(Received for publication, November 7, 1994)
From the
The Gal2 galactose transporter takes up galactose in yeast. A homologous glucose transporter from the same organism, Hxt2, was selected, and various chimeras between these two transporters were constructed by making use of homologous recombination in Escherichia coli. Comparison of the galactose transport activities of three series of chimeras enabled us to positively identify a crucial substrate recognition region of 101 amino acids that lies close to the carboxyl terminus of the Gal2 transporter.
Gal2 is a high affinity galactose transporter of yeast Saccharomyces cerevisiae, which transports galactose, glucose,
and other sugars via facilitated diffusion (1, 2, 3, 4, 5) . ()Hxt2 is one of several glucose transporters identified in
the same organism (1, 6) that transports glucose but
not galactose. Both sugar transporters exhibit high sequence homology
except for the NH
- and COOH-terminal regions. The amino
acid identity of the middle region (amino acid residues 70-547 of
Gal2, corresponding to amino acid residues 55-532 of Hxt2) is 71%
with no gaps imposed. These two transporters are members of a
transporter family designated in this paper as the Glut transporter
family, which includes a wide variety of transporters for sugars and
sugar analogs such as quinate, either active or facilitative, of
prokaryotes and eukaryotes(7, 8) . The Glut family
transporters belong to a larger membrane protein superfamily (mfs, for
major facilitator superfamily)(9) , which includes drug
resistance proteins, transporters for Krebs cycle intermediates,
transporters for organic phosphates, and transporters for
oligosaccharides. Thus, molecular studies on the Gal2 transporter are
expected to bring a general view applicable to a wide variety of
transporters.
Previous studies on the determination of functional domains in transporters employing localized mutagenesis have identified several amino acids, replacement of which impaired certain properties of transporters(10, 11) . With this approach, however, it is not easy to perform all the possible mutations, and the mutation sites selected are in many cases sporadic. Also, it is difficult to discern whether replacement directly changed a functional domain or changed a certain structure that then indirectly influenced the functional domain. In this study we have adopted a different approach in which a part of a sugar transporter is replaced in a systematic manner by the corresponding part of a homologous transporter having different substrate specificity. In this way it is possible to positively identify the substrate recognition site or at least the site in which substrates are differentially recognized in these two transporters. More importantly, the consecutive replacement of the protein sequence assures the localization of the functional site on a certain region and excludes the possible involvement of other regions. We have chosen Gal2 and Hxt2 transporters of yeast as closely related transporters and focused on galactose transport since Gal2 also transports glucose and the presence of several glucose transporters other than Hxt2 in S. cerevisiae(1) raises technical difficulties in analyzing glucose transport.
A DNA fragment containing GAL2 was cut out by EcoRI and PmaCI from a plasmid pS25(4) ,
provided by H. Ronne (Ludwig Institute for Cancer Research, Uppsala,
Sweden) and ligated to EcoRI and SmaI sites in a
multicloning site of pTV3 (YEp TRP1 bla)(12) ,
provided by J. Nikawa (Kyusyu Institute of Technology, Iizuka, Japan).
The nucleotide sequence immediately following the termination codon of GAL2, TAATGCGTT was modified to TAATCGATT by using PCR ()(13) to make a ClaI site. Then a single EcoRI site was disrupted by blunting with T4 DNA polymerase.
