Molecular Microbiology (1995) 16(1}, 157-167
Identification of novel HXT genes in Saccharomyces
cerevisiae reveals the impact of individual hexose
glycolytic flux
E. Reifenberger, K. Freidel, and IV
Institut fur Mikrobiologie, Heinrich-Heine-Universltat.
UniversitatsstraBe 1, W-40226 DOsseldorf, Germany.
Summary
In Saccharomyces cerevisiae, hexoss uptake is medi-
ated by HXT proteins which belong to a superfamily of
monosaccharide facilitators. We have identified three
more genes that encode hexose transporters {HXT5,
6, 7). Genes HXT6 and HXT7 are almost identical and
located in tandem 3 adjacent to HXT3 on chromo-
some IV. We have constructed a set of congenic
strains expressing none or any one of the seven
known WXr genes and followed growth and flux rates
for glucose utilization. The hxt null strain does not
grow on glucose, fructose or mannose, and both
glucose uptake and flux rate were below the detection
level. Expression of either HXT1, 2, 3, 4, 6 or 7 is basi-
cally sufficient for aerobic growth on these sugars. In
most of the constructs, glucose was the preferred
substrate compared to fructose or mannose. There Is
a considerabie variation in fiux and growth rates with
1% glucose, dependent on the expression of the indivi-
dual HXT genes. Expression of either HXT2, 6 or 7 in
the null background is sufficient for growth on 0.1%
glucose, while growth of strains with only HXT1, 3 or
4 requires higher (>1%) glucose concentrations.
These resuits demonstrate that individual HXT pro-
teins can function independently as hexose transpor-
ters, and that most of the metabolically relevant HXT
transporters from S. cerevisiae have been identified.
Introduction
Uptake of monosacoharides in baker's yeast, Saccharo-
myces cerevisiae, is mediated by carrier-mediated, facili-
fated diffusion, it has been proposed fhaf fhe process is
catalysed by two kineticaliy differenf systems, one with
high affinity tor the sugar and being dependent on the
Received 15 August, 1994; revised 3 December, 1994; accepted
14 December, 1994, ^'For correspondence, E-mail ciriacy@i..ini-
duesseldorf.de: Tel, (211) 3112778; Fax (211} 3115370. This work
is dedicated to F. K. Zimmermann, a pioneer in yeasi genetics, on
the occasion of his 60th birthday.
1995 BlackwGll Science Ltd
presence of a sugar kinase, and a constitutive, iow-affinity
coinponenf (for reviews, see Lagunas, 1993; Bisson et al.,
1993). The higivaffinity component is absent from cells
growing in high glucose (2%}. These conclusions have
largely been based on interpretations of biphasic Eadie-
Hofstee plots obtained from short-iime'Scale uptake
experiments. Further support for tliis view came from the
characterization of a mutant, snf3. winich was deficient in
expression of (he high-affinity component and in utilization
of low extracellular glucose concentrations (Bisson et a!.,
1987}, Tine SNF3 gene encodes a protein with structural
(and very limited sequence) homology to mammalian
glucose transporter genes (Celenza e( a/., 1988), How-
ever, the very low expression level of SNF3 did not
support the initial interpretation that the SNF3 protein func-
tions as the high-affinity transporter. Recent data suggest
that SNF3 may play some role in regulating glucose trans-
port (Ko etai, 1993},
Such interpretation of Eadie-Hofstee plots for giucose
uptake in S. cerevisiae has been questioned by Fuiirmann
and Volker (1992; 1993} on the basis of non-linear regres-
sion anaiysis of the data. On this basis, it became obvious
tiiat with the standard assay system a considerable part of
tiie apparent uptake can be described by first-order
kinetics and may be a consequence of passive diffusion
or, alternatively, mediated by a carrier with an extremely
low affinity (/<i,, = l-2iVl for glucose). By a modificafion of
the assay procedure. Walsh et al. (1994} were able lo
eliminate most of the passive diffusion moiety, which is
possibly caused by retention of glucose within the peripias-
mic space- From these data it also became obvious that
the overail kinetics of the uptake system does change
from low (Km = 26 mM) to high (Km = 1,6mM) affinity, prob-
ably because of the transition from high (2%) to very low or
zero glucose. Remarkably, the V^^^y of glucose uptake
remains constant during this transition.
Elucidation of the molecular basis of the glucose-
transport system has been begun only recently by the iso-
lation of putative hexose transporter genes {HXT1 to
HXT4) which were obtained as multicopy suppressors of
various uptake defects (Kruckeberg and Bisson, 1990;
Lewis and Bisson, 1991: Ko et ai. 1993; Ozcan e^ ai.
1993: Prior e; ai. 1993). /-/XT genes show remarkable
similarity to a previousiy identified gene, GAL2. which is
thought to be the major component of galactose uptake
 158 E. Reifenberger, K. Freidel and M. Ciriacy

