Portal de Periódicos da CAPES

Contribution to Substrate Recognition of Two Aromatic Amino Acid Residues in Putative Transmembrane Segment 10 of the Yeast Sugar Transporters Gal2 and Hxt2

1998; Elsevier BV; Volume: 273; Issue: 44 Linguagem: Inglês

10.1074/jbc.273.44.29106

1083-351X

Michihiro Kasahara, Mari Maeda,

Fermentation and Sensory Analysis

The comprehensive study of chimeras between the Gal2 galactose transporter and the Hxt2 glucose transporter of Saccharomyces cerevisiae has shown that Tyr446is essential and Trp455 is important for galactose recognition by Gal2. Consistent with this finding, replacement of the corresponding Phe431 and Tyr440 residues of Hxt2 with Tyr and Trp, respectively, allowed Hxt2 to transport galactose, suggesting that the two amino acid residues in putative transmembrane segment 10 play a definite role in galactose recognition (Kasahara, M., Shimoda, E., and Maeda, M. (1997) J. Biol. Chem. 272, 16721–16724). Replacement of Trp455 of Gal2 with any of the other 19 amino acids was shown to reduce galactose transport activity to between 0 and <20% of that of wild-type Gal2. The role of Phe431 in Hxt2 was similarly studied. Other than Phe, only Tyr at position 431 was able to support glucose transport activity, at the reduced level of <20%. In contrast, replacement of Tyr440 of Hxt2 with other amino acids revealed that most replacements, with the exception of Pro and charged amino acids, supported glucose transport activity. The importance of residue 431 in sugar recognition was more pronounced in a modified Hxt2 in which Tyr440 was replaced with Trp. Glucose transport was supported only by the aromatic amino acids Phe, Tyr, and Trp at position 431, and galactose transport was supported only by Tyr. These results suggest that an aromatic amino acid located in the middle of transmembrane segment 10 (Tyr446 in Gal2 and Phe431 in Hxt2) plays a critical role in substrate recognition in the yeast sugar transporter family to which Gal2 and Hxt2 belong. The comprehensive study of chimeras between the Gal2 galactose transporter and the Hxt2 glucose transporter of Saccharomyces cerevisiae has shown that Tyr446is essential and Trp455 is important for galactose recognition by Gal2. Consistent with this finding, replacement of the corresponding Phe431 and Tyr440 residues of Hxt2 with Tyr and Trp, respectively, allowed Hxt2 to transport galactose, suggesting that the two amino acid residues in putative transmembrane segment 10 play a definite role in galactose recognition (Kasahara, M., Shimoda, E., and Maeda, M. (1997) J. Biol. Chem. 272, 16721–16724). Replacement of Trp455 of Gal2 with any of the other 19 amino acids was shown to reduce galactose transport activity to between 0 and <20% of that of wild-type Gal2. The role of Phe431 in Hxt2 was similarly studied. Other than Phe, only Tyr at position 431 was able to support glucose transport activity, at the reduced level of <20%. In contrast, replacement of Tyr440 of Hxt2 with other amino acids revealed that most replacements, with the exception of Pro and charged amino acids, supported glucose transport activity. The importance of residue 431 in sugar recognition was more pronounced in a modified Hxt2 in which Tyr440 was replaced with Trp. Glucose transport was supported only by the aromatic amino acids Phe, Tyr, and Trp at position 431, and galactose transport was supported only by Tyr. These results suggest that an aromatic amino acid located in the middle of transmembrane segment 10 (Tyr446 in Gal2 and Phe431 in Hxt2) plays a critical role in substrate recognition in the yeast sugar transporter family to which Gal2 and Hxt2 belong. transmembrane polymerase chain reaction. The yeast Saccharomyces cerevisiae possesses nearly 20 homologous genes that potentially encode proteins belonging to the sugar transporter family (1Baldwin S.A. Biochim. Biophys. Acta. 1993; 1154: 17-49Crossref PubMed Scopus (280) Google Scholar, 2Bisson L.F. Coons D.M. Kruckeberg A.L. Lewis D.A Crit. Rev. Biochem. Mol. Biol. 1993; 28: 259-308Crossref PubMed Scopus (211) Google Scholar, 3Kruckeberg A.L. Arch. Microbiol. 