To make a cassette to replace GAL2 with HXT2 or
various chimeric genes, a new EcoRI site was introduced after
the initiation codon by using PCR. The nucleotide sequence,
ATGGCAGTTGAG was modified to ATGGCAGAATTC, which changed the deduced
amino acid sequence from Met-Ala-Val-Glu to Met-Ala-Glu-Phe. This
plasmid, GAL2-PTV3e, exhibited an unmodified galactose transport
activity when it was introduced to LBY416 (MAT
hxt2::LEU2
snf3::HIS3 lys2 ade2 trp1 his3 leu2 ura3)(6) , provided by
L. Bisson (University of California, Davis). Although the genotype of gal2 is not described in the original reference, we concluded
that LBY416 bears a gal2 mutation based on the following
observations. 1) Diploid constructed with a gal2 mutant,
STX7-3B (MATa gal2 met1 trp3 arg1 CUP
mal), obtained from the yeast genetic stock center
(University of California, Berkeley) showed Gal
phenotype and tetrad analysis led to
0Gal
:4Gal
segregation; 2) mating
with a GAL2 cell, W303-1a (MATa ade2-1
ura3-1 his3-11, 15 trp1-1 leu2-3, 112
can1-100), provided by Y. Nogi (Saitama Medical School,
Saitama, Japan) yielded Gal
diploid and tetrad
analysis of the diploid led to 2Gal
:2Gal
segregation; and 3) no appreciable high affinity galactose
transport in this cell was observed. The nucleotide sequence
immediately following the termination codon of HXT2 in plasmid
pAK5a, provided by L. Bisson, was modified from TAAGAGATT to TAATCGATT
to make a ClaI site. Since HXT2 has a EcoRI
site extending from the 7th to 12th nucleotide of the coding sequence, HXT2 is replaced with GAL2 in GAL2-pTV3e as a
cassette to make HXT2-pTV3e. Chimeras between HXT2 and GAL2 were created by the use of homologous recombination of E. coli except for H-G and G-H, which were constructed by PCR.
GAL2-pTV3e was cut with EcoRI and ClaI. The fragment
containing GAL2 was inserted between EcoRI and ClaI sites in the multicloning site of pGEM-7zf(+)
(Promega). An adaptor composed of CGGGCTCGAGCC was ligated to a ClaI site of HXT2-pTV3e to create a XhoI site, which
was digested with XhoI and SphI, and the fragment
containing HXT2 was inserted between XhoI and SphI sites of the above pGEM-7zf(+) containing GAL2 to create a plasmid containing HXT2 and GAL2 in
tandem. After cleaving the plasmid at the XhoI site situated
between HXT2 and GAL2, it was introduced to E.
coli JC8679(recBC sbcA)(14) , provided by T.
Ogawa (Osaka University). To isolate a single monomeric chimeric gene,
plasmids recovered from JC8679 were cleaved with SacI,
ligated, and introduced to E. coli F
Z
M15 recA(15) , provided by T. Fukasawa (Keio University),
which produced P series chimeras. Similarly, Q series chimeras was
obtained by using a plasmid containing GAL2 and HXT2 in tandem in pGEM-7zf(+). H series chimeras were made by the
use of a plasmid containing P74 of the P series and the latter half of HXT2 which was obtained with ApaI treatment. The
recombination site of each plasmid was first estimated by the
restriction mapping. The precise location of the recombination site of
each chimera was determined by a DNA sequencer (373A Applied
Biosystems) with a dye terminator method according to the
producer's protocol. A legitimate recombination was confirmed
with all the chimeric genes used in this study. Both strands of the
nucleotide sequence of the open reading frame was determined with GAL2, HXT2, H30, H-G, and G-H. One of the strands was
sequenced with other chimeras. Galactose transport activity was
measured with yeast cells grown in a synthetic medium (16) with
2% galactose as a carbon source supplemented with uracil, adenine, and
amino acids except for tryptophan. The cells were cultured at 30 °C
to an early log phase (OD
= 0.2-0.4) and
washed three times with a salt medium containing 0.1% KCl, 0.05%
MgSO
7H
O, 0.01% NaCl, and 0.01%
CaCl
2H
O. The initial rate of high
affinity galactose transport was assessed by the transport of 0.2
mMD-[
C]galactose (NEC-302X,
DuPont) for 15 s at 30 °C. The uptake reaction was started by the
addition of 20 µl of the isotope to 180 µl of the cell
suspension containing 0.8-6.7
10
cells in the
salt solution and terminated with the addition of 5 ml of cold salt
solution containing 0.5 mM HgCl
. The mixture was
filtered through a glass fiber filter (GF/F, Whatman), followed by a
20-ml wash with the cold salt solution containing HgCl
. The
radioactivity retained in the filter was measured with a liquid
scintillation counter. To assess the expression of Gal2, Hxt2, or
chimeric proteins, cells were washed with H
O and disrupted
by glass beads (Mini-Beadbeater, Biospec Products), and immunoblotting
of the cell homogenate was performed as described(17) .
