From the Universität Regensburg, Lehrstuhl für Zellbiologie und Pflanzenphysiologie, 93040 Regensburg, Germany
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ABSTRACT |
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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 increased Km value for D-glucose were isolated. The 40-fold increase in Km 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 in Km for D-glucose, respectively. An investigation of the individual mutations revealed that the substitutions I303N and F292L (both in helix VII) cause the Km 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 Km change for D-glucose is paralleled by a corresponding change for other hexoses tested, the exchange D44E exclusively alters the Km 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 for D-galactose 20-fold.
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INTRODUCTION |
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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 (1-4). Three cDNAs coding for highly homologous
Chlorella monosaccharide/H+ symporters were
cloned by differential screening (5, 6) and named
HUP13 (hexose uptake
protein). Their identities have been confirmed by
heterologous expression in Schizosaccharomyces pombe (6, 7).
Furthermore, the HUP1 transporter retains its uptake activity after
solubilization from the membrane of transgenic fission yeast,
purification to homogeneity, and reconstitution into
proteoliposomes (8, 9).
The HUP symporters belong to a large family of substrate transporters,
called the "major facilitator superfamily" (10). Members of this
major facilitator superfamily are thought to consist of 12 -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 (11) and N-glycosylation scanning mutagenesis
studies on the human glucose facilitator GLUT1 (12). 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 (13, 14) was carried out in a sugar uptake deficient S. pombe strain (15). Several mutants with an increased Km value for D-glucose uptake were found by site-directed mutagenesis (13) and by polymerase chain reaction random mutagenesis with subsequent selection for decreased sensitivity toward the toxic sugar 2-deoxyglucose (14). 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 (16, 17).
The symporters HUP1 and HUP2 differ significantly in their substrate specificity (6, 18). Especially, the affinity for D-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 (18).
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 Km 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 in D-galactose recognition?
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EXPERIMENTAL PROCEDURES |
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Strains and Growth Conditions--
All cloning steps were
carried out in E. coli DH5 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 (15) 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.
Transformation of S. pombe YGS-B25-- Wild-type, point-mutated, and chimeric transporter cDNAs were cloned via SacI/BamHI into the shuttle vector pEVP11 (19) or pART3 (20), the latter allowing significant higher expression. S. pombe YGS-B25 was transformed as described in Ref. 7.
Random Mutagenesis by Polymerase Chain Reaction-- The full-length cDNA of HUP1 was amplified by polymerase chain reaction under suboptimal conditions as described previously (14) 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.
Recovery and Sequencing of the Mutated HUP1
cDNAs--
Plasmid reisolation from RGY52 was performed by the
phenol/chloroform/isoamyl alcohol procedure (21) 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. coli DH5. 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.
Separation of the Mutations of the Double Mutants RMY126 and RGY52-- 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 HUP1 coding 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.
Site-directed Mutagenesis-- Preparation of the single-stranded HUP1 and HUP2 template DNA was performed as described previously (13, 18). Site-specific mutagenesis was carried out with the SculptorTM in vitro mutagenesis 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).
Construction of the Chimeric HUP1/2/1 cDNAs-- We previously reported (18) 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. The EcoRV 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 of HUP1 for that of HUP2 resulted in the chimeric cDNA C7. C8 was generated in the same way by replacement of the EcoRV/Asp700I fragment. All chimeric HUP1/2/1 cDNAs possess exactly the same 5'-untranslated sequence as the wild-type HUP1 and were cloned via SacI/BamHI into the expression vector pEVP11.
Transport Assays-- 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 the Km and Vmax 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 were D-[U-14C]compounds purchased from Amersham.
Isolation of Total Membranes, SDS-Polyacrylamide Gel
Electrophoresis, and Immunoblotting--
S. pombe cells of
a 30-ml culture (OD578 1) were pelleted by
centrifugation. Their membranes were isolated as described (13). The
protein content was assayed by the method of Bradford (22).
SDS-polyacrylamide gel electrophoresis was carried out according to
Laemmli (23); proteins were transferred electrophoretically to
nitrocellulose and incubated overnight with polyclonal anti-HUP1-A antibody (13). 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).
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RESULTS |
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Random Generation of HUP1 Mutants with an Increased Km Value for D-Glucose-- The Km value is an expression independent measure of the affinity of the HUP1 transporter for its sugar sustrate(s). Therefore Km 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 Km mutants (14). It is based on a 1000-fold increase in 2-deoxyglucose sensitivity upon transformation of a sugar uptake deficient S. pombe strain with the HUP1 cDNA. A pool of randomly mutated HUP1 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. Four Km mutants had been obtained in this way (14). In the meantime further use of this strategy has been made and three additional mutants have been isolated (Table I).
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Separation of the Mutations in the HUP1 Double Mutants-- In order to elucidate whether the Km 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 the Km 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 identical Km 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 a Km 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.