1996; 166: 283-292Crossref PubMed Scopus (205) Google Scholar, 4Nelissen B. De Wachter R. Goffeau A. FEMS Microbiol. Rev. 1997; 21: 113-134Crossref PubMed Google Scholar). This family includes both Hxt2, a major high affinity glucose transporter and Gal2, a major high affinity galactose transporter that also transports glucose with almost the same affinity (2Bisson L.F. Coons D.M. Kruckeberg A.L. Lewis D.A Crit. Rev. Biochem. Mol. Biol. 1993; 28: 259-308Crossref PubMed Scopus (211) Google Scholar, 5Nishizawa K. Shimoda E. Kasahara M. J. Biol. Chem. 1995; 270: 2423-2426Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). We have previously taken a three-step comprehensive approach to identify the amino acid residues responsible for substrate recognition in Gal2 and Hxt2 (5Nishizawa K. Shimoda E. Kasahara M. J. Biol. Chem. 1995; 270: 2423-2426Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 6Kasahara M. Shimoda E. Maeda M. FEBS Lett. 1996; 389: 174-178Crossref PubMed Scopus (22) Google Scholar, 7Kasahara M. Shimoda E. Maeda M. J. Biol. Chem. 1997; 272: 16721-16724Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In the first step (5Nishizawa K. Shimoda E. Kasahara M. J. Biol. Chem. 1995; 270: 2423-2426Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), three types of systematic chimeras between Gal2 and Hxt2 were constructed with the use of the Escherichia coli homologous recombination system. The site responsible for differential recognition of galactose and glucose was localized to a COOH-terminal region of 101 amino acids. In the second step (6Kasahara M. Shimoda E. Maeda M. FEBS Lett. 1996; 389: 174-178Crossref PubMed Scopus (22) Google Scholar), the 101-amino acid region was subdivided into transmembrane (TM)1 segment 10, TM11, TM12, and the proximal half of the COOH-terminal hydrophilic tail by introducing five restriction enzyme sites into the corresponding segment of each gene, without changing the encoded amino acids. By analyzing the 16 clones encoding all possible combinations of subdomains, we identified TM10 as the segment responsible for differential recognition of galactose and glucose. In the third step (7Kasahara M. Shimoda E. Maeda M. J. Biol. Chem. 1997; 272: 16721-16724Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), a mixture of ∼25,000 distinct plasmids that encoded all possible combinations of the 12 amino acids in TM10 that differ between Gal2 and Hxt2 was produced. All the 19 galactose transport-positive clones selected on galactose-limited agar plates encoded transporters containing the Tyr446 residue of Gal2 (see Fig. 8). Fourteen of the 19 clones also encoded Trp455 of Gal2 and the other five encoded Cys455, a residue not found in either Gal2 or Hxt2. To confirm that Tyr446 is important for substrate recognition, we replaced this residue of Gal2 with each of the other 19 amino acids. None of the resulting transporters exhibited galactose transport activity. Replacement of Phe431 and Tyr440 of Hxt2 with the corresponding Tyr and Trp of Gal2 allowed the modified Hxt2 to transport galactose. These results demonstrated that Tyr446is essential and Trp455 is important for the discrimination of galactose and glucose by Gal2. The present study was designed to answer the following questions: (i) Is the apparently essential role of Tyr446 in substrate recognition by Gal2 attributable to a contribution to the affinity of the transporter for galactose? (ii) Which amino acid residues can substitute for Trp455 of Gal2? (iii) Is Phe431of Hxt2 essential for glucose recognition? (iv) Is Tyr440of Hxt2 important for glucose recognition? We obtained the results indicating that Tyr446 is exclusively required for galactose recognition and two aromatic amino acids in TM10 play important roles in substrate recognition both in Gal2 and Hxt2. The cassette vector GAL2-pTV3e was constructed as described previously (5Nishizawa K. Shimoda E. Kasahara M. J. Biol. Chem. 1995; 270: 2423-2426Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Briefly,GAL2 (8Nehlin J.O. Carlberg M. Ronne H. Gene (Amst.). 