Polyclonal rabbit antibody to Gal2 or Hxt2 was produced by using the
COOH-terminal 13 or 14 oligopeptide, respectively, which was coupled to
keyhole limpet hemocyanin.
A multicopy plasmid possessing GAL2, GAL2-pTV3e, was
constructed and introduced into LBY416, a strain of S. cerevisiae in which a high affinity galactose transport activity is not
observed and two major glucose transporter genes, HXT2 and SNF3, have been disrupted. Gal2 was expressed under control of GAL2 promoter in the presence of galactose. Galactose
transport in this transformant (GAL2 cells) is approximately linear for
2 min (Fig. 1A). HgCl inhibited the activity,
with an ID
of 15 µM. The cells harboring the
vector only (pTV3e cells) showed a low level of galactose transport,
which was slightly more than the background estimated by the
measurement in the presence of HgCl
. The cells harboring
HXT2-pTV3e (HXT2 cells) showed a low level of galactose transport
similar to pTV3e cells, indicating Hxt2 is not capable of transporting
galactose (data not shown). The initial rates of high affinity
galactose transport at 0.2 mM galactose expressed as
pmol/10
cells/15 s (n = 10) were 48.1
± 8.7 (mean ± S.D.) (GAL2 cells), 4.4 ± 1.1 (pTV3e
cells), and 4.1 ± 1.1 (HXT2 cells). Details of sugar transport
by Gal2 and Hxt2 will be reported elsewhere.
Figure 1:
Galactose
transport in yeast cells possessing the Gal2 or chimeric transporter.
The Gal2 or chimeric galactose transporter of S. cerevisiae was maximally expressed using the GAL2 promoter in a
multicopy plasmid, pTV3e, in a galactose transport-deficient cell,
LBY416. A, galactose transport in cells harboring the
multicopy plasmid GAL2-pTV3e (GAL2 cells) () or cells harboring
vector only (pTV3e cells) (
). Galactose transport in the presence
of 0.5 mM HgCl
in GAL2 cells (
) or pTV3e
cells (
) indicates the background of transport. B,
Eadie-Hofstee plots of galactose transport in GAL2 cells (
), H30
cells (
), or pTV3e cells (
). H30 transporter contains the
minimum Gal2 region among galactose transport-active chimeric
transporters. The lines are drawn with the use of parameters obtained
by a nonlinear least squares regression analysis (the program supplied
with KaleidaGraph, Synergy Software), assuming two independent
saturable transport systems.
Three types of chimeras
were constructed. The first series of chimeras, named P series, was
made by combining the 5`-portion of HXT2 and the 3`-portion of GAL2 (Fig. 2A). We have selected 13 chimeras,
recombination sites of which covered the entire open reading frame of
1722 bp. The galactose transport activities of these chimeric
transporters were significant but somewhat variable from P23 to P81.
Two chimeras, P28 and P39, did not exhibit a significant galactose
transport activity. These results suggest that the galactose
recognition requires the structure contained in the region coded by the
sequence from bp 1358 to bp 1722 (Ala-Glu
of Gal2) or, with a conservative view, bp 1303 to bp 1722
(Met
-Glu
of Gal2). The possibility
that the lack of galactose transport activity in P28 and P39 cells is
due to the lack of chimeric transporters in these cells was negated by
immunoblotting analysis of these cells (Fig. 3A). The level
of chimeric transporters are not equal, but a significant amount of
chimeric transporters are synthesized and retained in the P series
cells. The second set of chimeric transporters, the Q series, was
constructed by the reversion of the order, the 5`-portion of GAL2 and the 3`-portion of HXT2 (Fig. 2B). In
this series, only two chimeras, Q18 and Q9, showed galactose transport
activities. In contrast to the previous P series, chimeric transporters
were not detected by immunoblotting in most Q series cells except for
Q18, Q9, and Q14 cells (data not shown). Although it is difficult to
draw a conclusion from the results of the Q series chimeras alone, when
combined with previous data on the P series chimeras, a simple
hypothesis emerges that the galactose recognition site lies in the
region coded by the sequence extending to bp 1303-1620
(Met
-Pro
of Gal2). To test this
hypothesis, we constructed the H series chimeras containing limited
regions of GAL2 by making chimeras using P74 and HXT2 (Fig. 2C). The galactose transport activity of
these chimeras indicated that the region coded by bp 1303-1605 of GAL2 (Met
-Glu
) recognized
galactose as a substrate. The levels of expression of the chimeric
proteins were significant in all cases (Fig. 3B).
