. 2), which would contrib-ute only marginally to the global glucose uptake rate in thewild type. If this weak glucose uptake is due to a yet-unknownsugar carrier, its gene would not have a sequence closely re-lated to that of HGT1 or RAG1.As mentioned above, the rag1 strains fail to grow on en-

riched medium with 5% glucose in the presence of antimycinbut grow well in its absence. hgt1 strains grow well in this typeof medium, and hgt1 does not restore antimycin insensitivity toa rag1 strain (data not shown).In minimal medium (without antimycin) (Fig. 6), the Dhgt1

FIG. 5. Low-stringency Southern blot analysis of the HGT1 and SNF3 genes. Genomic DNA of strains 2359/152 of K. lactis (lanes 3 and 5) and W303-1A of S.cerevisiae (lanes 2 and 4) were digested with EcoRI (lanes 2 and 3) or EcoRI plus PstI (lanes 4 and 5), electrophoresed on an 0.8% agarose gel, and transferred to anitrocellulose membrane. Lane 1 contains l DNA digested with HindIII. (A) The 630-bp ClaI fragment of HGT1 was radiolabelled by random priming and hybridizedto filter-bound DNA in 63 SSC–50% formamide at 428C for 15 h (13 SSC is 0.15 M NaCl–0.015 M sodium citrate). The blot was washed twice with 23 SSC–0.1%sodium dodecyl sulfate (SDS) at room temperature for 20 min and then twice with 13 SSC–0.1% SDS for 20 min at room temperature. (B) The same Southern blotas in panel A was stripped and then hybridized with the radiolabelled 2.4-kb BamHI-EcoRI from plasmid pBL8 containing the whole coding sequence of the SNF3gene of S. cerevisiae.



.org/D

ownloaded from

http://jb.asm.org/

null mutant (MW270-7B/Dh2) had a doubling time of 11 hcompared with 6 h for the parental strain MW270-7B on 0.1%glucose medium, while growth on 2% glucose was indistin-guishable from that of the wild type. Drag1 (MW270-7B/Dr)grew on 2% glucose-containing medium with a doubling timesimilar to that of the wild type, ca. 3.5 h, and also grew nor-mally on 0.1% glucose medium (doubling time, ca. 6 h). On0.1% glucose medium, the double null mutant (MW270-7B/Dh2 Dr) appeared to have an extended growth lag and partic-ularly low yield; on 2% glucose, it had a doubling time of 4 hbut lower yield. Thus, the double mutant still grew quite wellon glucose (implying adequate transport) at 2% concentrationand was not totally impaired even on 0.1% glucose.In summary, the present weight of the evidence suggests that

HGT1 likely governs or specifies a major apparent high-affinityglucose uptake system in K. lactis and that RAG1 specifies amajor or sole low-affinity glucose uptake system. The situationappears to be less complex than that in S. cerevisiae. However,the relatively rapid growth of the double mutant on 2% glucoseimplies other as yet undetected components of glucose uptake.Expression of uptake in K. lactis is also complicated, with therole of hexokinase (rag5) being of particular interest.

ACKNOWLEDGMENTS

We thank Reinhard Fleer for useful discussions and David Law-rence for critical reading of the manuscript. We also thank MarianCarlson for giving us the pBL8 plasmid.This research was supported in part by the European Union contract

BIOT CT91-0287. Sandrine Menart and Patrick Billard were respec-tively the recipients of pre- and post-doctoral Rhone-Poulenc-Rorerfellowships.

REFERENCES

1. Bisson, L. F., D. M. Coons, A. L. Kruckeberg, and D. A. Lewis. 1993. Yeastsugar transporters. Crit. Rev. Biochem. Mol. Biol. 28:259–308.

2. Bisson, L. F., L. Neigeborn, M. Carlson, and D. G. Fraenkel. 1987. The SNF3gene is required for high-affinity glucose transport in Saccharomyces cerevi-siae. J. Bacteriol. 168:1656–1662.

3. Celenza, J. L., L. Marshall-Carlson, and M. Carlson. 1988. The yeast SNF3

gene encodes a glucose transporter homologous to the mammalian protein.Proc. Natl. Acad. Sci. USA 85:2130–2134.

4. Chen, X. J., M. Saliola, C. Falcone, M. M. Bianchi, and H. Fukuhara. 1986.Sequence organization of the circular plasmid pKD1 from the yeastKluyveromyces drosophilarum. Nucleic Acids Res. 14:4471–4481.