in S. cerevisiae (Tsctiopp etai, 1986; Nehiin etai, 1989).
HXT./GAL2 genes belong to a superfamily of mono-
saccharide facilitators which aiso includes mammalian
glucose transporters (Glut: for reviews, see Bisson et al.,
1993; Henderson, 1993).
The significance of individual HXT transporters to
hexose transport or hexose utilization proved to be difficult
to assess in vivo. Mutants deficient in expression of any
/-/XT gene did not show any growtii phenotype, nor were
dramatic changes in uptake kinetics observed in such hxt
single mutants. A recent and more detailed analysis of
HXT2 protein expression revealed a complex pattern in
response to giucose (Wendell and Bisson, 1994). By
means of appropriate hxt muiliple mutants it has been
demonstrated that genes HXT1 through to HXT4, when
expressed individually, allow normal growth on glucose,
and it has been speculated thaf an hxt1 to hxt4 quadruple
mutant is unable to tai<e up gluoose (Ko et ai, 1993). We
became interested in yeast glucose transport in two
classes of regulatory mutants {HTR1. grr1) which showed
significant aiterations in glucose repression, glycolytic fiux
and expression of HXTgenes (Ozcan etal., 1993: 1994),
One speculation was that hexose transport triggers
signals for a segment of glucose-repressible functions,
possibly in combination with the activity of hexokinase
Pll. Although tiieie are alternative and equivalent explana-
tions for the HTR1/grr1 effect, we decided to explore
the nature of yeast hexose transporters in a systematic
way and to assess their functional significance in flux
control and in regulatory circuits of yeast carbohydrate
metabolism. Upon isolation of three novel HX7-related
genes {HXT5, HXT6. and HXT7) we constructed a mutant
strain defective in seven HXT genes. This mutant was
nearly unable to metabolize glucose, fivctose or man-
nose, whereas strains expressing any of the genes
HXTt 2, 3. 4. 6. 7 can utilize glucose at considerable
rates, largely by aerobic fermentation.
Results
Isolation of genes HXT6 and HXT7
In S. cereWs/ae there are at least five genes which encode
putative glucose transporters. Four genes (HXT1 to
HXT4) belong to a family of oloseiy related sequences
whicti presumably encode bona fide hexose transporters
upon structural and functional criteria. Ko etal. (1993)
have shown that ceils deficient in all fouf HXT genes are
severeiy affected in glucose growth. We have recently
identified a gene [HTRi) which is involved in transcription
of HXTgenes: in dominant HTR1 mutants, expression of
several HXT genes is shut off, and such mutants display
a growth phenotype similar to that of quadruple hxt
mutants (Ozcan ef ai, 1993). A fifth gene. SNF3, may
also encode a hexose transporter (Celenza et ai, 1988).
There are hints that ttie SNF3 protein has a reguiatory
function rather than being directly involved in glucose
transport (Ko et ai, 1993: Walsh et ai, 1994). in an
attempt to dissect the catabolic and possible regulatory
functions of the diverse HXT proteins in more detaii, we

HBgB X HEE MN XH MKH E Bg H MN

1/1 1 1 1 / " II II III LJ I I I
HXT3 HXT7

Xm / i Xm
HIS3 hxt7::HIS3

l]xt3::LEU2

hxi3A::LEU2::Alixt6

pT54

p303

p21

0
SI S2 S3 1kb

Fig. 1, Genetic orcjanizalion and restriction map ot the HXT cluster on chromosome IV containing genes HXT3, HXT6 and HXT7. Open
leaclincj frames are indicated by arrows. Thick lines indicale cloned sequences (plasmids pT54, p303 and p21]. LEU2 and HISS insertions into
HAT genes are shown by boxes. The reslriction fragments indicated were used for chromosomal replacement of the respective wild-type gene.
The hxt3::LBU2 Iragment integrated in one case out ot 12 transformants in HXT3/HXT6, as marked by the asterisk. Tight linkage ol H.XT3I
HXT6 and HXT7was first demonstrated by letrad analysis using marked HXT3 and HXT7 genes (26 parental ditypes, one lelratype among 27
teltads). The location of ihe H.XT7 done (p2l) was inferred from Southern hybridi^alion of genomic tfa!,}men)s. Probes S2 (0.3)(l-i XhoUHindlW
fragment from p303) and S3 (0.5kb EcoRUBglW fragmeni from done p21) labelled a common 3.5kb XhoVBglW fragment. The S3 probe aiso
marked a 1.6 kb H/ndlll,'eg/ll fragmeni. Furthermore, ali three genes appear to be located on a single BarnHl Iragment. The Barri\-\\ and
Hndlll sites downsiream ot HXT/wBve interred from genomic Soulhern hybridizations (broken line). Restriction sites: B, SamHl- Bg BglW F
I; H, Hndlll; M, Msc\; N, Ncoi\ X, Xho\; Xm, Xmn\.

