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• W2088263411 abstract "The cDNAs HUP1 and HUP2 of Chlorella kessleri code for monosaccharide/H+ symporters that can be functionally expressed in Schizosaccharomyces pombe. By random mutagenesis three HUP1 mutants with an increasedK m value for d-glucose were isolated. The 40-fold increase in K m of the first mutant is due to the amino acid exchange N436I in putative transmembrane helix XI. Two substitutions were found in a second (G97C/I303N) and third mutant (G120D/F292L), which show a 270-fold and 50-fold increase inK m for d-glucose, respectively. An investigation of the individual mutations revealed that the substitutions I303N and F292L (both in helix VII) cause theK m shifts seen in the corresponding double mutants. These mutations together with those previously found support the hypothesis that helices V, VII, and XI participate in the transmembrane sugar pathway.Whereas for most mutants obtained so far the K m change for d-glucose is paralleled by a corresponding change for other hexoses tested, the exchange D44E exclusively alters the K m for d-glucose. Moreover the pH profile of this mutant is shifted by more than 2 pH units to alkaline values, indicating that the activity of the transporter may require deprotonation of the corresponding carboxyl group.Chimeric transporters were constructed to study the 100-fold lower affinity for d-galactose of the HUP1 symporter as compared with that of the HUP2 protein. A crucial determinant for the differential d-galactose recognition was shown to be associated with the first external loop. The effect could be pinpointed to a single amino acid change: replacement of Asn-45 of HUP1 with isoleucine, the corresponding amino acid of HUP2, yields a transporter with a 20 times higher affinity for d-galactose. The reverse substitution (I47N) decreases the affinity of HUP2 ford-galactose 20-fold. The cDNAs HUP1 and HUP2 of Chlorella kessleri code for monosaccharide/H+ symporters that can be functionally expressed in Schizosaccharomyces pombe. By random mutagenesis three HUP1 mutants with an increasedK m value for d-glucose were isolated. The 40-fold increase in K m of the first mutant is due to the amino acid exchange N436I in putative transmembrane helix XI. Two substitutions were found in a second (G97C/I303N) and third mutant (G120D/F292L), which show a 270-fold and 50-fold increase inK m for d-glucose, respectively. An investigation of the individual mutations revealed that the substitutions I303N and F292L (both in helix VII) cause theK m shifts seen in the corresponding double mutants. These mutations together with those previously found support the hypothesis that helices V, VII, and XI participate in the transmembrane sugar pathway. Whereas for most mutants obtained so far the K m change for d-glucose is paralleled by a corresponding change for other hexoses tested, the exchange D44E exclusively alters the K m for d-glucose. Moreover the pH profile of this mutant is shifted by more than 2 pH units to alkaline values, indicating that the activity of the transporter may require deprotonation of the corresponding carboxyl group. Chimeric transporters were constructed to study the 100-fold lower affinity for d-galactose of the HUP1 symporter as compared with that of the HUP2 protein. A crucial determinant for the differential d-galactose recognition was shown to be associated with the first external loop. The effect could be pinpointed to a single amino acid change: replacement of Asn-45 of HUP1 with isoleucine, the corresponding amino acid of HUP2, yields a transporter with a 20 times higher affinity for d-galactose. The reverse substitution (I47N) decreases the affinity of HUP2 ford-galactose 20-fold. The green alga Chlorella kessleri possesses an inducible transport system, capable of accumulative uptake of a variety of monosaccharides using an electrochemical proton gradient as driving force (1Tanner W. Biochem. Biophys. Res. Commun. 1969; 36: 278-283Crossref PubMed Scopus (81) Google Scholar, 2Komor E. FEBS Lett. 1973; 38: 16-18Crossref PubMed Scopus (97) Google Scholar, 3Komor E. Tanner W. J. Gen. Physiol. 1974; 64: 568-581Crossref PubMed Scopus (100) Google Scholar, 4Komor E. Tanner W. Eur. J. Biochem. 