Journal of Biological Chemistry · 1995 · 126 citations · 32 references
Life SciencesBiochemistryMolecular BiologyEnzyme MechanismEnzymologyStructural BiologyEnzyme EvolutionEnzyme FunctionEnzyme InhibitionMembrane PermeabilityMembrane-bound EnzymesStructural EnzymologyActive TransportActive Transport InhibitionBase SubstitutionsEnzyme Active Site MutationsEscherichia ColiMembrane Protein MotifsMembrane Transporter FamiliesTurn Propensity
A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been identified among a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane. To determine the importance of this motif within the lactose permease of Escherichia coli, a total of 28 site-directed mutations at the conserved first, fifth, sixth, eighth, ninth, and tenth positions were analyzed. A dramatic inhibition of activity was observed with all bulky mutations at the first-position glycine. Based on these results, together with sequence comparisons within the superfamily, it seems likely that small side chain volume (and possibly high β-turn propensity) may be structurally important at this position. The acidic residue at the fifth position was also found to be very important for transport activity and even a conservative glutamate at this location exhibited marginal transport activity. In contrast, many substitutions at the eighth-position glycine, even those with a high side chain volume and/or low β-turn propensity, still retained high levels of transport activity. Similarly, none of the basic residues within the motif were essential for transport activity when replaced individually by nonbasic residues. However, certain substitutions at the basic residue sites as well as the eighth-position glycine were observed to have moderately reduced levels of active transport of lactose. Taken together, the results of this study confirm the importance of the conserved loop 2/3 motif in transport function. It is suggested that this motif may be important in promoting global conformational changes within the permease. A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been identified among a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane. To determine the importance of this motif within the lactose permease of Escherichia coli, a total of 28 site-directed mutations at the conserved first, fifth, sixth, eighth, ninth, and tenth positions were analyzed. A dramatic inhibition of activity was observed with all bulky mutations at the first-position glycine. Based on these results, together with sequence comparisons within the superfamily, it seems likely that small side chain volume (and possibly high β-turn propensity) may be structurally important at this position. The acidic residue at the fifth position was also found to be very important for transport activity and even a conservative glutamate at this location exhibited marginal transport activity. In contrast, many substitutions at the eighth-position glycine, even those with a high side chain volume and/or low β-turn propensity, still retained high levels of transport activity. Similarly, none of the basic residues within the motif were essential for transport activity when replaced individually by nonbasic residues. However, certain substitutions at the basic residue sites as well as the eighth-position glycine were observed to have moderately reduced levels of active transport of lactose. Taken together, the results of this study confirm the importance of the conserved loop 2/3 motif in transport function. It is suggested that this motif may be important in promoting global conformational changes within the permease. An important mechanism for the uptake of solutes by living cells involves membrane proteins known as cation/substrate cotransporters or symporters. These types of transport systems are widely found in many organisms including bacteria(1West I. Biochem. Biophys. Res. Commun. 1970; 41: 655-661Crossref PubMed Scopus (147) Google Scholar, 2Wilson T.H. Wilson D.M. Elson E. Frazier W. Glaser L. Cell Membranes: Methods and Reviews. Plenum, New York1983: 1-39Google Scholar), fungi(3Eddy A.A. Nowacki J.A. Biochem. J. 1971; 122: 701-711Crossref PubMed Scopus (72) Google Scholar, 4Slayman C.L. Slayman C.W. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 1935-1939Crossref PubMed Scopus (173) Google Scholar), plant(5Komor E. FEBS Lett. 1973; 38: 16-18Crossref PubMed Scopus (103) Google Scholar, 6Hofer M. Misra P.C. Biochem J. 1978; 172: 15-22Crossref PubMed Scopus (77) Google Scholar), and animal cells(7Crane R.K. Physiol. Rev. 1960; 40: 784-825Crossref Scopus (265) Google Scholar, 8Crane R.K. Fed. Proc. 1962; 21: 891-895PubMed Google Scholar). During uptake, symporters simultaneously couple the inwardly directed cation electrochemical gradient to the transport of solute so that secondary active transport can be achieved with regard to the solute(9Crane R.K. Rev. Physiol. Biochem. Pharmacol. 