5. Chen, X. J., M. Wesolowski-Louvel, and H. Fukuhara. 1992. Glucose trans-port in the yeast Kluyveromyces lactis. II. Transcriptional regulation of theglucose transporter gene RAG1. Mol. Gen. Genet. 33:97–105.

6. Coons, D. M., R. B. Boulton, and L. F. Bisson. 1995. Computer-assistednonlinear regression analysis of the multicomponent glucose uptake kineticsof Saccharomyces cerevisiae. J. Bacteriol. 177:3251–3258.

7. Deshler, J. O., G. P. Larson, and J. J. Rossi. 1989. Kluyveromyces lactismaintains Saccharomyces cerevisiae intron-encoded splicing signals. Mol.Cell. Biol. 9:2208–2213.

8. Dohmen, R. J., A. W. M. Strasser, C. B. Honer, and C. P. Hollenberg. 1991.An efficient transformation procedure enabling long-term storage of com-petent cells of various yeast genera. Yeast 7:691–692.

9. Geever, R. F., L. Huiet, J. A. Baum, B. M. Tyler, V. B. Patel, B. J. Rutledge,M. E. Case, and N. H. Giles. 1989. DNA sequence, organization and regu-lation of the qa gene cluster of Neurospora crassa. J. Mol. Biol. 207:15–34.

10. Goffrini, P., A. A. Algeri, C. Donnini, M. Wesolowski-Louvel, and I. Ferrero.1989. RAG1 and RAG2: nuclear genes involved in the dependence/indepen-dence on mitochondrial respiratory function for the growth on sugars. Yeast5:99–106.

11. Goffrini, P., M. Wesolowski-Louvel, I. Ferrero, and H. Fukuhara. 1990.RAG1 gene of the yeast Kluyveromyces lactis codes for a sugar transporter.Nucleic Acids Res. 18:5294.

12. Gould, G. W., and G. I. Bell. 1990. Facilitative glucose transporters: anexpanding family. Trends Biochem. Sci. 15:18–23.

13. Hawkins, A. R., H. K. Lamb, M. Smith, J. W. Keyte, and C. F. Roberts. 1988.Molecular organisation of the quinine acid utilization (QUT) gene cluster inAspergillus nidulans. Mol. Gen. Genet. 214:224–231.

14. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation ofintact yeast cells treated with alkali cations. J. Bacteriol. 153:163–168.

15. Ko, C. H., H. Liang, and R. F. Gaber. 1993. Role of multiple glucosetransporters in Saccharomyces cerevisiae. Mol. Cell. Biol. 13:638–648.

16. Kruckeberg, A. L., and L. F. Bisson. 1990. The HXT2 gene of Saccharomycescerevisiae is required for high-affinity glucose transport. Mol. Cell. Biol.10:5903–5913.

17. Lagunas, R. 1993. Sugar transport in Saccharomyces cerevisiae. FEMS Mi-crobiol. Rev. 104:229–242.

18. Lewis, D. A., and L. F. Bisson. 1991. The HXT1 gene product of Saccharo-myces cerevisiae is a new member of the family of hexose transporters. Mol.Cell. Biol. 11:3804–3813.

19. Maniatis, F., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: alaboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Har-bor, New York.

FIG. 6. Growth curves of the wild type, Drag1, Dhgt1, and Drag1Dhgt1 null mutants. Strains were grown at 288C on minimal medium (0.7% yeast nitrogen basewithout amino acids) completed with respect to auxotrophic markers and containing glucose. Growth was monitored by measuring A600 at the times indicated. (A)Growth on 2% glucose-containing medium. (B) Growth on 0.1% glucose-containing medium. Symbols: F, MW270-7B (RAG1 HGT1); Ç, MW270-7B/Dr (rag1::URA3HGT1); E, MW270-7B/Dh2 (RAG1 hgt1::LEU2); h, MW270-7B/Dh2Dr (rag1::URA3 hgt1::LEU2).



.org/D

ownloaded from

http://jb.asm.org/

20. Marshall-Carlson, L., J. L. Celenza, B. C. Laurent, and M. Carlson. 1990.Mutational analysis of the SNF3 glucose transporter of Saccharomyces cer-evisiae. Mol. Cell. Biol. 10:1105–1115.

21. Meilhoc, E., J. M. Masson, and J. Teissie. 1990. High-efficiency transforma-tion of intact yeast cells by electric field pulses. Bio/Technology 8:223–227.

21a.Menart, S. 1993. Ph.D. thesis, Universite de Paris XI. Orsay, France.22. Nehlin, J. O., M. Carlberg, and H. Ronne. 1991. Control of yeast GAL genes

by MIG1 repressor: a transcriptional cascade in the glucose response.EMBO J. 10:3373–3377.