1976; 70: 197-204Crossref PubMed Scopus (125) Google Scholar). Three cDNAs coding for highly homologousChlorella monosaccharide/H+ symporters were cloned by differential screening (5Sauer N. Tanner W. FEBS Lett. 1989; 259: 43-46Crossref PubMed Scopus (126) Google Scholar, 6Stadler R. Wolf K. Hilgarth C. Tanner W. Sauer N. Plant Physiol. 1995; 107: 33-41Crossref PubMed Scopus (53) Google Scholar) and namedHUP1−3 (hexose uptakeprotein). Their identities have been confirmed by heterologous expression in Schizosaccharomyces pombe (6Stadler R. Wolf K. Hilgarth C. Tanner W. Sauer N. Plant Physiol. 1995; 107: 33-41Crossref PubMed Scopus (53) Google Scholar, 7Sauer N. Caspari T. Klebl F. Tanner W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7949-7952Crossref PubMed Scopus (67) Google Scholar). Furthermore, the HUP1 transporter retains its uptake activity after solubilization from the membrane of transgenic fission yeast, purification to homogeneity, and reconstitution into proteoliposomes (8Opekarová M. Caspari T. Tanner W. Biochim. Biophys. Acta. 1994; 1194: 149-154Crossref PubMed Scopus (13) Google Scholar, 9Caspari T. Robl I. Stolz J. Tanner W. Plant J. 1996; 10: 1045-1053Crossref PubMed Scopus (17) Google Scholar). The HUP symporters belong to a large family of substrate transporters, called the “major facilitator superfamily” (10Marger M.D. Saier Jr., M.H. Trends Biochem. Sci. 1993; 18: 13-20Abstract Full Text PDF PubMed Scopus (756) Google Scholar). Members of this major facilitator superfamily are thought to consist of 12 α-helical transmembrane segments connected by internal and external loops. Support for this topological model comes from alkaline phosphatase fusion protein analysis of the Escherichia coli lactose permease lacY (11Calamia J. Manoil C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4937-4941Crossref PubMed Scopus (232) Google Scholar) and N-glycosylation scanning mutagenesis studies on the human glucose facilitator GLUT1 (12Hresko R.C. Kruse M. Strube M. Mueckler M. J. Biol. Chem. 1994; 269: 20482-20488Abstract Full Text PDF PubMed Google Scholar). However, hard structural data on the nature of the binding sites and translocation pathways of substrates and cosubstrates have not been obtained. Since no three-dimensional structure of a transporter is in sight, one has to be content with indirect evidence, deduced for example from mutagenesis studies. Structure-function analysis of the HUP1 transporter (13Caspari T. Stadler R. Sauer N. Tanner W. J. Biol. Chem. 1994; 269: 3498-3502Abstract Full Text PDF PubMed Google Scholar, 14Will A. Caspari T. Tanner W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10163-10167Crossref PubMed Scopus (29) Google Scholar) was carried out in a sugar uptake deficient S. pombe strain (15Milbradt B. Höfer M. Microbiol. 1994; 140: 2617-2623Crossref PubMed Scopus (31) Google Scholar). Several mutants with an increased K m value ford-glucose uptake were found by site-directed mutagenesis (13Caspari T. Stadler R. Sauer N. Tanner W. J. Biol. Chem. 1994; 269: 3498-3502Abstract Full Text PDF PubMed Google Scholar) and by polymerase chain reaction random mutagenesis with subsequent selection for decreased sensitivity toward the toxic sugar 2-deoxyglucose (14Will A. Caspari T. Tanner W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10163-10167Crossref PubMed Scopus (29) Google Scholar). The affected amino acids cluster in the middle of the transmembrane helices V (Gln-179), VII (Gln-298 and Gln-299), and XI (Val-433 and Asn-436), with the exception of Asp-44 putatively located at the beginning of the first external loop (Fig. 1). The fact that predominantly acidic amino acids and their amides were identified correlates well with the finding that binding sites of periplasmic sugar-binding proteins are built up by such residues (16Quiocho F.A. Vyas N.K. Nature. 1984; 310: 381-386Crossref PubMed Scopus (237) Google Scholar, 17Vyas N.K. Vyas M.N. Quiocho F.A. Science. 1988; 242: 1290-1295Crossref PubMed Scopus (331) Google Scholar). The symporters HUP1 and HUP2 differ significantly in their substrate specificity (6Stadler R. Wolf K. Hilgarth C. Tanner W. Sauer N. Plant Physiol. 