1977; 78: 99-159Crossref PubMed Google Scholar, 10Mitchell P. Biochem. Soc. Symp. 1963; 22: 142-168Google Scholar). The lactose permease of Escherichia coli has provided a model system in which to investigate secondary active transport(11Roepe P.D. Consler T.G. Menezes M.F. Kaback H.R. Res. Microbiol. 1990; 141: 290-308Crossref PubMed Scopus (31) Google Scholar, 12Brooker R.J. Res. Microbiol. 1990; 141: 309-316Crossref PubMed Scopus (26) Google Scholar). This protein is found in the E. coli cytoplasmic membrane and couples the transport of H+ and lactose. From the cloning and nucleotide sequence of the lacY gene, the lactose permease contains 417 amino acids with a molecular weight of 46,504 (Refs. 13Teather R.M. Muller-Hill B. Abrutsch U. Aichele G. Overath P. Mol. & Gen. Genet. 1978; 159: 239-248Crossref PubMed Scopus (109) Google Scholar and 14Buchel D.E. Gronenborn B. Muller-Hill B. Nature. 1980; 283: 541-545Crossref PubMed Scopus (286) Google Scholar). Hydropathicity plots as well as genetic studies are consistent with a secondary structural model in which the protein contains 12 hydrophobic segments which traverse the membrane in an α-helical manner(15Foster D.L. Boublik M. Kaback H.R. J. Biol. Chem. 1983; 258: 31-34Abstract Full Text PDF PubMed Google Scholar, 16Calamia J. Manoil C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4937-4941Crossref PubMed Scopus (232) Google Scholar, 17King S.C. Hansen C.L. Wilson T.H. Biochim. Biophys. Acta. 1991; 1062: 177-186Crossref PubMed Scopus (131) Google Scholar). Recently, analyses of sequences among a variety of transporters has led to the conclusion that a large and diverse group of solute transporters are evolutionarily related to one another(18Henderson P.J.F. J. Bioenerg. Biomembr. 1990; 22: 525-569Crossref PubMed Scopus (142) Google Scholar, 19Griffith J.K. Baker M.E. Rouch D.A. Page M.G.P. Skurray R.A. Paulsen I.T. Chater K.F. Baldwin S.A. Henderson P.J.F. Curr. Opin. Cell Biol. 1992; 4: 684-695Crossref PubMed Scopus (273) Google Scholar, 20Marger M.D. Saier Jr., M.H. Trends Biochem. Sci. 1993; 18: 13-20Abstract Full Text PDF PubMed Scopus (773) Google Scholar). At the functional level, this superfamily includes proteins that transport a wide array of substrates such as sugars, Kreb's cycle intermediates, and antibiotics. For example, the lactose permease of E. coli, the glucose transporter of red blood cells, and bacterial tetracycline exporters are members. Mechanistically, this group is rather variable and includes proteins that catalyze uniport, symport, or antiport. This superfamily has been referred to as the USA1( 1The abbreviation used is: USAuniport, symport, antiport.) superfamily and as MFS (major facilitator superfamily)(20Marger M.D. Saier Jr., M.H. Trends Biochem. Sci. 1993; 18: 13-20Abstract Full Text PDF PubMed Scopus (773) Google Scholar, 21Goswitz V.C. Brooker R.J. Protein Sci. 1995; 4: 534-537Crossref PubMed Scopus (82) Google Scholar). Like the lactose permease, secondary structural models for members of the USA superfamily are commonly found to depict a membrane protein with 12 transmembrane segments traversing the membrane in an α-helical manner. It has been proposed that this arrangement of 12 transmembrane segments may have arisen by a gene duplication/fusion event of an ancestral gene encoding a protein with six transmembrane segments(22Maiden M.C.J. Davis E.O. Baldwin S.A. Moore D.C.M. Henderson P.J.F. Nature. 1987; 325: 641-643Crossref PubMed Scopus (333) Google Scholar). More recently, we have proposed a three-dimensional model based on the structural characteristics of transmembrane domains, hydrophilic loops, putative helical interactions in the lactose permease, and rotational symmetry between the two halves of the protein(21Goswitz V.C. Brooker R.J. Protein Sci. 1995; 4: 534-537Crossref PubMed Scopus (82) Google Scholar). uniport, symport, antiport. When comparing the sequences among distant relatives within the USA superfamily, it is frequently the case that relatively little overall homology is observed. Therefore, the superfamily has been divided into several groups (see Refs. 19Griffith J.K. Baker M.E. Rouch D.A. Page M.G.P. Skurray R.A. Paulsen I.T. Chater K.F. Baldwin S.A. Henderson P.J.F. Curr. Opin. Cell Biol. 1992; 4: 684-695Crossref PubMed Scopus (273) Google Scholar and 20Marger M.D. Saier Jr., M.H. Trends Biochem. Sci. 1993; 18: 13-20Abstract Full Text PDF PubMed Scopus (773) Google Scholar). It is extraordinarily striking, however, that although members within different groups may bear little overall sequence homology, a motif, GXXXD(R/K)XGR(R/K), has been conserved throughout the entire superfamily(19Griffith