1995 Blaokwell Science Ltd, Molecular Microbioiogy, 16, 157-167


decided to reconstruct an hxt1-hxt4 quadrupie mutant as
described by Ko etal. (1993), We had previously obtained
genes HXT1. 3 and 4 as multicopy suppressors of the
HTR1-23 mutation from a YEp13-based genomic library
(Ozcan etai, 1993). One such clone (pT54, Fig, 1) con-
tained the HXT3 gene, as verified by sequencing of its 5'
end; hybridization experiments suggested that this clone
contained an HXrs-related sequence approximately
1,2 kb downstream of HXTS. This was oonfirmed by
sequencing: 493 bp revealed 85% identity to the HXT3
N-terminal half; we designated this gene HXT6. In order
to isolate the entire HXT6 gene we identified appropriate
genomic fragments using HXT3 as a probe. Surprisingly,
we detected another HXT3-\\ke sequence which we desig-
nated HX77 (Fig. 1). It is noteworthy that the hybridization
patterns obtained with HXT3 as well as with other HXT
genes as probes were identical for the set of strains
used here, and for strain S288C (Fig, 2, lane 2), a strain
used in many yeast iaboratories.
Gene HX7"7was obtained as a 5.2 kb EcoRI fragment
(p21, see Figs 1 and 2, iane 1), while isolation of the
entire HXT6 was rather difticult because of some plasmid
instability in Escherichia eoti transformants. At the end,
we obtained an HXT6 H/ndlll fragment (p303. Fig. 1)
which overlapped the pT54 clone. There were two hints
that HXT7 is closely linked to HXT3-HXT6: (i) in hetero-
zygous crosses with mari<ed HXT3. HXT6, and HXT7
genes we observed, almost exciusivoiy, parental segrega-
tion (see the legend ot Fig. 1); (ii) ali three sequences are
apparentiy iocated on a single BamHI fragment. Mapping
of appropriate genomic fragments by Southern hybridiza-
tion led us to propose an arrangement for HXTS and
/-/XTT"as depicted in Fig. 1.
Sequences of HXT6 and HXT7
Sequencing of HXTS and HXT7 revealed two almost
1 2 3 4
HXT3
HXT7 HXT3 —
. .- 3 hiit7::HIS3
HXT6
HXT4
Hindlll
;o 1995 Blackwell Science Lid, Molecular Microbiology. '16, 157-167
Hexose transport in yeast 159
identical open reading frames of 570 eodons each
(Fig. 3A). The five nucleotide exchanges result in two
rather consen/ed exchanges for amino acid residues 293
(Val— He) and 556 (Thi — Aia) which are iocated outside
the 12 putative membrane-spanning domains according
to the structure of mammalian glucose transporters pre-
dicted by Mueckler et ai (1985), and none of lhese resi-
dues appears as conserved within the HXT gene family.
The HXTSIHXT7 similarity is also obvious within the
96 bp upstream of the putative start codon (Fig. 3B).
Further upstream, there is no obvious similarity between
both genes. This may indicale that these genes are differ-
ently regulated in their expression ievels. Preliminary
results from Northern blot experiments with various hxt6/
7 mutant strains do suggest that HXT7 transcripts are
more abundant than HXTG transcripts (data not shown).
A comparison of the predicted HXT7 protein with other
known HXT proteins revealed identities of 72,4% wifh
HXT1, 69,1% with HXT2, 76,1% with HXT3, 83,5% with
HXT4 and 72,7% witii GAL2, a transporter required for
galactose utilization (Tschopp et ai. 1986; Nehiin et ai,
1989). The identity between all known eight HXT/GAL2
protein sequences ranges from 64% (HXT2/3) to 99%
(HXT6/7),
Construction of defined hxt mutants
It has previously been shown that null mutations in indi-
vidual HXTgenes do not result in distinct growth pheno-
types. Only the concomitant elimination of HXT1 to HXT4
function resulted in reduced growth rates on glucose
especially in the absence of oxidative phosphorylation
(Ko etai. 1993), However, even such an /7xf7,2,3,4 quad-
rupie mutant strain is apparently able to utilize giucose
(see below). In order to clarify the roles of individual HXT
transporters in hexose utilization we constructed a set of
strains expressing none or any one of the seven HXT
Fig. 2. Genomic Soiilhern biol anafysis of
HXTgenes in wild-type and diverse tixt
mulant strains.
5 A. EcoB\ Iragments.
B, Hiiid\\\ fragments. Lanes: 1, MC996A (wild
type); 2, S288C (wild type); 3, hxt7::HIS3
— h!(!3::L£U2 mutant; 4, hxtSA::LEU2:: Ahxl6 mutanV. 5,
hxt3:\LEU2 mutant. HYTsequences were
probed wilh a digoxigfinin-labelled 1.5 kb
EcoRI/Wcol fragmen! of HXr3 (see Fig. 1,
Si) at 60 C. Both HXT3 and HXW genes are
located on a 5.8 kb EcoRI iragmeni, and
HXTZon a 5,2 kb EcafW fragmenf. The 5.6 kb
ECDRI fragmeni (weak signal) indicates the
HXT1-HXT4 cluster on chromosome VIII, and
the 3.6kb H/ndlll fragment indicates the HXr3
gene, HXT7\s located on a 2,85 kb HirnM
fragment and HXT6 on a 1.75 kb HiiKlWi
fragment. The weak signals uf 4.1 kb and
1.6 kb indicate genes HXT1 and HXT4.
respectively.
160 E. Reifenberger, K. Freidel and M. Ciriacy

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Cl 1995 Blackwell Science Ltd, Molecular Microbiology. 16. 157-167

 Hexose transport in yeasf 161

genes. We included in this study a hitherto unknown HXT- Table 1. S. cerevisiae strains.
like gene adjacent to the HXT1-HXT4 couple on chromo- Genotype"
Strain
some VIK. We called this gene HXT5 (EMBL gene bank
accession number X77961; £. Reifenberger etai, sub- MC996A t^ATa ura3-52 tiis3-11.15 leu2-3.112 MAL2 SUC2
GAL MEL
mitted). Disruptions/deletions of HXT genes were carried
WIC996B MAT:-/ ura3-52 his3-} 1.15 Ieu2-3.112 MAL2 SUC2
out as described in detail in the Experimental pro- GAL MEL
cedures. Construction of null alleles for HXT3, HXTS and RE101B MATr, hxtr.:HIS3
HXT7 is shown in Fig, 1, and the correct replacement of RE102B MAT-t hxt2A::HIS3
RE103A M,ATa hxi3::LEU2
the corresponding wild-type sequences has been verified RET05A MATa hx!5::LBU2
by Southern blotting of genomic Hi'ntiiW fragments of RE201A MATa hxt3A::LEU2::Ahxt6
yeast transformants (Fig. 2B, lanes 3-5). The strategy RE302B MATri hKt1A::HI.S3::Ahxt4
RE203A MATa hxf3;.LEU2 hxt7::HIS3
for construction of a strain (RE700A) deficient in ali RE204e MAT-/, hxti ::HIS3 hxt2A::HIS3
seven HXTgenes is outlined in Fig. 4, and a list of strains RE301A MATa hx!3A::LEU2:: .Ahxt6 hxt7::HiS3
generated by this procedure is given in Table 1. We have RE301B MAT'f. hxt3A::LBU2:\ Ahxt6 hxl7\\HIS3
RE302A MATa hxni-.-.HfS3:-.Afi>;!4 (ix!a\';.H(S3
taken advantage of the fact that HXT1, 4, 5 and HXT3, 6, 7 RE303A MATa hxt1A::HtS3::AtJxt4 hxl3::LEU2
are arranged in two clusters which segregate as single RE304B MATy. hxt2A::HIS3 hxl3A::LEU2::Ahxt6
genes during meiosis allowing isolation of the desired RE306A MATsi hxti ::HIS3 hxtSA ::LEU2::Mix!6
RE307B MATT hKt1 ::HIS3 h.y.t5::LEU2 hKt2A ::HIS3
strains In a relatively easy way. Southern blot analysis con- RE308B MATzf. hxt2.\..HIS3 hx(3::LEU2 hxl7::HIS3
firmed in each case the genotype deduced. While most of RE401A MATa hxt2A\\H!S3 hxt3A ::LEU2:: Ahxt6 hxt?:\HIS3
the strains oanying just one wild-type HXT gene were RE401B MAT'j hxl2.A::HIS3 hxt3A::LEU2::.\hxt6 lixl7::HIS3
RE402A MATa hxt1A::HiS3::AI-ixl4 hxl3A ::LEU2:: .\hxt6
RE403A MATa hxt1A::HIS3::.Mixt4 hxt5::LEU2 lixt2A •.:HIS3
RE700A MATa hxt1A::HIS3::Ahxt4 hxt5::LEU2 hxt2,\ ::HIS3
hxt3A ::LEU2:: Ahxtd nxt7::HIS3
RE201A RE202A RE601A MATa hxt1A::HIS3::.\hx!4 hxt5::LEU2 hxt2A::HIS3
t!rt3A::LEU2::Ahiiie hxtSA :.LBU2.\ Ahxt6 hxt7::HIS3 ura3-52::Ylp5-
I HXT1
hXl7::HIS3 RE602B MATn hxt1A::HIS3::Anxl4 hx13::LEU2
hxt3A .±£02.: Ahxt6 hxl7::HIS3
RE603A MATa hxt1A::HIS3::Ahxt4 hxt5::LEU2 hxl2i\ ::HIS3
hxt3A .±£02:: Ahxt6 hxt7..HlS3 i/ra3-52::Ylp5-
I-IXT3
RE604A MATa hxti :.HIS3 hxt5:±EU2 hxl2A ::HIS3
hxt3\\±EU2\\.Ahxte hxt7::HI.S3
RE605A MATa hxn.\::HIS3::Ahxt4 hxl2A::HIS3
hxl3A :: LEU2:: Ahxt6 iixt7::HIS3
RE606A MATa hxt1A::HIS3::A!lxt4 hxt5.±EU2 hxl2A: :HIS3
iixt3::LEU2hxt7::HiS3
RE607A MATa hxt1A::HlS3::Ahxl4 hxt5:±EU2 hxt2A •. :HIS3
nxt3A:±EU2::Ahx!6