1995; 107: 33-41Crossref PubMed Scopus (53) Google Scholar, 18Will A. Tanner W. FEBS Lett. 1996; 381: 127-130Crossref PubMed Scopus (16) Google Scholar). Especially, the affinity ford-galactose is more than 100 times higher for the HUP2 protein. The amino acids of the HUP1 protein probably involved in substrate recognition (see above) are also present in the HUP2 transporter. The different substrate specificities of the two transporters must, therefore, be determined by differing residues at still unidentified positions. Recently, a study using chimeric proteins revealed that the exchange of a 30-amino acid span at the beginning of the first extracellular loop of HUP1 for that of HUP2 increases the affinity for d-galactose by about 15-fold (18Will A. Tanner W. FEBS Lett. 1996; 381: 127-130Crossref PubMed Scopus (16) Google Scholar). The present work tries to find answers to the following questions. 1) Do additional residues exist in the HUP1 symporter, which give rise to an increased K m value for d-glucose uptake upon replacement? 2) Do all these HUP1 mutants also exhibit decreased affinities for other sugars, or do some of them show substrate specific effects? 3) Is it possible to narrow down the segment of the first external loop of HUP2 participating ind-galactose recognition? All cloning steps were carried out in E. coli DH5α with the plasmid vector pUC18.E. coli TG1 served as host for the phagemid pUC118 and the helper virus M13KO7 in site-directed mutagenesis. The Leu−and sugar uptake deficient strain S. pombe YGS-B25 (15Milbradt B. Höfer M. Microbiol. 1994; 140: 2617-2623Crossref PubMed Scopus (31) Google Scholar) used for heterologous expression of the various transporter cDNAs was grown in 2% gluconate, 2% yeast extract. Transformed S. pombe cells were cultivated in minimal medium containing 2% gluconate and 0.67% yeast nitrogen base without amino acids. Wild-type, point-mutated, and chimeric transporter cDNAs were cloned viaSacI/BamHI into the shuttle vector pEVP11 (19Russel P. Nurse P. Cell. 1986; 45: 145-153Abstract Full Text PDF PubMed Scopus (714) Google Scholar) or pART3 (20McLeod M. Stein M. Beach D. EMBO J. 1987; 6: 729-736Crossref PubMed Scopus (187) Google Scholar), the latter allowing significant higher expression. S. pombe YGS-B25 was transformed as described in Ref. 7Sauer N. Caspari T. Klebl F. Tanner W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7949-7952Crossref PubMed Scopus (67) Google Scholar. The full-length cDNA of HUP1 was amplified by polymerase chain reaction under suboptimal conditions as described previously (14Will A. Caspari T. Tanner W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10163-10167Crossref PubMed Scopus (29) Google Scholar) in order to achieve one error per cDNA fragment on average. The pool of randomly mutagenized cDNAs was ligated into pEVP11 or pART3 and introduced in S. pombe cells leading to the RMY and RGY transformants, respectively. Plasmid reisolation from RGY52 was performed by the phenol/chloroform/isoamyl alcohol procedure (21Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. 2nd Ed. Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology. John Wiley & Sons, New York1992: 13-43-13-44Google Scholar) with the following addition. The aqueous phase containing the recovered plasmids was purified and precipitated by successive treatment with phenol, diethyl ether, and ethanol prior to transformation of E. coliDH5α. However, this procedure for plasmid isolation from yeast cells as well as several others failed in the cases of RMY126 and RMY254. Therefore one big colony of each of these transformants was picked and directly applied to a standard polymerase chain reaction. Plasmids were released from the cells due to preincubation at 94 °C for 10 min. The mutated HUP1 cDNAs were amplified afterward using flanking primers that bind in the promotor and the polylinker region of pEVP11. Then they were subcloned via SacI/BamHI into pUC18 and their nucleotide changes were determined by sequence analysis using the T7SequencingTM kit (Pharmacia Biotech) and synthetic oligonucleotides. The transformants RMY126 and RGY52 both exhibited two point mutations in the HUP1 gene (see “Results”). These mutations could be separated using a unique KpnI restriction site lying in between (Fig. 1). The SacI/KpnI fragment and the KpnI/BamHI fragment coding for the N- and C-terminal part were ligated to the respective missing sequences