J.K. Baker M.E. Rouch D.A. Page M.G.P. Skurray R.A. Paulsen I.T. Chater K.F. Baldwin S.A. Henderson P.J.F. Curr. Opin. Cell Biol. 1992; 4: 684-695Crossref PubMed Scopus (273) Google Scholar). Within secondary structural models, this peptide sequence is found in the hydrophilic loop between transmembrane segments 2 and 3, and repeated again between transmembrane segments 8 and 9. Although some variability is found within this motif, it has been fairly well conserved among all members of the superfamily. Indeed, it would appear that the presence of this sequence within loop 2/3 and 8/9 in a membrane protein is indicative of its appropriate placement within the superfamily. Upon careful inspection of both loop 2/3 and 8/9 among more members of the superfamily, it seems reasonable to suggest that the motif is D/E at the fifth position and R/K at the ninth position. Only a few previous studies have involved perturbations in the structure within the loop 2/3 motif of the lactose permease. McKenna et al.(23McKenna E. Hardy D. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11954-11958Crossref PubMed Scopus (57) Google Scholar) inserted two or six histidine residues between Leu-72 and Arg-73 within the loop 2/3 motif. These insertional mutants exhibited a marked defect in the downhill and uphill accumulation of lactose. Very recently, the role of glycine residues within the lactose permease has been investigated(24Jung K. Jung H. Colacurcio P. Kaback H.R. Biochemistry. 1995; 34: 1030-1039Crossref PubMed Scopus (54) Google Scholar). Even though none of the glycine residues within the permease were found to be essential for sugar transport, bulky substitutions at the glycine in the first position of loop 2/3 were found to be inhibitory. Finally, Wrubel et al. (25Wrubel W. Stochaj U. Sonnewald U. Theres C. Ehring R. J. Bacteriol. 1990; 172: 5374-5381Crossref PubMed Google Scholar) examined the ability of portions of the lactose permease to be synthesized independently of each other and then reassemble into a functional permease. A mutant lacking amino acids 4 through 69 was coexpressed with a truncated form of the permease encoding amino acids 1 through 71. The results showed that these two pieces of the lactose permease were able to reassemble and produce a functional protein. More detailed analyses of amino acids within loop 2/3 have been conducted on the tetracycline antiporter of E. coli (26-28). With regard to the conserved charged residues, it has been found that the negatively charged residue at the fifth position is critical for transport function. Of the three basic residues, only the one at the ninth position in the motif appeared to be important. The conserved glycines at the first and eighth positions were both found to be important for function with the first glycine being significantly more critical than the eighth. In the current study, a similar approach has been undertaken to examine the functional significance of the conserved amino acids within the loop 2/3 motif of the lactose permease. Lactose (O-β-D-galactopyranosyl-[1,4]-α-D-glucopyranose) and melibiose (O-α-D-galactopyranosyl-[1,6]-α-D-glucopyranose) were purchased from Sigma. [14C]Lactose was purchased from Amersham Corp. The remaining reagents were analytical grade. The bacterial plasmids are described in Table I.TABLE IExpression levels in mutant strains Open table in a new tab Plasmid DNA was isolated by the NaOH-sodium dodecyl sulfate method (29Birnboim H.C. Doly J. Nucleic Acids Res. 1979; 7: 1513-1523Crossref PubMed Scopus (10202) Google Scholar) and introduced into the appropriate bacterial strain by the CaCl2 transformation procedure of Mandel and Higa(30Mandel M. Higa A. J. Mol. Biol. 1970; 63: 154-162Google Scholar). Stock cultures of cells were grown in YT media (31Miller J. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 433Google Scholar) supplemented with tetracycline (0.01 mg/ml). For transport assays, cells were grown to in YT media tetracycline and to the of the lactose permease. For the of Table cells were in and and in the to a of of were then at and sugar was At appropriate were and a membrane The was then with of by For the uphill transport was in the to the lactose permease and sugar the of the a the was also for sugar uptake in to an for sugar transport of and mutant strains Open table in a new tab For the H+ transport of cells were grown to and with The cells were then in and to a of of of cells were in a with a for the of a the of and the of were a of for at To H+ transport, an lactose was to a of The in was with a and in were on a which been to the of changes to a a a in the The was with to a the entire lacY This was to the in such a that the of the lacY gene was with the of the was then by the method of and M. Methods 1983; PubMed Scopus