meiolic meiolic a. All stvains lisled ate uniformly ura3-52 fijs3-T-!, !5 feu2-3,112 MAL2
segregalion segregalion SUC2 GAL MEL as indicated wilh lhe HXTwild-type strains, MC396A
I I and MC996B. The designalion of HXT1 to HXT4 is according lo Ko et
ai (1993).
RE401B RE403A
hxt2A::HIS3 fiXl2A::HIS3
tixt3A:±EU2::Ahxl6~tixt7::HIS3 hx t5::LEU2- hxnA::HIS3::A hxta obtained by the construction scheme outlined in Fig. 4,
genes HXT1 and HXT3, respectively, had to be reintro-
duced in strain RE700A (for simplicity, we call it the 'hxt
nuir strain). This was achieved by integrative transforma-
meiotic tion of RE700A with plasmids Ylp5-HXT1 or Ylp5-HXT3
(for details, see the Experimentat procedures) resulting in
single-copy integration at the URA3 locus.
RE700A
hxl2A:;HfS3
hxtB::LeU2-hxtiA::HIS3::Ahxl4 Hexose utilization, glycolytic flux and HXT functions
hxi3A::iEU2::Ahxte--hxl7::HIS3
Fig. 4. Strategy for consiruction of strain RE700A deficient in ail We determined the growth characteristics of the HXTcon-
seven HXTgems. Note thai genes HXT1, 4, 5 and HXT3, 6, 7are structs on solid media with various hexoses (gluoose, fruc-
arranged in two clusters, whioh segregate as single genes during
meiosis. The genotypes deduced from meiolic segregation patterns tose, mannose, galactose, maltose, sucrose and raffinose)
were verified by Southern analysis in ali cases. as the major (YEP-media) or sole (YNB-media) carbon