from the wild-type clone. This resulted in HUP1coding regions carrying either the one or the other mutation. Those originating from RMY126 were resubcloned into pEVP11, those originating from RGY52 into pART3. S. pombe YGS-B25 was transformed as described above. Preparation of the single-stranded HUP1 and HUP2 template DNA was performed as described previously (13Caspari T. Stadler R. Sauer N. Tanner W. J. Biol. Chem. 1994; 269: 3498-3502Abstract Full Text PDF PubMed Google Scholar, 18Will A. Tanner W. FEBS Lett. 1996; 381: 127-130Crossref PubMed Scopus (16) Google Scholar). Site-specific mutagenesis was carried out with the SculptorTM in vitromutagenesis system (Amersham) according to the instructions of the manufacturer. The sequences of the synthetic oligonucleotides used were as follows (changed bases are underlined): (a) K59Q/K60M, 5′-CTGGGAAGAACATCTGCTCAAAGGCCTC-3′; (b) R144L, 5′-ACCAAGCAGCACGAGGCCGACAATCAGCA-3′; (c) R204L, 5′-GACCCAGGGACAGAAGCCACCCGTTCTCC-3′; (d) N45I, 5′-CCAGTCACACCGATATCATAGCC-3′; (e) V52T, 5′-CCTCCAGGGAGGTGACGCCACCAG-3′; (f) I47N, 5′-CCAGTCACACCGTTGTCATAGCCG-3′. Sequencing the whole length of the cDNA verified the desired mutation(s) and excluded the possibility of additional changes. Correct clones were subcloned into pART3 (a–c) or pEVP11 (d–f). We previously reported (18Will A. Tanner W. FEBS Lett. 1996; 381: 127-130Crossref PubMed Scopus (16) Google Scholar) the construction of a chimeric HUP1/2/1 transporter (C6), which consists mainly of HUP1 sequence. Only the front part of the first extracellular loop is derived from the HUP2 symporter. The HUP1 fragment coding for this loop section was excised by EcoRV/BsgI digestion (see Fig. 1) and replaced with the equivalent HUP2 fragment. TheEcoRV restriction site had first to be introduced into the cDNAs of the wild-type transporters without changing the amino acid sequences. Approximately in the middle of the exchanged loop segment exists a unique Asp700I restriction site (Fig. 1) at homologous positions in HUP1 and HUP2. Substitution of the Asp700I/BsgI fragment ofHUP1 for that of HUP2 resulted in the chimeric cDNA C7. C8 was generated in the same way by replacement of theEcoRV/Asp700I fragment. All chimericHUP1/2/1 cDNAs possess exactly the same 5′-untranslated sequence as the wild-type HUP1 and were cloned viaSacI/BamHI into the expression vector pEVP11. Five to 20 ml of S. pombe cells (OD578 = 1.0) were harvested, washed once in 5 ml of 100 mm potassium phosphate buffer, pH 6.0, and resuspended in the same buffer to a final volume of 1 ml. Cells were optimally energized by adding ethanol to a final concentration of 120 mm. After 2 min of shaking at 30 °C the test was started by adding radioactive sugar. Samples were withdrawn at given intervals, filtered through nitrocellulose filters (0.8 μm pore size), and washed once with distilled water. Incorporation of radioactivity was determined by scintillation counting. In order to obtain theK m and V max values, initial uptake rates were measured at different substrate concentrations and plotted according to Lineweaver-Burk. Since nontransformed S. pombe cells (YGS-B25) do not show measurable hexose uptake activity, transport rates of mutated HUP gene products as low as 0.1% of that of wild-type HUPs expressed in S. pombe could reliably be measured. When the effect of external pH was tested, cells were washed and resuspended in McIlvaine buffer (adjusted to a given pH in the range from pH 3 to 7 by mixing 100 mm citric acid with 200 mm Na2HPO4) or 50 mm Tris/HCl buffer (adjusted to a given pH in the range from pH 7 to 9). All radioactive sugars wered-[U-14C]compounds purchased from Amersham. S. pombe cells of a 30-ml culture (OD578 ≈ 1) were pelleted by centrifugation. Their membranes were isolated as described (13Caspari T. Stadler R. Sauer N. Tanner W. J. Biol. Chem. 1994; 269: 3498-3502Abstract Full Text PDF PubMed Google Scholar). The protein content was assayed by the method of Bradford (22Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217450) Google Scholar). SDS-polyacrylamide gel electrophoresis was carried out according to Laemmli (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207509) Google Scholar); proteins were transferred electrophoretically to nitrocellulose and incubated overnight