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Substrate Specificity of Several Mutated HUP1
Transporters--
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 Km values for the
uptake of D-glucose, D-mannose, D-fructose, D-xylose, and
D-galactose turned out to be in the range of 1.5 × 105, 1.5 × 10
4, 3 × 10
4, 1.5 × 10
3, and 3 × 10
3 M, respectively (Table
III). In previous publications (13, 14), as well as in this paper, several HUP1 mutants with an increased Km 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.
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Characterization of Position Asp-44-- 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 (24). 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 (13), 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 (Table IV). The ability to take up D-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 Km 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.
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Determination of Substrate Specificities of HUP1 and
HUP2--
Despite the high homology (74%), the Chlorella
symporters HUP1 and HUP2 differ significantly in substrate specificity
(6, 18). Whereas both carriers efficiently transport
D-glucose with comparable affinities, there exists a large
difference concerning D-galactose. This hexose is only
poorly accepted by HUP1 and favored by HUP2. The Km
values for D-galactose uptake differ from one another by 2 orders of magnitude (3 × 103 M
versus 2.5 × 10
5 M). In a
study using chimeric proteins it was recently shown that exchanging
only the front part of the first extracellular loop of HUP1 for that of
HUP2 (chimera C6), gives rise to a 15-fold higher affinity for
D-galactose as compared with that of the wild-type HUP1
(18). A sequence alignment of the interchanged segments demonstrates
that HUP1 and HUP2 differ in 16 out of 29 positions in this region
(Fig. 3).
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DISCUSSION |
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The HUP proteins of Chlorella catalyze the uptake of several monosaccharides in co-transport with protons. How transport function is accomplished by the Chlorella symporters or, for that matter by transporters of any other organism, is an open and intriguing question. Regarding the nature of the sugar-binding site and the translocation pathway, it is assumed that residues directly interacting with the substrate should not be replaceable without causing a significant change in the affinity of the transporter for the substrate. Several HUP1 mutants with an increased Km value for D-glucose were found previously (13, 14). Apart from Asp-44, which will be discussed later, the following amino acids were affected: Gln-179 (in helix V), Gln-298 and Gln-299 (both in helix VII), Val-433 and Asn-436 (both in helix XI).
Studies on the human glucose facilitator GLUT1 support the notion that at least some of these residues are intimately involved in substrate binding, e.g. glutamine 161, which is homologous to Gln-179 in HUP1 (25), and glutamine 282, which corresponds to Gln-298 of HUP1 (26). There is also good evidence, that helix XI of GLUT1 interacts directly with D-glucose (27).
Here, three additional randomly generated HUP1 mutants with decreased affinity to D-glucose have been reported (Table I). The N436I mutation confirms previous findings concerning this special asparagine (14). The fact that the same amino acid has been found repeatedly in random mutagenesis studies indicates that the screen for Km mutants approaches saturation. The HUP1 symporter of mutant RMY126 revealed two amino acid exchanges, G97C (in helix II) and I303N (in helix VII), but only the latter proved to be responsible for the detected Km effect (Table II). A parallel situation exists in mutant RGY52, where only F292L (in helix VII) induces a Km shift identical to that in the double mutant. However, the efficiency of D-glucose binding is also influenced by the second exchange, G120D in helix III, but the insertion of a new negative charge in a non-polar transmembrane domain may more likely perturb the secondary structure of the protein.
Taken together, the new Km mutants strengthen the importance of residues within helices VII and XI, which have previously been suggested to play a role in D-glucose binding. Remarkably, residues Phe-292, Gln-298, Gln-299, and Ile-303 cluster on one face of putative helix VII when viewed on a helical wheel plot (Fig. 5). One might speculate therefore that Phe-292, Gln-299, and Ile-303, which are located in closest vertical proximity, are probably guiding the sugar substrate along the translocation path. Stacking of hydrophobic patches of the glucopyranose ring with aromatic residues is clearly visible in the binding pocket of the D-glucose-binding protein of E. coli (17). Considering phenylalanine 292, the same could hold for D-glucose binding of HUP1.
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By x-ray crystallography it was shown that sugar binding in periplasmic binding proteins is mediated mainly by charged amino acids and their amides via hydrogen bonds (16, 17). It is striking that most of the HUP1 residues identified as good candidates for sugar interaction are amides (Gln-179, Gln-298, Gln-299, and Asn-436). In addition, there is a charged residue, Asp-44, in HUP1 that cannot be changed without affecting D-glucose binding. The topological model of HUP1 (Fig. 1) puts Asp-44 outside the hydrophobic membrane, but a location within helix I is imaginable as well. Supposing it were so, then helices I, V, VII, and XI would participate in sugar binding. Although it might be a coincidence that the first and fifth transmembrane spanning domains of the N- and C-terminal half of HUP1 would then have been identified, it is nevertheless remarkable, since the 12-helix arrangement of the major facilitator superfamily transporters most likely has arisen by a gene duplication event of an ancestral gene encoding a protein with six transmembrane spans (28). Helices with functional importance may very well be corresponding ones in each half of the protein.