1995 Slackwell Science Ud, Moiecular Microbiology. 16. 157-167

162 E. Reifenberger, K. Freidel and M. Ciriacy
source, and also on media containing various concentra-
tions (0,1%, 1%, 5%) of glucose, frucfose or mannose.
Sucrose and raffinose are hydrolysed outside the cell, pro-
viding very low extracellular concentrations of glucose and
fructose and of galactose (note that the strains used here
are melibiose-positive). An example from such an analy-
sis for selected media is shown in Fig, 5, As expected,
we did not observe clear effects of the various H X r geno-
types on growth on 2% ethanol (non-fermentable carbon
source: not shown) or on maltose, which is taken up by a
separate transport system (Serrano, 1977). in contrast,
the hxt nuli strain did not grow within 2d on any of the
media containing glucose, fructose or mannose. This sug-
gested that most, if not all, of the metabolically relevant
HXT transporters were absent in this strain. Expression
of individual HXT genes in this null background should
therefore allow a first and rough identification of their role
in hexose metabolism. With the HXT1 strain it was note-
worthy that growth was strongiy dependent on sugar con-
centration, i.e. there was almost no growth at 0.1% but
there was wild-type growth at 5% glucose, A similar,
although less pronounced, effect of the sugar concentra-
tion was observed with cells expressing only HXT3 or
HXT4. HXT3 cells grew very slowly on 1% mannose, but
on 5% mannose growth was almost normal. Strains with
only HXT2 or HXT7 showed wild-type growth on low
(0,1%) glucose, and high (5%) glucose caused some
growth inhibition. The HATgstrain displayed a growth pat-
tern similar to that of the latter two strains; however, growth
was impaired on all media. HXT6 cells were clearly sensi-
tive to anti'mycin A on giucose (antimycin A bfocks mito-
chondrial eieotron transport/oxidative phosphorylalion,
and renders the yeast cell entirely dependent upon glyco-
lytic ATP synthesis). This would be consistent with the
strong similarity of HXTS and HXT7 proteins (which may
indicate identical transport functions) and with the lower
expression level of HXT6 mentioned above, A strain
expressing only gene HXT5 was not significantly different
from the null strain (data not shown).
With galactose as sole carbon source we did not
obseive clear effects in the various constructs (Fig, 5),
In the presence of antimyoin A, however, galactose
growth was impaired in cells lacking genes HXT3, HXTS
and HXT7. With 2% raffinose as sole carbon source the
effects of hxt mutations were less clear. Sinoe the strains
were melibiose-positive, the galactose freed from the meli-
biose can in turn switch on galaotose metabolism. In the
absence of oxidative phosphorylation (antimycin A), how-
ever, all mutants except the HX72and the /-/XT7 construct
failed to grow on raffinose. This growth pattern was also
observed for 2% sucrose/antimycin A- It has been
reported that in S. cerevisiae sucrose oan be taken up by
H*/sucrose symport (Santos ef ai, 1982). We did observe
a slight increase in growth rale on sucrose in the hxt null
2% mairose 2% galactose 1 % mannose
0.1% glucose 1% glucose 5% glucose
Fig, 5. Growth phenotypes ot wilcl-lype strain MC996A (wl) and
niuianl strains RE700A (hx!), RE601A (HXT^). RE602B {HXT2).
RE603A {HXT3). RE604A [HXT4). RE606A {HXT6) and RE607A
{HXT7). The complete genotypes of the strains are hsted in Table
1. Ceils were pre-grown on YEP 2% mallose for 2d and streaked
on YNB medium with the indicated carbon sources. The plates
were incubated at 30 C for 2-3 d before being photograplied.
strain, and this may be attributed to specific sucrose
uptake. Nevertheless, this sucrose uptake is olearly limit-
ing for metabolism as indicated by the antimycin sensitiv-
ity. The raffinose/sucrose growth patterns may suggest
that HXT2 and HXT7 are better suited than the other
HXT proteins to the utilization of very low external hexose
leveis. To summarize, these kinds of analyses revealed
that expression of the various HXT genes had distinct
anci profound effects on the utilization of glucose, fructose
and mannose. We did not obsenye clear differences
between individual HXT functions on the specificity for glu-
cose, fructose or mannose. It was notable, however, that
growth on either fructose or mannose in strains with only
HXT1, HXT3 or HXT4 required higher sugar concentra-
tions than on glucose.
Such analyses are not very precise, in order to relate the
functions of the various HXTgenes on a quantitative scale
we performed a more detailed analysis of glucose growth
rates (pmax) and flux rates (1/GIC) ^or selected HXT strains
(Table 2). It is remarkable that expression of HXT2 or
HXT3 results oniy in a very modest decrease (4-6%) in
the glycolytic flux on 1% glucose relative to wild-type
cells. With HXTt HXT4 and HXT7 strains, \/GIC was
reduced more significantly. An even more severe defect
in glucose utilization was seen with the HA re strain. The
hxt null strain showed, as expected, the most drastic
!(., 1995 Blackwell Science Ltd, Molecular Microbiology. 16, 157-167
 Hexose transport in yeast 163
Table 2, Growth rate {|i) and glucose-consumption rafe {VQ,I.) in wild- rates. This indicates that in the various HXT constructs
type and diverse HX7 mutant strains,""
the glycolytic flux limits ihe maximal specific growth rate.