Google Scholar) as by et al. R.A. Methods 1987; PubMed Scopus Google Scholar) the the appropriate were identified by DNA (see The form DNA was then isolated and with to produce the the lacY This was then into the of transformation of E. coli strain cells plasmids with a lacY were identified by of The were to the of the lacY gene on the Only those which the lacY gene and the tetracycline gene in the were The mutant plasmids were throughout the entire lacY sequence to the presence of the and to be certain that other secondary mutations At two for each mutant were for DNA was by the method S. Proc. Natl. Acad. Sci. U. S. A. 1977; PubMed Scopus Google Scholar) which within the lacY DNA was isolated from and to et R. J. Google Scholar). For cells the appropriate were grown as described for the sugar transport of cells were by low in and at were in of 4 were in and were then three for were by and in of of total membrane protein was to dodecyl were to and then an that the of the lactose permease. The of lactose permease was then by a conserved motif is found within hydrophilic loop 2/3 and 8/9 among members of the USA superfamily. 1 the loop 2/3 motif in members of the USA superfamily. to an V.C. Brooker R.J. Protein Sci. 1995; 4: 534-537Crossref PubMed Scopus (82) Google Scholar), the first three or amino residues are commonly to within transmembrane At the first and eighth a glycine residue side chain high β-turn propensity) is conserved although amino acids with a small side chain volume or or amino acids with a high or or are found in a few amino acids are found at the and An acidic residue is frequently at the fifth position basic residues are conserved at the sixth, ninth, and tenth position. It be that an amino is into this motif In the case of the lactose permease, an residue is found between the glycine at the eighth position and the at the ninth position. from the tetracycline antiporter has that the conserved glycine residues, acidic and one of the basic residues, are very important for transport A. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, A. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, A. J. Biochem. PubMed Scopus Google Scholar). To these conserved residues are also important for the function of the lactose permease, we have the site-directed mutants described in Table I. To determine these mutations permease levels of the and mutant strains were by and in Table the of the mutants showed levels that were similar to the In it seems likely the variability among the mutants is to in this rather than in the of permease. However, it was that both of the substitutions at positions and showed very low levels of permease Therefore, these mutants were with regard to transport activity. The of bacterial on can the of cells that are able to transport and the sugar form red a defect in this results in the of In a lactose permease mutant be in transport to a red When the and mutant strains were on lactose or melibiose some were observed. in Table mutations at the first glycine in the lactose or at the fifth acidic residue were found to be in sugar A few mutations at the glycine were none of the mutants at the basic residues or appeared to be even though nonbasic substitutions were at these on Open table in a new tab The of Table that the and mutants are in ability to transport the other mutants are or a In the of Table in transport were to a more of the uptake In Table the and mutant strains were for ability to transport In transport assays, plasmids the lacY gene or mutant were introduced into an E. coli strain that is Upon into the lactose is so that the lactose than the G. G. J. Google Scholar). Therefore, these lactose transport is downhill or with its In the transport the plasmids were into a bacterial strain that is from the results of Table of the first position mutants were very in both the downhill and uphill transport of (see Table Only the mutant was similar to the strain in downhill transport and uphill the the were observed in the strain which showed transport that were than the These results are consistent with the of residues at this position. from glycine, and are the two residues that are likely to be at the first position in this motif (see Very different results were at the eighth-position glycine. With regard to downhill lactose transport, all of the site-directed mutants at this position were observed to have transport levels that are similar to the strain (see Table These results the of transport a glycine at the eighth position is for transport activity appear essential for the and/or of the lactose permease the levels of of the position mutants were found to be similar to (see Table These the high of that at the eighth position. However, it a that an eighth-position glycine may be important for