©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Substrate Recognition Domain of the Gal2 Galactose Transporter in Yeast Saccharomyces cerevisiae as Revealed by Chimeric Galactose-Glucose Transporters (*)

(Received for publication, November 7, 1994)

Kazuhisa Nishizawa Eriko Shimoda Michihiro Kasahara (§)

From the Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, Tokyo 192-03, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) . (^1)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(2)- 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.


EXPERIMENTAL PROCEDURES

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 (^2)(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 (MATalpha 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 CUPmal), 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 FZM15 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(4)bullet7H(2)O, 0.01% NaCl, and 0.01% CaCl(2)bullet2H(2)O. The initial rate of high affinity galactose transport was assessed by the transport of 0.2 mMD-[^14C]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 times 10^7 cells in the salt solution and terminated with the addition of 5 ml of cold salt solution containing 0.5 mM HgCl(2). 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(2). 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(2)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.


RESULTS AND DISCUSSION

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) (circle) or cells harboring vector only (pTV3e cells) (up triangle). Galactose transport in the presence of 0.5 mM HgCl(2) in GAL2 cells (bullet) or pTV3e cells () indicates the background of transport. B, Eadie-Hofstee plots of galactose transport in GAL2 cells (circle), H30 cells (up triangle), 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(max) 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/10^7 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(max) of the high affinity component were 4.7 ± 1.1 mM (n = 3) and 0.74 ± 0.04 nmol/10^7 cells/15 s, respectively. Thus, the K of H30 is about 70% higher than that of Gal2 and the V(max) 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 alpha-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(2)-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 alpha-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.


FOOTNOTES

*
This work was supported by grants from the Ministry of Education, Science and Culture of Japan and Ono Pharmaceutical Co. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, Tokyo 192-03, Japan. Tel.: 81-426-78-3261; Fax: 81-426-75-0025.

(^1)
M. Kasahara and E. Shimoda, unpublished observation.

(^2)
The abbreviations used are: PCR, polymerase chain reaction; TM, transmembrane segment; ATB-BMPA, 2-N-4-(1-azi-2,2,2-trifluorethyl)benzoyl-1,3-bis(D-mannos-4-yloxyl)-2-propylamine; bp, base pair(s).


ACKNOWLEDGEMENTS

We thank T. Fukasawa for discussion and E. coli cell and L. Bisson, J. Nikawa, Y. Nogi, T. Ogawa, and H. Ronne for E. coli cells, plasmids, and/or yeast cells.


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