1989; 85: 313-319Crossref PubMed Scopus (76) Google Scholar) was introduced into the YEp-based vector pTV3 (YEpTRP1 bla) (9Rose M.D. Broach J.R. Methods Enzymol. 1991; 194: 195-230Crossref PubMed Scopus (214) Google Scholar). After the disruption of an EcoRI site situated at the boundary of GAL2 and the vector by blunting, a new EcoRI site was introduced immediately downstream of the initiation codon by PCR (10Pont-Kington G. BioTechniques. 1994; 16: 1010-1011PubMed Google Scholar), which resulted in a change in the encoded amino acid sequence from Met-Ala-Val-Glu to Met-Ala-Glu-Phe. A new ClaI site was also introduced immediately downstream of the termination codon of GAL2. Since HXT2 contains a single EcoRI site at the position corresponding to that of the newly created site in GAL2, it was necessary to introduce only a newClaI site at the position corresponding to that in GAL2. These two sites were used to replace the open reading frame of GAL2 in GAL2-pTV3e with that of HXT2, yielding HXT2-pTV3e. Both GAL2-pTV3e and HXT2-pTV3e were further modified to create five restriction enzyme sites (SacI,MluI, SpeI, StuI, and NcoI) in the distal half of each gene (6Kasahara M. Shimoda E. Maeda M. FEBS Lett. 1996; 389: 174-178Crossref PubMed Scopus (22) Google Scholar), yielding GAL2d-pTV3e and HXT2d-pTV3e, respectively (the StuI site in GAL2 and the NcoI site in HXT2 are preexisting sites). Residues Tyr446 and Trp455 of Gal2 and the corresponding Phe431 and Tyr440 of Hxt2 were targeted for by each of the other 19 amino acids. The nucleotide sequence of Gal2 coding for Trp455 was randomly modified by PCR with a degenerate primer. A forward primer 5′-CACGG TAAAA GCCAG CCGAG CTCTA AAGGT GCCGG TA encompassing the SacI site and a degenerate reverse primer 5′-TTCGA CTTGA CGCGT AGTGG GAATG ATTCT GCTGT GATGA CNNNG GCA AC TGGGG CCCAG GT that creates a new ApaI site at a position corresponding to that in HXT2 were used for long PCR (Ex Taq, Takara). Twenty-five cycles of 95 °C for 30 s, 50 °C for 2 min and 72 °C for 1 min were performed with a thermocycler (model 2400, Perkin-Elmer). The PCR products were digested with SacI and MluI and introduced into the corresponding position of GAL2d-pTV3e. A total of 40 clones was sequenced, and 14 encoding the desired amino acids were selected (Table I). The remaining five clones were generated with the use of a specific primer for each amino acid. This series of clones was designated Gal2(Y-X). Replacement of Tyr446 with each of the other 19 amino acids was performed in a similar manner, with this series of clones designated Gal2(X-W). For the Hxt2 transporter, four series of clones, designated Hxt2(X-Y), Hxt2(F-X), Hxt2(X-W), and Hxt2(Y-X) according to the same nomenclature were produced.Table ICodon usage for mutation of GAL2 and HXT2Gal2(X-W)Gal2(Y-X)Hxt2(X-Y)Hxt2(F-X)Hxt2(X-W)Hxt2(Y-X)X446CodonW455Y446X455CodonX431CodonY440F431X440CodonX431CodonW440Y431X440CodonGGGGGGGCGGGAGGGGGGGAGGGGAGCUAGCCAGCGAGCUAGCGAGCUVGUGVGUCVGUGUAUVGUUVGUGVGUULCUULUUGLUUGLCUALUUGLUUAIAUAIAUAIAUAIAUUIAUAIAUUFUUUFUUUFUUCUACUUUFUUUFUUUFUUUWUGGWUGGWUGGWUGGWUGGWUGGMAUGMAUGMAUGMAUGMAUGMAUGCUGUCUGUCUGUCUGUCUGUCUGUPCCUUGGUAUPCCCPCCAPCCUPCCUUGGUAUPCCUSAGUSUCCSAGUSUCUSUCGSUCUTACUTACUTACUTACGTACUTACUYUAUYUACYUAUUAUUUCYUACYUAUYUAUNAACNAAUNAAUNAAUNAAUNAAUQCAAQCAAQCAAQCAGQCAAQCAGDGAUDGAUDGAUUUUDGAUDGAUDGAUEGAAEGAAEGAAEGAAEGAAEGAAHCAUHCAUHCAUHCAUHCAUHCAUKAAAKAAAKAAAKAAAKAAAKAAARCGURAGARCGARAGARCGARAGAThe nucleotide sequences of GAL2 and HXT2 were modified to encode Gal2(X-W), Gal2(Y-X), Hxt2(X-Y), Hxt2(F-X), Hxt2(X-W), and Hxt2(Y-X) transporters as described under "Experimental Procedures." The original codons are UAU(Y446) in GAL2 and UUC(F431) and UAC(Y440) in HXT2. Open table in a new tab The nucleotide sequences of GAL2 and HXT2 were modified to encode Gal2(X-W), Gal2(Y-X), Hxt2(X-Y), Hxt2(F-X), Hxt2(X-W), and Hxt2(Y-X) transporters as described under "Experimental Procedures." The original codons are UAU(Y446) in GAL2 and UUC(F431) and UAC(Y440) in