Relevant genotype'^ Per ceni Per cent Finally, we performed a glucose-uptake assay according
to the assay protocol of Walsh ef ai (1994) with wild-type
Wild type 0,396 100 2,49 100
and null mutant cells preincubated on YEP 0.1% glu-
hxt 0.038 10
HXT1 0,287 73 1.50 60 cose, and we found that uptake was undetectable over a
HXT2 0,389 98 2.39 96 wide concentration range (0.2-200 mM glucose) in the
HXTS 0.386 97 2.34 94 null strain.
HXT4 0.315 80 1,84 74
HXT6 0,136 34 0.59 24
HXT7 0,357 90 2.17 87
Discussion
a. Each culture was pre-grown to the mid-log phase on YEP 2%
mailose, washed with YEP medium, and then transferred to YEP We have identified in S. cerews/aethree genes with signifi-
• i % glucose.
cant similarity to four known HXTgenes encoding hexose
b. The HATgenolype refers lo (he indicated wild-type HXTgene in an
othenAiise hxt null background. transporters. We designated these novel genes HXTS,
0. Maximum specific growth rate. Liruft* ( h " ' ) . HXTS and HXT7. and analysed the latter two in this
d. Calculation oi 1/GIC (in grains per hour per gram of dry weigfit) was work. Genes HXT1 to HXT4 were isolated previously as
based on a decrease in glucose concentration from 1 % io approxi-
mately 0.5% at j.l,„c^,; for the calculation, see the Experimental pro- suppressors of various uptake defects (Kruckeberg and
cedures. Mean vaiues ot two independent cultures are given. Bisson, 1990; Lewis and Bisson, 1991; Ko etal., 1993;
e. ND, not detectable. Ozcan ef a/., 1993; Prior et ai, 1993). The HXTS gene
was detected on an HXr3 clone, and HXT7was identified
reduction in the flux rate; because of the very low amounts because of its strong similarity Eo HXT6 on a genomic frag-
we were unable to calculate reproducible flux rates for the ment. Likewise, gene HXTSwas identified by its similarity
null strain. We estimate that 1/GIC in this strain was clearly to HX7"7 and was located immediately upstream of HXT1-
less than 15% of the wild-type rate. In hxt null cells HXT4 on chromosome VIII (E, Reifenberger et ai, sub-
grown on maltose, concentrated to high cell densities mitted). This arrangement of genes HXT1. 4, 5 has
(7g dry weight per litre; that is about a 50-fo(d amount recently been confirmed by sequencing of chromosome
of cells in comparison to the standard assay) and shifted VIII in its entirety (Johnston et ai, 1994). Thus, S. cere-
to 2% glucose we did obseive glucose consumption visiae contains two clusters of three HXT genes each;
after a 2h lag phase. The I/QIC was 0.35gg ^h~\ i.e, the HXT2 gene (Kruckeberg and Bisson, 1990) is unique
approximately 20% of the wild-type rate under these in that it is apparently not associated with other HXT
conditions. sequences. In order to verify whether the functions of
We also included in this study an hxt1-hxt4 quadruple these genes are indeed implicated in hexose transport
mutant which was previously described as a strain with a we constructed a series of strains which carried none or
severe defect in glucose utilization, although this has not any one of the seven HXT" genes and determined hexose
been verified in terms of flux rates or uptake rates (Ko ef utilization in the various constructs. This strategy is similar
ai, 1993), In the strain background used here, the quad- to the approach of Ko et ai (1993); however, we did not
ruple mutant resembled the HXT7 strain in glucose consider SA/F3 function in this study. SNF3\s expressed
growth; V^u, was about 80% of the wild type value (results at a very low ieve! (Marshall-Carlson ef ai, 1990; Ozcan
not shown). Thus we could not confirm, in this strain back- et ai. 1994), and a regulatory role for the SNF3 protein
ground, the original description of the quadruple mutant is more likely than its direct involvement in sugar transport.
strain as giucose negative. This discrepancy is presum- We found that expression of any one of the genes HXT1,
ably caused by the fact that the quadruple hxt1-hxt4 2, 3, 4, 6, 7 is suificient to allow substantial glucose utiliza-
strain described by Ko ef ai (1993) contains a chimera tion, although to a very different degree. Strains expressing
made of the 5' end of HXT6 and the 3' end of HXT7 HXT2, HXT3or HXT7 had glucose flux rates on 1% glu-
instead of two distinct genes (R. F, Gaber, personal cose that were very similar to wild-type cells, HXT3 is
communication). expressed in wild-type cells at moderately high leveis
In mosl of the HXT strains 1 % glucose was utilised even in the absence ot external glucose (Ozcan et ai,
largely (80%) by fermentation, as could be calculated 1994), and this may explain the nearly wild-type growth
from ethanol yields; this is not significantly different from of the HXT3 strain. With the HXT^strain this is more difficult
wild-type cells. No ethanol was found in the null strain, to explain. Wendell and Bisson (1994) have reported fhat
and in the HXTS strain, only 40% of the glucose was the HXT2 protein is abundant only in cells grown on low glu-
used lermentatively. It is noteworthy that there is rather a cose (0.05%). These authors have suggested thai the HXT2
strong correlation between the maximal specific growth gene is induced at low glucose levels and repressed at high
rates (|.imax; Table 2, first two columns) and the fiux (2%) glucose levels, although this has not been verified by