Therefore, H18 and H19 seem to be inactive in galactose transport.
Preliminary studies indicated that H18 and H19 are also inactive in
glucose transport. The possibility that H18 and H19 chimeric
transporters are active, but are retained in an intracellular organelle
and do not contribute to galactose transport, is not excluded in this
study.
Figure 2: Production of chimera between GAL2 and HXT2 and galactose transport activity. GAL2 and HXT2 are aligned using the malign program in ODEN package(36, 37, 38) . Since the open reading frame of GAL2 is longer than that of HXT2 and no gap is imposed, the nucleotide number of GAL2 is used as a reference. With each chimera, the first nucleotide of the recombination site is indicated. Galactose transport activities of chimeric transporters Gal2, Hxt2, or the vector only were assessed by measuring galactose transport in LBY416 transformed with the plasmid containing the corresponding gene. After subtracting the background obtained with pTV3e cells, the galactose transport activity was expressed relative to GAL2 cells. Transport activities shown are the average of two to five experiments. A, P series chimeras created with the 5`-portion of HXT2 and the 3`-portion of GAL2. B, Q series chimeras composed of the 5`-portion of GAL2 and the 3`-portion of HXT2. C, H series chimeras produced with P74 of the P series and HXT2.
Figure 3:
Detection of chimeric transporters with
immunoblotting. Gal2, Hxt2, or chimeric transporters in cell
homogenates are immunoblotted with antibodies to the Gal2 or Hxt2 COOH
terminus. Autoradiography of I-protein A (IM 144,
Amersham Corp.) was performed with imaging plates (BAS2000, Fuji Film). A, P series chimeras. For each lane, 3 µg of homogenate
was applied. Expression of a significant amount of chimeric
transporters was observed in all the cells harboring chimeric genes.
The origin of the low molecular weight cross-reacting material is not
known, but it disappeared when anti-Gal2 antibody was preincubated with
the COOH-terminal peptide used to raise the antibody. B, H
series chimeras. Five µg of homogenates were immunoblotted with
anti-Hxt2 COOH-terminal antibody.
Kinetic parameters of Gal2 and H30 were measured. Gal2
exhibited apparently two components of galactose transport that have
been observed in a previous study (3) (Fig. 1B). A high affinity component was on
the order of several millimolar and a low affinity component was on the
order of more than 20 mM, which was difficult to measure
accurately under the present assay conditions. The K and V
of the high affinity component
of Gal2-mediated transport were 2.8 ± 0.7 mM (mean
± S.D., n = 4) and 1.6 ± 0.4
nmol/10
cells/15 s, respectively. Among galactose
transport-active chimeric transporters so far obtained, H30, which
possessed a minimal Gal2 region, showed similarly two components of
galactose transport. The K
and V
of the high affinity component were 4.7
± 1.1 mM (n = 3) and 0.74 ± 0.04
nmol/10
cells/15 s, respectively. Thus, the K
of H30 is about 70% higher than that of
Gal2 and the V
of H30 is about 50% lower, which
is rather remarkable when we consider the fact that H30 retains only
18% of the Gal2 sequence. Substrate specificities of galactose
transport in GAL2 and H30 cells were studied by the addition of a
200-fold excess of several sugars. The order of inhibition was the same
in two cells: D-Gal > D-Glc >
2-deoxy-D-Glc, D-Fru > D-Man, D-Fuc, 3-o-methyl-D-Glc, D-Xyl,
6-deoxy-D-Glc. The degrees of inhibition were weaker with H30
cells than GAL2 cells, which may be due to lowered affinities for
substrates in H30 transporter.