HXT2. The nucleotide sequences of the substituted fragments and the surrounding regions in each clone were verified by sequencing both strands with a DNA sequencer (model 373A, Perkin-Elmer). Yeast cells were cultured as described (5Nishizawa K. Shimoda E. Kasahara M. J. Biol. Chem. 1995; 270: 2423-2426Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 6Kasahara M. Shimoda E. Maeda M. FEBS Lett. 1996; 389: 174-178Crossref PubMed Scopus (22) Google Scholar, 7Kasahara M. Shimoda E. Maeda M. J. Biol. Chem. 1997; 272: 16721-16724Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Transport of galactose or glucose in yeast strain LBY416(MATα hxt2::LEU2 snf3::HIS3 gal2 lys2 ade2 trp1 his3 leu2 ura3) harboring the various Gal2 or Hxt2 plasmids was measured at 30 °C for 5 s (7Kasahara M. Shimoda E. Maeda M. J. Biol. Chem. 1997; 272: 16721-16724Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Transport activities with 0.1 mmgalactose or glucose were expressed as picomoles/107cells/5 s. For comparison, the background obtained with control cells harboring the empty vector was subtracted from transport activity obtained with each clone, and the activity was normalized by expression as a percentage of that obtained with cells expressing Gal2 (galactose transport) or Hxt2 (glucose transport). Although in some instances, values of <10% were significant by Student's t test (p < 0.05), we considered only values of >10% as significant, since cell numbers determined with Burker-Turk cell counters were somewhat variable, and some transporters showed activities that were less than the control values, sometimes as low as −5 to −10%. Immunoblot analysis of cell homogenates was performed as described previously (5Nishizawa K. Shimoda E. Kasahara M. J. Biol. Chem. 1995; 270: 2423-2426Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Briefly, polyclonal rabbit antibody to Gal2 or Hxt2 was produced by using the COOH-terminal 13 or 14 oligopeptide, respectively, that was coupled to keyhole limpet hemocyanin. Yeast cells grown to an early log phase were washed with H2O and disrupted with glass beads. The homogenate was subjected to SDS-gel electrophoresis and blotted onto a polyvinylidine difluoride membrane (Immobilon PSQ, Millipore), followed by incubation with125I-protein A (IM144, Amersham Pharmacia Biotech) overnight. Autoradiography was performed with imaging plates (BAS2000, Fuji Film). Under the present conditions, a linear relation of amounts of protein and radioactivity was observed. These antibodies reacted with Gal2 or Hxt2, but not with other homologous proteins revealed by the yeast genome sequence (5Nishizawa K. Shimoda E. Kasahara M. J. Biol. Chem. 1995; 270: 2423-2426Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 6Kasahara M. Shimoda E. Maeda M. FEBS Lett. 1996; 389: 174-178Crossref PubMed Scopus (22) Google Scholar). To evaluate the possible degradation of modified Hxt2 transporters in vivo and in vitro, we used the protease-deficient strain BJ3505 (MAT a pep4::HIS3 prb1-Δ1.6R his3 lys2 trp1 ura3 gal2 can1) in place of LBY416 or added a mixture of protease inhibitors consisting of 5 mm EDTA, pepstatin A (2 μg/ml), leupeptin (2 μg/ml), aprotinin (100 units/ml), and 2 mm phenylmethylsulfonyl fluoride (final concentrations) to the homogenization solution. Neither approach appeared to affect the amounts of these transporters. Our previous studies with systematic series of chimeras of Gal2 and Hxt2 revealed that Tyr446 in Gal2 is essential for the differential recognition of galactose and glucose (5Nishizawa K. Shimoda E. Kasahara M. J. Biol. Chem. 1995; 270: 2423-2426Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 6Kasahara M. Shimoda E. Maeda M. FEBS Lett. 1996; 389: 174-178Crossref PubMed Scopus (22) Google Scholar, 7Kasahara M. Shimoda E. Maeda M. J. Biol. Chem. 1997; 272: 16721-16724Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). To characterize further the role of Tyr446 in substrate recognition, we measured transport of galactose or glucose by Gal2(X-W) transporters at a substrate