When the proposed helix packing of HUP1 is compared with that of the intensively studied lactose permease of E. coli a number of parallels like the postulated neighborhood of helices V, VII, and XI can be seen (29). The importance of helices I and VII of lactose permease is, furthermore, underpinned by sugar specificity mutants (Refs. 30 and references therein). Only for helix X, potentially playing a major role in lactose transport (30, 31), corresponding evidence for HUP transporters is missing.
Oppositely charged residues in transmembrane segments sometimes neutralize each other by forming a salt bridge (24). Provided that Asp-44 is located in helix I, it might also be paired with a basic amino acid. Candidates that could possibly act as positive counterions for Asp-44 were selected and replaced by neutral residues (Table IV). Evidence that Asp-44 is linked to a basic amino acid via a salt bridge has not been obtained, however. The shift in the pH optimum from pH 4.5 of the wild-type HUP1 protein to pH 7.0 of the D44E transporter indicates that the carboxyl group of the mutant is located in a drastically changed environment (increased hydrophobicity), leading to such a large increase in its pKa. The shift also suggests that a negative charge is required at this position for transport activity; in the case of the mutant one would have to assume that the proton dissociates only under the more alkaline condition. Of course, this is in accordance with the observation that transport in the mutant D44N is abolished (13).
Ten monosaccharide/H+ symporters with proven transport
activity have been cloned from plants so far: HUP13 from C. kessleri (5, 6), STP1
4 from Arabidopsis thaliana
(32)1, MST1 from
Nicotiana tabacum (33), HEX3 from Ricinus
communis (34), and MTST1 from Medicago truncatula (35).
A sequence comparison of these transporters reveals that HUP1 residues
that most likely contribute to sugar binding (Asp-44, Gln-179, Gln-298, Gln-299, Ile-303, Val-433, Asn-436) are absolutely conserved, the only
exception being Phe-292. On the one hand, this fact clearly emphasizes
the great importance of these residues. On the other hand, additional
positions must be postulated to explain the differing substrate
specificities of the highly homologous transporters. The HUP2
symporter, for example, transports D-galactose more
efficiently and with a 100 times higher affinity than the HUP1
symporter does. Various chimeras between these two transporters were
previously constructed in order to identify crucial site(s) for the
differential D-galactose recognition (18). A chimera
consisting of the N-terminal half of HUP2 and the C-terminal half of
HUP1 shows a Km value for D-galactose
uptake indistiguishable from that of the high affinity transporter
HUP2, indicating that the critical positions lie within the N-terminal
half (18). In the chimera C6 only the first 29 amino acids of putative
loop 1 were removed from HUP1 and replaced by the corresponding
residues of HUP2 (Fig. 3). This chimera still has a 15-fold increased
affinity for D-galactose, implying that there are at least
two separate determinants in the N-terminal half of HUP2, one inside
the interchanged loop segment and one outside.
By use of additional chimeras and site-directed mutants it has been proven that one single exchange within loop 1, N45I, causes the improved D-galactose affinity seen in C6 (Table V). A HUP2 mutant carrying the reverse substitution, I47N, reveals a 20-fold increased Km value for D-galactose uptake and therefore strongly supports the notion that the exchanged position is a crucial determinant.
Recently the construction of various chimeras between two closely related facilitators of S. cerevisiae have been reported, i.e. the Gal2 protein, which transports D-galactose and D-glucose, and the Hxt2 protein, which is specific for D-glucose (36, 37). In contrast to the findings for HUP2, a 35-amino acid segment around helix X was identified as the D-galactose recognition domain of Gal2. Taking into account that the corresponding affinities of the Chlorella and S. cerevisiae transporters differ by orders of magnitude, this could mean that the mechanism of D-galactose binding varies among different members of the major facilitator superfamily. However, one should be aware of the fact that only those regions within the HUP2 and the Gal2 protein can be detected by the chimera method, which differ in sequence between the homologous partners. In other words, some positions that are essential but not sufficient for good D-galactose binding may be conserved in HUP1 and HUP2 as well as in Gal2 and Hxt2, respectively. If such conserved regions in the two Chlorella transporters in question differ from those conserved in the two S. cerevisiae transporters, the method of constructing chimeras may very well uncover different parts of a related binding pocket. Therefore, residues within the two identified regions of the proteins may cooperate in D-galactose binding and the pocket may in principal be similarly constructed in the HUP2 and the Gal2 protein.
Finally, it should be mentioned that an interesting model for ligand specificity of closely related opioid receptors was recently proposed (38). In this case extracellular loops are thought to act as selective barriers that control which ligands can enter the rather unspecific transmembrane binding pocket common to all receptor subtypes. If applied to HUP1, asparagine 45 (and also Asp-44) might actually be located in the first external loop, where it may restrict the passage of D-galactose, not, however, that of D-glucose.
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ACKNOWLEDGEMENT |
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We thank Arabel Vollmann for help in some of the experiments.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB521.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: University of Sussex, MRC Cell Mutation Unit,
Falmer Brighton, BN1 9RR, UK.
§ To whom correspondence should be addressed. Tel.: 49-941-943-3018; Fax: 49-941-943-3352.
1 K. Baier, unpublished results.
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REFERENCES |
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