[£.• 1995 Blackwetl Science Ltd, Molecular Microbiology, 16, 157-167

164 E. Reifenberger. K- Freidel and M. Ciriacy
HXT2 transcript levels. Current analysis of HXT transcript
levels indicates that in the wild-type strain used here,
HXT2 is also induced by 2% glucose. We could not rule
out strain differences in the expression patterns of other
HXTgenes, In this context, it is noteworthy, however, that
we did not find any difference in the presence and the
gross stmcture of genomic HXT fragments among three
laboratory strains (MC996A; S288C; ENY.WA1-1A, from
K. D. Entian) and a commercial wine yeast (Siha 3,
Begerow GmbH, Langenlonsheim/Germany),
Growth of the HXT7strain was almost like that of the wild
type on 0.1 % or t % glucose, but higher glucose concentra-
tions were inhibitory to some degree. We do not believe that
this is due to HX77 repression since preliminary character-
ization of glucose uptake kinetics revealed, in 2% glucose-
grown HXT7 cells, a very high-affinity uptake (E, Reifen-
berger ef al., unpublished). Thus the effect of high gluoose
on HXT7 ceils can hardly be due to the absence of a
glucose-transport system under such conditions, In con-
trast, the HXT1 strain required high (>2%) giucose for
normal growth. Cells transferred from maltose to high
glucose had an unusually long lag phase (6-8 h). This is
probably because of transcriptional regulation of HXT1
which requires external glucose for induction (Ozcan ef
ai, 1994) and high (22%) glucose levels for maximal
expression (S. Ozcan, personal communication, and our
observations). Thus, the flux rates determined for the
HXT1 strain on 1% glucose are presumably submaximal.
Some of the constructs were antimyoin-sensitive on 1 %
giucose {HXTS) or growth was impaired in the presence of
antimycin, although there was considerable glucose
utilization (especially obvious with HXT1 and HXT4. cf.
Table 2). This indicates that the glycolytic flux and hence
glycolytio ATP synthesis Is insufficient to maintain cellular
growth. In this context it is noteworthy that the reduced
glycolytic ATP synthesis apparently cannot be fully
compensated by enhanced oxidative phosphorylation,
although the growth conditions used here should allow suf-
ficient oxygen supply. The decrease in ffux rates in all con-
structs clearly parallels the decrease In the maximum
specific growth rate, ^inia^c This could indicate that in S,
cerevisiae the respiratory capacity is limiting, as pre-
viously suggested (for a review of this issue, see Kappeli.
1986; Gancedo and Serrano. 1989). it could also be
speculated that, in the presence of high glucose, oxidative
phosphorylation is inefficient. Also, we cannot rule out the
possibility that high external glucose can still induce a vari-
ety of glucose-sensitive functions in hxt strains; in the
absence of sufficient glucose uptake this may disturb
metabolism and cell growth.
Does this prove that the seven predicted HXT proteins
contain ail metabolically relevant glucose transporters?
This suggestion can only be made with caution. Although
the hxt null strain does not take up detectable amounts
of giucose under the standard assay conditions, we
observed slow glucose utilization (approximately 20% of
wild type under such conditions) at very high cell densities
in 2% glucose. The nature of the glucose transport under
these specitic conditions is unclear. It is possible that
under severe oxygen limilation unknown HXT genes are
induced. We have preliminaty evidence for the existence
of three more HXT-related sequences in S. cerevisiae. At
least one of these genes, when overexpressed in ihe hxt
null strain, can restore its glucose growth defect (the
other two were cloned in an incomplete form only). It is
possible that these HX7 genes are expressed at very low
levels under the conditions tested here, as is obvious for
HXT5{E. Reifenberger etai, submitted). Also, we cannot
rule out the possibility that some of the HXT transporters
described here controi the expression of uncharacterized
HXT genes, in opposition to the view of Walsh et ai
(1994), who interpret HXT functions as mere modulators
of an unidentified hexose transporter, we assume that
HXT proteins are authentic hexose transporters; the
modulation of kinetic parameters according to environ-
mental conditions is due to the properties of individual
transporters, their differential expression and perhaps
interactions between individual HXT proteins. We are cur-
rently testing these hypotheses on the basis of the various
constructs described here.
Experimental procedures
Yeast strains, media, and growth conditions
Isogenic wild-type strains used in this study were MC996A
[tVlATa ura3-52 ivs3-11,15 Ieu2-3112 MAL2 SUC2 GAL
MEL) and MC996B {MAT^, otherwise identical to MC996A}.
Both strains were closely related to a set of strains used pre-
viously (Ozcan et ai, 1993). A set of MC996-CQngenic strains
was constructed by one-step gene replacement and meiotic
segregation (for detaiis, see below). Standard yeast genetic
techniques for mating, sporulation, and tetrad analysis were
used as previously described (Sherman, 1990). Yeast
cells were grown at 30 C in YEP media (2% Difco Bacfo
Peptone, 1% Ditco yeast extract) that contained glucose,
fructose, mannose, galactose, maltose, sucrose, or raffinose
at the concentrations indicated, or 2% ethanol as a non-
fermentable carbon source. Sugars were of analytical grade.
Nutritional requirements were scored on media consisting of
2% maltose and 0.67% Difco yeast nitrogen base (YNB) sup-
plemented with the appropriate amino acids, adenine, and
uracil. Antimycin A {final concentralion, 10|.!t\/i) was u.sed as
a respiration inhibitor. Plasmid transformations of yeast ceils
were carried out by the freeze method (Dohmen &t ai,
1991). E. CO//DH5aF' was transformed tiy the rubidium chlor-
ide method (Hanahan, 1985).
DNA and RNA isolation, eleofrophoresis, and
hybridization
Plasmid DNA from E coli was prepared as described by
;c.i 1995 Blackwell Science Ltd, Molecular Microbioiogy. 18. 157-167