with polyclonal anti-HUP1-A antibody (13Caspari T. Stadler R. Sauer N. Tanner W. J. Biol. Chem. 1994; 269: 3498-3502Abstract Full Text PDF PubMed Google Scholar). The blot was immunodetected with the ECL kit of Amersham. Expression of mutant cDNAs was compared with that of wild-type HUP1 cloned in the same vector (pEVP11 or pART3). The K m value is an expression independent measure of the affinity of the HUP1 transporter for its sugar sustrate(s). Therefore K m mutants should lead to the identification of amino acid residues most probably involved in substrate binding. Recently we reported an unbiased functional screening for such K m mutants (14Will A. Caspari T. Tanner W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10163-10167Crossref PubMed Scopus (29) Google Scholar). It is based on a 1000-fold increase in 2-deoxyglucose sensitivity upon transformation of a sugar uptake deficient S. pombestrain with the HUP1 cDNA. A pool of randomly mutatedHUP1 cDNAs was generated by polymerase chain reaction and used for transformation. Transformants with intermediate 2-deoxy-d-glucose sensitivity were selected and tested for decreased affinity for d-glucose. FourK m mutants had been obtained in this way (14Will A. Caspari T. Tanner W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10163-10167Crossref PubMed Scopus (29) Google Scholar). In the meantime further use of this strategy has been made and three additional mutants have been isolated (TableI).Table Id-Glucose transport characteristics of three additional S. pombe strains transformed with randomly mutated Chlorella HUP1 cDNAsS. pombe strainNucleotide exchangeAmino acid exchangeLocationK mV maxTransport proteinM%wt-HUP11.5 × 10−5100100RMY254TAC→TATY81YLoop 16 × 10−42≤1AGG→CGGR197RLoop 5AAC→ATCN436IHelix XIRMY126GGT→TGTG97CHelix II4 × 10−375ATC→AACI303NHelix VIIRGY52GGT→GATG120DHelix III7 × 10−423TTT→TTAF292LHelix VII Open table in a new tab The HUP1 cDNA isolated from RMY254 exhibits three nucleotide changes, but only one of them affects the primary structure of the HUP1 protein. The substitution of the asparagine residue at position 436 in helix XI for isoleucine decreases the affinity ford-glucose 40-fold. The cDNAs of the otherK m mutants carry only two point mutations, which both result in amino acid changes. Thus, the HUP1 symporter of RMY126 contains cysteine and asparagine instead of glycine 97 (helix II) and isoleucine 303 (helix VII), respectively. The K m value for d-glucose is dramatically increased by a factor of about 270. The point mutations in the HUP1 cDNA of RGY52 alter the transport protein in the following way. Glycine 120 (helix III) is replaced by an aspartic acid and phenylalanine 292 (helix VII) is changed to leucine. As a consequence theK m value for d-glucose rises approximately 50-fold as compared with that of the wild-type. These K m mutants also show a dramatic decrease inV max value, which correlates well, however, with the poor level of expression (Table I). A reduced amount of transport protein is not unusual for clones originating from the random mutagenesis approach, since a lower uptake rate also contributes to higher 2-deoxy-d-glucose resistance (14Will A. Caspari T. Tanner W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10163-10167Crossref PubMed Scopus (29) Google Scholar). It is not really understood, however, why these mutations lead to such a low expression of transport protein. The possibility that mutations affect protein secondary structure and/or protein stability, leading to indirect effects on K m values, unfortunately cannot be ruled out. Although, in principle this is true for all mutations described here or elsewhere, particular caution is required for those reducing the expression level. In order to elucidate whether the K m change found in RMY126 and RGY52 is brought about by both substitutions acting additively or whether it is simply caused by one of them, the mutations were separated from each other as described under “Experimental Procedures.” Table II lists theK m values of the single mutants. Obviously, the effect of substituting Gly-97 for Cys on the d-glucose affinity of HUP1 is negligible. The nearly identicalK m values of mutant and wild-type transporter manifest that glycine 97 is not important for the interaction