concentration (10 mm) 100 times that used previously (Fig. 1). Even at this high substrate concentration, substantial galactose transport was mediated only by Gal2(Y-W); Gal2(F-W) showed 12% of the galactose transport activity of Gal2, whereas the other Gal2(X-W) transporters showed no significant activity (Fig. 1 A). Glucose transport at the high substrate concentration was mediated by many of the Gal2(X-W) transporters (Fig. 1 B). Gal2(Y-W) and Gal2(F-W) were most active, with Gly, Ala, Val, Leu, Met, Cys, Ser, Thr, and Asn substitutions also conferring transport activity at the high glucose concentration. Comparison of glucose transport activities between substrate concentrations of 0.1 and 10 mm indicates that the transporters with glucose transport activities of ∼20% at 10 mm glucose possess K m values of ∼100 mm. The differences in transport activity were not due to differences in the extent of expression of Gal2(X-W) transporters, as revealed by immunoblot analysis of cell homogenates (Fig. 2 A).Figure 2Expression of Gal2(X-W) and Gal2(Y-X) transporters as detected by immunoblot analysis. LBY416 cells harboring plasmids containing GAL2, each of 20 Gal2(X-W) genes (A), or each of 20 Gal2(Y-X) genes (B) were cultured to early log phase and homogenized. A portion of each homogenate (10 μg of protein) was subjected to immunoblot analysis with antibodies to the COOH terminus of Gal2 and125I-labeled protein A (5Nishizawa K. Shimoda E. Kasahara M. J. Biol. Chem. 1995; 270: 2423-2426Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The 53,000 (53k) recombinant transporters were detected by autoradiography with imaging plates (BAS2000, Fuji Film).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We have previously shown that Trp455 of Gal2 is important for the differential recognition of galactose and glucose (7Kasahara M. Shimoda E. Maeda M. J. Biol. Chem. 1997; 272: 16721-16724Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The replacement of Trp455 with any of the other 19 amino acids markedly reduced galactose transport to Trp, as well as with Gal2(X-W) transporters, which showed the same rank order of preference at the low glucose concentration. These observations suggest that the glucose recognition process is similar in both transporters. The apparently different roles of the middle aromatic site in galactose and glucose transport might be indicative of multiple functions of the amino acid at this site (see below). With regard to the cytoplasmic aromatic site, galactose transport by Gal2(Y-X) transporters was preferentially supported by Trp, with several other amino acids, including Tyr > Cys ≈ Met ≈ Thr ≈ Ile, also conferring activity. Glucose transport by Hxt2(F-X) transporters was supported by most amino acids, excluding Pro and charged residues. Similar patterns of glucose transport activities were observed with Hxt2(Y-X) and Gal2(Y-X) transporters, although Tyr at the middle aromatic site appeared to reduce the activities of these series. These results indicated that the interaction of the cytoplasmic site with glucose may be similar in Gal2 and Hxt2. It should be mentioned that the variable level of expression of Hxt2(F-X) and Hxt2(Y-X) transporters revealed by immunoblot analysis may not necessarily reflect the amounts of the transporters in intact cells, since the expression level of Gal2(Y-X) transporters was not variable and yet they showed a pattern of glucose transport activity similar to those of the Hxt2(F-X) and Hxt2(Y-X) transporters. A total of 18 closely related sugar transporters has been identified in S. cerevisiae, the cluster I of sugar permease homologs (4Nelissen B. De Wachter R. Goffeau A. FEMS Microbiol. Rev. 1997; 21: 113-134Crossref PubMed Google Scholar) that is equivalent to the hexose transporter family (3Kruckeberg A.L. Arch. Microbiol. 