We have constructed chimeric genes made by intramolecular recombination between two homologous genes using E. coli recombination system(14) . As noted previously(18, 19) , a problem in chimera studies is the fact that the chimeric proteins are not always expressed in cells. We have selected GAL2 and HXT2 as two homologous genes and produced three sets of chimeras. Of these, the P and H series chimeras were all successfully expressed and used for localizing a galactose recognition domain. The reason for our success is not clear at present. It may well be that the homology between GAL2 and HXT2 is high and that both are derived from and expressed in the same organism. It is intriguing to find out that Gal2 changes to a Hxt2-type glucose transporter when the COOH-terminal region is replaced with the corresponding region of Hxt2. One prerequisite to doing so is to look for a glucose analog that is recognized by Hxt2 but not by Gal2. Furthermore, refinement of the experimental system to decrease glucose transport by other glucose transporters in yeast is necessary.
Our present results clearly indicate that the limited GAL2 region of 303 bp coding for 101 amino acids that lies close to the
COOH terminus is important for the recognition of galactose. The region
contains putative TMs 10-12 and about 18 amino acid residues of
the hydrophilic COOH-terminal tail. The homology of this region is
relatively high so that only 30 out of 101 amino acid residue differ
between Gal2 and Hxt2. Thus, a few amino acids among these 30 may be
responsible for the recognition of galactose. Previous studies on the
transporters of the Glut family and the major facilitator superfamily
have pointed out functionally important amino acids or sequence in this
region: the replacement of Pro, Trp
,
Trp
, or Asn
(Pro
,
Trp
, Trp
, or Gly
in Gal2) in
Glut1 of animal
cells(20, 21, 22, 23) , the
replacement of Trp
in Glut3 (Trp
in
Gal2)(24) , deletion of the COOH-terminal tail of
Glut1(25) , and the replacement of the COOH-terminal tail of
Glut1 with the corresponding part of Glut2(26) . In contrast,
truncation of TM12 and the COOH-terminal tail did not abolish the
transport activity of the
-ketoglutarate transporter of E.
coli(27) . Particularly interesting observations have been
reported for Glut1. Truncation of the COOH-terminal 37 amino acids or
mutation of Pro
caused inhibition of glucose transport
and ATB-BMPA binding but no significant change in cytochalasin B
binding(20, 25) , which was interpreted as indicating
that Glut1 is locked into an inward-facing form(25) . The
opposite situation was observed with mutation of Trp
or
Asn
, which caused inhibition of glucose transport and
cytochalasin B labeling but no appreciable inhibition of
4,6-o-ethylidene-D-glucose or ATB-BMPA binding (22, 23) . This combination of impairment was
interpreted that Glut1 is locked in an outward facing
conformation(28) . Although, as noted in the Introduction,
these results obtained with localized mutagenesis are mostly loss of
function and are not all consistent with each other, it seems that
these results support the idea that this region plays important roles
on several aspects of transport other than the substrate recognition,
namely a pivotal role in the transition of conformational change
concomitant with substrate translocation, pore formation capability, or
inhibitor binding.
The mutation that impairs transport activity is
not limited to the COOH-terminal region; TMs 4, 5, 7, and 8 and the
NH-terminal or other hydrophilic regions are also
implicated in studies on Glut1 and Glut2 of mammalian glucose
transporters(28, 29, 30, 31, 32, 33) ,
Hup1 glucose transporter of Chlorella(34) , Snf3
glucose transporter of S. cerevisiae(35) , and KgtP
-ketoglutarate transporter of E. coli(27) . In
view of these results, it seems reasonable to assume as a working
hypothesis that more than one region is necessary for the substrate
recognition and that the COOH-terminal region we identified may
contribute to the recognition of the structure around C-4 of hexoses,
where D-glucose and D-galactose are epimeric. It is
also possible that there are two substrate binding sites(11) ,
the COOH-terminal region and the other region (possibly including TMs
4, 5, 7, and 8), where the former site recognizes C-4 position but the
latter site recognizes different portion(s) of hexoses. If this is the
case, Hxt2 would have provided in place of Gal2 the other binding site
in transport-active chimeric transporters. A wide variety of chimera
analysis using transporters of different substrate specificities will
contribute to clarify this essential subject in transport studies.