with the substrate. On the other hand mutant I303N exhibits aK m value very similar to that of the double mutant RMY126. Therefore, it is suggested that the isoleucine residue in helix VII is involved in d-glucose binding.Table IId-Glucose transport characteristics of S. pombe strains expressing HUP1 transporter with the single mutations G97C, I303N, G120D, or F292LMutantK mV maxTransport proteinM%wt-HUP11.5 × 10−5100100G97C5 × 10−53≤5I303N6 × 10−3120NDaND, not determined.G120D3 × 10−4915F292L7 × 10−41315a ND, not determined. Open table in a new tab In the case of RGY52 the results are more complex. Both substitutions in question generate effects on d-glucose affinity, but they are not additive. The replacement F292L causes a 50-fold increase of the K m value equal to that of the double mutant. In the G120D mutant the K m value is raised somewhat less but still significantly (about 30-fold). It is not clear why the effects are not additive to some extent. It seems that the structural aberration caused by F292L includes the one induced by G120D. Since G120D is a much more drastic change when compared with F292L, the results are taken as indication that the phenylalanine residue in helix VII is important for correct substrate binding. Finally, it should be emphasized that for all single mutants the V maxvalue correlates very well with the amount of transport protein detected by immunoblotting (Table II). The Chlorella HUP1 symporter enables transformed S. pombe cells to take up a great number of monosaccharides. The affinities for the particular sugar substrates differ widely, however. Thus, the K m values for the uptake of d-glucose, d-mannose,d-fructose, d-xylose, andd-galactose turned out to be in the range of 1.5 × 10−5, 1.5 × 10−4, 3 × 10−4, 1.5 × 10−3, and 3 × 10−3m, respectively (TableIII). In previous publications (13Caspari T. Stadler R. Sauer N. Tanner W. J. Biol. Chem. 1994; 269: 3498-3502Abstract Full Text PDF PubMed Google Scholar, 14Will A. Caspari T. Tanner W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10163-10167Crossref PubMed Scopus (29) Google Scholar), as well as in this paper, several HUP1 mutants with an increasedK m value for d-glucose were described. It was of interest to find out whether these mutants show less efficient binding also of other substrates, i.e. whether sugar specific effects or more general ones are produced by the mutations. For this purpose the mutated HUP1 transporters listed in Table III were chosen for a detailed analysis of their substrate specificities and compared with that of the wild-type protein.Table IIISubstrate specificity of S. pombe strains expressing various mutated HUP1 transportersMutantd-Glucosed-Mannosed-Fructosed-Xylosed-Galactosewt-HUP11.5 × 10−51.5 × 10−43 × 10−41.5 × 10−33 × 10−3D44E2 × 10−42.5 × 10−46 × 10−42 × 10−32 × 10−3Q179EaThis mutant is actually a double mutant, but the second exchange, F497S, is in all probability unimportant, since it is located in the C-terminal tail, that can be deleted without affecting substrate binding (14).1.5 × 10−47.5 × 10−33.5 × 10−3n.d.n.d.Q298N1.5 × 10−43 × 10−47.5 × 10−4≥2 × 10−2≥5 × 10−2I303N6 × 10−32.5 × 10−21.5 × 10−2n.d.n.d.N436Y≥5 × 10−2≥5 × 10−2≥5 × 10−2n.d.n.d.N436Q3.5 × 10−45.5 × 10−44 × 10−4≥5 × 10−2≥5 × 10−2b N.D., not determined.a This mutant is actually a double mutant, but the second exchange, F497S, is in all probability unimportant, since it is located in the C-terminal tail, that can be deleted without affecting substrate binding (14Will A. Caspari T. Tanner W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10163-10167Crossref PubMed Scopus (29) Google Scholar). Open table in a new tab b N.D., not determined. Three mutations, Q179E, I303N, and N436Y, equally diminish the affinities for all sugars tested (d-glucose,d-mannose, and d-fructose). In the case of Q298N and N436Q the K m values ford-glucose, d-xylose, andd-galactose transport are significantly increased while those for d-mannose and d-fructose uptake are only mildly affected. The conservative exchange D44E, however, influences the substrate specificity in an extraordinary way. As previously pointed out, this substitution increases theK m for d-glucose uptake by more than 10 times (13Caspari T. Stadler R. Sauer N. Tanner W. J. Biol. Chem. 1994; 269: 