1996; 166: 283-292Crossref PubMed Scopus (205) Google Scholar), excluding Snf3 and Rgt2, which appear to be glucose sensor (11Ozcan S. Dover J. Rosenwald A.G. Wolfl S. Johnston M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12428-12432Crossref PubMed Scopus (347) Google Scholar). Of these 18 homologs, only Gal2 contains Tyr at the middle aromatic site and Trp at the cytoplasmic site. All the other 17 homologs, including Hxt2, contain Phe at the middle site. At the cytoplasmic site, Tyr is present in most homologs and Phe in Hxt4, Hxt12, and Hxt14. Together with our demonstration that Phe was the second most active residue in Hxt2(F-X) transporters in terms of glucose transport, these observations are consistent with the idea that Gal2 is the only galactose transporter and that the other 17 transporters are glucose transporters. If this is the case, low affinity galactose transport noted in previous studies (2Bisson L.F. Coons D.M. Kruckeberg A.L. Lewis D.A Crit. Rev. Biochem. Mol. Biol. 1993; 28: 259-308Crossref PubMed Scopus (211) Google Scholar) may be attributable to one or more of the glucose transporters carrying galactose with low affinity. This notion is also consistent with the selection of galactose transport-positive clones fromgal2 mutants (7Kasahara M. Shimoda E. Maeda M. J. Biol. Chem. 1997; 272: 16721-16724Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) and the selection of glucose transport-positive clones from strains carrying gal2 and multiple hxt mutations in the presence of antimycin (12Liang H. Ko C.H. Herman T. Gaber R.F. Mol. Cell. Biol. 1998; 18: 926-935Crossref PubMed Scopus (26) Google Scholar). Several functions for the two aromatic sites in galactose transport by Gal2 have been proposed (7Kasahara M. Shimoda E. Maeda M. J. Biol. Chem. 1997; 272: 16721-16724Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), including roles in steric hindrance, hydrogen bonding, a stacking effect with the sugar, and structural changes in other amino acids that interact directly with the sugar. In addition, the possibility that the two amino acids function independently has also been suggested. Recent structural studies on porins (13Dutzler R. Wang Y.F. Rizkallah P. Rosenbusch J.P. Schirmer T. Structure. 1996; 4: 127-134Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 14Forst D. Welte W. Wacker T. Diederichs K. Nat. Struct. Biol. 1998; 5: 37-46Crossref PubMed Scopus (209) Google Scholar) may be worth mentioning in this respect. The three-dimensional structures of maltoporin and the sucrose-specific porin SrcY analyzed by x-ray crystallography indicate that maltose and sucrose passes through the corresponding porins via a relay of aromatic amino acids. Since most galactose and glucose molecules are in the form of β- and α-pyranose in aqueous solution (15Vyas M.N. Vyas N.K. Quiocho F.A. Biochemistry. 1994; 33: 4762-4768Crossref PubMed Scopus (71) Google Scholar), differential recognition of galactose and glucose requires differentiation between α- and β-anomers and C4 epimers. Crystallographic studies of binding proteins in the periplasm of bacteria (16Quiocho F.A. Biochem. Soc. Trans. 1993; 21: 442-448Crossref PubMed Scopus (71) Google Scholar) and of lectins (17Weiss W.I. Drickamer K. Annu. Rev. Biochem. 1996; 65: 441-448Crossref PubMed Scopus (1017) Google Scholar), in addition to the porin studies, have shown that the binding of sugars to these proteins is mediated by hydrogen bonding to various amino acids and H2O and stacking of the sugars with aromatic amino acids. Thus, further insight into the molecular mechanism of substrate recognition by Gal2 and Hxt2 should be provided by identification of the amino acid residues that presumably form hydrogen bonds with galactose and glucose located at the middle site. In addition, it will be important to determine whether other aromatic amino acids in these transporters contribute to substrate binding. We thank L. Bisson (University of California, Davis) for yeast strain LBY416 and the HXT2-containing plasmid pAK5a; H. Ronne (Uppsala University, Sweden) for theGAL2-containing plasmid pS25; J. Nikawa (Kyushu Institute of Technology, Iizuka, Japan) for plasmid pTV3; and Y. Ohsumi (National Institute for Basic Biology, Okazaki, Japan) for yeast strain BJ3505.