Birnboim and Doly (1979). Total yeast DNA or RNA was iso-
lated from S. cerevisiae as described (Kotter ef a/., 1990;
Sherman, 1990). Southern blot analysis was carried ouf wifb
the DNA Labelling Detection and Non radioactive Kit (Boeb-
ringev Mannheim) at 60 C. Foi' Northern blot analysis,
poly(A)"'' RNA was isolated witb tbe mRNA Purification Kit
(Pbarmacia), denatured witb glyoxaL and separated on 1 %
agarose gels, DNA probes were labelled by nick translation.
Cloning and sequencing of genes HXT6 and HXT7
The HXTdand HXT7ger\es were isolated by colony bybridiza-
tion of partial genomic libraries. For construction of sucb
libraries, genomic DNA of strain MC996A was digested witb
EcoRI or H/ndlll, respectively. EcoRI fragments about 4 . 0 -
8.0 kb in size were isolated and Iigated into vector YEp352
(Hill etal.. 1986). H/ndlil fragments of about 1.4-1.9kb were
isolated and Iigated into vector pBR322 (Bolivar et aL.
1977). The libraries were screened by colony hybridization
(approximately 4000 colonies eacb) at 60-C {6x SSPE,
0.2% SDS) with the '^^P-labelled 1.5 kb EcoRI/A/col fragment
containing HXT3. Altogether, 21 positive clones were obtained
and further analysed by Soutbern blotting and restriction
analysis. Among tbese, plasmid p21 contained tbe HXTZ
gene on a 5.2 kb EcoRI fragment. Plasmid p306 contained a
part of tbe HXT6 gene on a 1.75 kb H/ndlll fragment in
pBR322. Tbe 5' region of HXT6 was identified on plasmid
pT54 (see Fig. 1). Plasmid piO contained tbe HXT5 gene
on a 4.6 kb EcoRI fragment in YEp352 and plasmid p17 con-
tained HXT1 and HXT4 on a 5.7 kb EcoRI fragment. Double-
stranded piasmids were sequenced by tbe dideoxy method
using the T7-Sequencings'^ Kit (Pbarmacia) and [cf-^^S]-
dATP (Amersham) on botb strands. Nested deletion clones
of HXT6 and HXT7were geneiated witb the Nested Deletion
Kit (Pbarmacia). Additional sequence data were obtained by
using otigonucleotide primers syntbesized by MWG.
Piasmids and constructions ot disruption mutants
Tbe nomenclature for genes HXT1 to HXT4 is according to Ko
ef al. (1993). Piasmid pT32, containing tbe HXT1 gene and
part of tbe HXT4 gene, and pT54, containing tbe HXT3 gene
and part of tbe HXT6 gene, have been isolated as multicopy
suppressors of tbe HTR1-23 mutation from a YEp13~based
genomic library (Ozcan et a!., 1993). Plasmid pHXT3-1 con-
tained tbs HXT3 gene on a 4.2 kb H/ndlll fragment of pT54
cloned into pT7T3-18U (Pbarmacia). Tbe 574 bp Sspl frag-
ment of pHXT3-1 was used to localize the HXT6 gene on
pT54.
For disruption of HXT1 (Lewis and BIsson, 1991), the
1.66kb Ecof\i/Nsi\ fragment of HXT1 was cloned into vector
pT7T3-1 SU. Tbe resulting plasmid was linearized by Msc\ and
Iigated witb the Klenow-treated 1.76 kb SamHI fragment, con-
taining the HtS3 gene, of ptasmid GS15 (Lee et al., 1988)
yielding plasmid p D I . To generate the /7xf?::H/S3 disruption
ailele, pDI was digested with EcoRI and Nco\ and trans-
formed into yeast strain MC996A. Tbe hxt2A:HIS3 ailele
was constructed as follows. Tbe i.74kb EcoH\/Xba\ frag-
ment of HXT2 (Kruckeberg and Bisson, 1990) was cloned
into pT7T3 (plasmid pT-HXT2). pT-HXT2 was linearized by
Mscl and Iigated witb the Klenow-treated 1,76 kb SamHI
• o 1995 Blackwell Science Ltd, Molecular Microbiology. 16. 157-167
Hexose transport in yeasi 165
HIS3 fragment of plasmid GS15, resulting in plasmid pD2.
Tbe EcoRI/A/col fragment of pD2 was transformed into
MC996B by selection for H(s^ Plasmid pT-HXT3, containing
the HX7"3 gene on a 3.6kb H//idlll fragment, was linearized
by Msc\ and ((gated with the Klenow-trealecl 2.26 kb Smal/
Sal\ fragment of pUC-LEU2. pUC-LEU2 contains tbe 2.22 kb
Xho\/Sal\ fragment of YEp13 (Broach et al., 1979) in
pUC18. Tbe resulting plasmid (pD3) was digested witb H/ndlll
and A/col and transformed into MC996A by selection for Leu*.
In one ot 12 transtormants, recombination between HXT3and
HXT6 sequences occurred. This resulted in tbe genotype
hxl3Ay.LEU2y.Ahx!6 in str.ain RE201A. To generate the
deletion allele, hxt1A::HIS3::Ahxt4 plasmid pi 7, containing
a 5.7 kb EooRI fragment of HXT1 and HXT4 (isolated by col-
ony hybridization; see above), was digested witli Mscl. The
resulting 7.2 kb Mscl fragment was iigated with the 1.37 kb
Klenow-treated EcoRV/Smal fragment of pUC21-HIS3.
PUC21-HIS3 contains the 1.3 kb BamH\/Xho\ fragment of
GS15 in pUC21. The resulting plasmid, pD14. was digested
with XmnI and transiormed into strains MC996A and
MC996B (RE202A and RE202B). To construct a disruption
of HXT5. plasmid piO-H containing the 3.5kb H/ndlll frag-
ment of p10 in YEp352 was linearized with Mscl and Iigated
with the 2.22 kb Klenow-treated Bam\~\\/Sal\ fragmanf of
pUC-LEU2. The 5.8 kb Hindlli fragment of the resulting pias-
mid, pD5, was transformed into strains RE302A (RE403A)
and RE204B (RE307B). To generate the hxtJwHISS allele,
plasmid p21 was linearized with Mscl and iigated with the
1.37kb EccPiyiSma\ fragment of ptJC21-HIS3 {pD7). The
2.43 kb Xmnl tragment of" pD7 was transformed into
strain RE103A (yielding RE203A) and into strain RE201A
(yielding RE301A). Piasmids Ylp5-HXT1 and Ylp5-HXT3
were constructed to reinfroduce genes HXT1 and HXT3 into
strain RE700A. The 4.2 kb H/ndlll fragment of plasmid pT32
containing HXT1 was oloned inlo vector Ylp5 (Struhl Bi a\..
1979). The resulting plasmid, Ylp5-HXT1. contained 1.2kb
of fhe promoter region of HXT1. The 3.9 kb Xho\ fragment of
plasmid pT54 containing the HXT3 gene was cloned into
Y!p5, resulting in Yip5-HXT3. This construct contains 0.85 kb
of the HXT3 promoter region. Targeted integration at the
chromosomal ura3'52 Q.\\G\e was achieved by linearizafion ol
the piasmids with Stu\ within the URA3 sequence. Correct
replacements in yeast cells were confirmed in all cases by
Southern blot analysis.
Fermentation studies and glucose-uptake assay
Growth rates and sugar-consumption rates were determined
in steady-state batch cultures. I.e., al low cell densities
(5-; 10'^^ to 3 x 1 0 ^ ceils m l " ' ) . Cells were pre-grown on YEP
2% maltose, washed with YEP. and transferred to YEP with
1% giucose. Strain RE601A was pre-grown in YEP 1 % glu-
cose because tbis strain showed a very long lag pbase on
glucose when transferred from maltose media. Samples
were removed at intervals of 3 0 - 6 0 min. Enzymatic methods
for glucose and eEhanol were applied as described previously
(Ozcan et al..^ 1993). Sugar-consumption rates were deter-
mined according to Walsh et al. (1991). I/yield values were
obtained from the (linear) slope of plots of giucose versus
mg dry weight. I/R,^ is i / y x j i ^ g ^ (maximum specific growth
rate h " ' ) expressed in mg glucose per mg diy weight per
166 E. Reifenberger, K. Freidel and M. Ciriacy
hour. The glucose-uptake assays were performed as
described previously (Ozcan et al.. 1993) with modifications
according to Walsh etal. (1994).
Acknowledgements
We thank S. Ozcan (St. Louis, USA) and Astrid Bolten (this
laboratory) for some hints on regulation of HXT genes, and
R. F. Gaber (Evanston, USA) for communicating some results
prior to publication. This work is part of a Ph.D. thesis by E.R.
Financiai support came, in part, from the Bundesministerium
fQr Forschung und Technofogfe (Grant 031670aA/B2 to M.C.).
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