3498-3502Abstract Full Text PDF PubMed Google Scholar). Surprisingly, none of the K m values for other sugar substrates is altered in the D44E mutant significantly,i.e. by more than a factor of 2. These results suggest a special role of aspartate 44 in the glucose transport process. Therefore, this amino acid position has been investigated more thoroughly. In the transporter model of Fig. 1 aspartate 44 is situated at the very beginning of the first extracellular loop of HUP1. This is based on the simple energetic reason that charged amino acids are expected to avoid the lipophilic environment of the membrane. In the lactose permease of E. coli, however, two aspartate residues are thought to exist in transmembrane helix VII. They both are neutralized by forming salt bridges with lysine residues in helix X and XI, respectively (24Sahin-Tóth M. Dunten R. Gonzalez A. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10547-10551Crossref PubMed Scopus (136) Google Scholar). It is a characteristic feature of these charge pairs that simultaneous replacement of both partners by neutral amino acids does not impair the translocation process, whereas the exchange of only one partner, leaving the other one unpaired, inactivates the permease completely. Since the mutation D44N leads to a total inactivation of the HUP1 transporter (13Caspari T. Stadler R. Sauer N. Tanner W. J. Biol. Chem. 1994; 269: 3498-3502Abstract Full Text PDF PubMed Google Scholar), it might be suspected that the aspartic acid residue likewise is involved in a salt bridge. The primary structure of the Chlorella transporter provides several basic amino acids that might act as the positively charged counterpart. Some of them were replaced individually or in combination by neutral residues (TableIV). The ability to take upd-glucose is maintained in the mutants R144L, R204L, and K59Q/K60M, albeit with dramatically reduced overall activities. These reductions, however, primarily reflect the low expression levels. There is no indication that the catalytic activity per se is impaired. Moreover d-glucose transport tests of all three mutants yield K m values nearly identical to that of wild-type HUP1. These results make it unlikely that one of the basic amino acids at position 59, 60, 144, or 204 in the transport protein interacts with aspartate 44 via a salt bridge.Table IVd-Glucose transport characteristics of S. pombe strains expressing HUP1 transporter with the site specific mutations K59Q/K60M, R144L, or R204LMutantK mV maxTransport proteinM%wt-HUP11.5 × 10−5100100K59Q/K60M2.5 × 10−5410R144L1.5 × 10−5<1<1R204L1.5 × 10−5210D44NNo measurable activity80 Open table in a new tab Previous studies not only demonstrated the absolute necessity of a carboxyl group at position 44 but also the importance of its precise location for the transporter to be active. Thus, increasing the side chain length by changing aspartic into glutamic acid (D44E) decreases the V max of d-glucose uptake by 90% and raises the K m by 15-fold as compared with the wild-type, although similar amounts of transport protein are present in the cells (13Caspari T. Stadler R. Sauer N. Tanner W. J. Biol. Chem. 1994; 269: 3498-3502Abstract Full Text PDF PubMed Google Scholar). In order to elucidate whether the potential for protonation/deprotonation of Glu-44 as compared with that of Asp-44 is affected, the influence of extracellular pH on the translocation reaction catalyzed by the two symporters has been examined. Thed-glucose uptake activity of HUP1 is optimal at about pH 4.5 and declines steeply toward lower and higher values (Fig.2). The D44E mutant shows a completely different pH dependence. As ambient pH is increased from 3.0 to 9.0,d-glucose uptake is accelerated gradually, reaching an optimum at about pH 7.0. Despite the high homology (74%), the Chlorellasymporters HUP1 and HUP2 differ significantly in substrate specificity (6Stadler R. Wolf K. Hilgar" @default.
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• W2088263411 date "1998-05-01" @default.
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• W2088263411 title "Alteration of Substrate Affinities and Specificities of theChlorella Hexose/H+ Symporters by Mutations and Construction of Chimeras" @default.
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