23. Neigeborn, L., P. Schwartzberg, R. Reid, and M. Carlson. 1984. Null muta-tions in the SNF3 gene of Saccharomyces cerevisiae cause a different pheno-type than do previously isolated missense mutations. Mol. Cell. Biol. 6:3569–3574.

24. Ozcan, S., and M. Johnston. 1995. Three different regulatory mechanismsenable yeast hexose transporter (HXT) genes to be induced by differentlevels of glucose. Mol. Cell. Biol. 15:1564–1572.

24a.Prior, C. 1993. Ph.D. thesis. Universite de Parix XI, Orsay, France.25. Prior, C., H. Fukuhara, J. Blaisonneau, and M. Wesolowski-Louvel. 1993.

Low-affinity glucose carrier gene LGT1 of Saccharomyces cerevisiae, a ho-molog of Kluyveromyces lactis RAG1 gene. Yeast 9:1373–1377.

26. Prior, C., P. Mamessier, H. Fukuhara, X. J. Chen, and M. Wesolowski-Louvel. 1993. The hexokinase gene is required for the transcriptional regu-lation of the glucose transporter gene RAG1 in Kluyveromyces lactis. Mol.Cell. Biol. 13:3882–3889.

27. Reifenberger, E., K. Freidel, and M. Ciriacy. 1995. Identification of novelHXT genes in Saccharomyces cerevisiae reveals the impact of individualhexose transporters on glycolytic flux. Mol. Microbiol. 16:157–167.

28. Rose, M. D., P. Novick, J. H. Thomas, D. Botstein, and G. R. Fink. 1987. ASaccharomyces cerevisiae genome plasmid bank based on a centromere-containing shuttle vector. Gene 60:237–243.

29. Sherman, F., G. R. Fink, and J. B. Hicks. 1986. Laboratory course manualfor methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold

Spring Harbor, New York.30. Theodoris, G., N. M. Fong, D. M. Coons, and L. F. Bisson. 1994. High-copy

suppression of glucose transport defects by HXT4 and regulatory elements inthe promoters of HXT genes in Saccharomyces cerevisiae. Genetics 137:957–966.

31. Thomas, B. J., and R. Rothstein. 1989. Elevated recombination rates intranscriptionally active DNA. Cell 56:619–630.

32. Walsh, M. C., H. P. Smits, M. Scholte, and K. van Dam. 1994. Affinity ofglucose transport in Saccharomyces cerevisiae is modulated during growth onglucose. J. Bacteriol. 176:953–958.

33. Wendel, D. L., and L. F. Bisson. 1994. Expression of high-affinity glucosetransport protein Hxt2p of Saccharomyces cerevisiae is both repressed andinduced by glucose and appears to be regulated posttranslationally. J. Bac-teriol. 176:3730–3737.

34. Wesolowski, M., A. A. Algeri, P. Goffrini, and H. Fukuhara. 1982. KillerDNA plasmids of the yeast Kluyveromyces lactis. I. Mutations affecting thekiller phenotype. Curr. Genet. 150:137–140.

34a.Wesolowski-Louvel, M. Unpublished data.35. Wesolowski-Louvel, M., and H. Fukuhara. 1995. A map of the Kluyveromyces

lactis genome. Yeast 11:211–218.36. Wesolowski-Louvel, M., P. Goffrini, I. Ferrero, and H. Fukuhara. 1992.

Glucose transport in the yeast Kluyveromyces lactis. I. Properties of an in-ducible low-affinity glucose transporter gene. Mol. Gen. Genet. 33:89–96.

37. Wesolowski-Louvel, M., C. Prior, D. Bornecque, and H. Fukuhara. 1992.Rag2 mutations involved in glucose metabolism in yeast: isolation and ge-netic characterization. Yeast 8:711–719.

38. Wesolowski-Louvel, M., C. Tanguy-Rougeau, and H. Fukuhara. 1988. Anuclear gene required for the expression of the linear DNA-associated killersystem in the yeast Kluyveromyces lactis. Yeast 4:71–81.

39. Zhang, C. C., M. C. Durand, R. Jeanjean, and F. Roset. 1989. Molecular andgenetic analysis of the fructose-glucose transport system in the cyanobacte-rium Synechocystis PCC 6803. Mol. Microbiol. 3:1221–1229.



.org/D

ownloaded from

http://jb.asm.org/

glucoseuptakein kluyveromyceslactis:roleofthe hgt1 gene ... ·...

Documents