transport activity and/or that are different from those described in this it be that some of the such as and exhibited uphill accumulation levels that were than that of the that substitutions at this position can the transport The acidic residue at the fifth position to be critical for transport activity. in Table all of the mutants at this position were found to have than of the downhill of lactose The results with the mutant it the group and a glutamate is commonly found at the fifth position in the loop 8/9 motif. the results with the lactose permease a rather for an residue at position Finally, the role of the basic residues was examined by changes at all three basic residues. of the basic residues were found to be for downhill transport function. These results are different from those with the tetracycline antiporter it was found that the basic residue at the ninth position to Arg-73 in the lactose is essential for transport A. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). at the basic residues in the lactose permease were to have only in uphill accumulation from for the strain to for the mutant (see Table the residue at position is it was of to the position mutants are able to couple H+ and sugar To a was used to H+ transport the of lactose. These results are in When lactose was the strain exhibited a of the to the of H+ and lactose into the bacterial the and mutant strains also exhibited a the of the was than that of the strain these mutants have a of lactose transport (see Table The of a motif within a large superfamily is indicative of an important functional and/or structural With regard to the lactose permease, the described in this study this The dramatic results were from mutations at the first-position glycine and the A variety of amino substitutions were to in or of transport activity. the eighth-position glycine and the three basic residues to be essential although many mutations at these showed in the uphill accumulation of lactose. However, the be that these residues may be important for transport activity and/or that are different from the current The role of glycine residues within loop 2/3 may be related to different of the glycine side In this side chain has the volume and a high β-turn or both of these may be important with regard to transport function. At the first is to suggest that both may a In the current and other it that only amino acids with a side such as and to a are able to for glycine. These results are consistent with the that side chain volume an important This conclusion also with the that and are the likely amino other than glycine, to at the first position among members of the USA superfamily. However, it is also to that β-turn propensity) and β-turn propensity) are found at the first position even though these residues have significantly side chain In loop 8/9 of the lactose permease, a is found in the first position of this motif. Therefore, it is to that a of side chain volume and β-turn may to the of amino substitutions at the first position. In some such as the mutant in the lactose permease, the of a large side chain volume may the of high β-turn At the eighth the role of the glycine side chain with large side and/or low β-turn propensity, showed relatively high transport activity. the position mutants with low uphill transport activity and Table with high side chain volume or low β-turn Therefore, based on these transport the results suggest that side chain volume or β-turn are critical at the eighth position. This conclusion seems very that the glycine at the eighth position is the conserved amino in the loop 2/3 motif and it is conserved in the loop 8/9 motif. substitutions of the at position including the conservative glutamate with levels of transport activity. However, it is that an acidic residue at position is essential for sugar The and mutants were able to catalyze the downhill and uphill accumulation of to a an acidic residue at this position appear to be for cation the and mutants were to transport H+ the of lactose. the USA superfamily, an is conserved in the loop 2/3 motif. However, it is rather that an acidic residue or is at the fifth position of this motif at one of the two repeated The only is a gene from Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In it is that a in one of the two repeated has been conserved throughout be to determine this is an important for transport among this superfamily of the motif is conserved among proteins that transport a variety of different of it seems that it is involved with solute of for the motif can be For example, this motif is found the cytoplasmic of it is to that this of the protein may be a critical that of the solute into the these the conserved motif may be important in global conformational changes solute in 3, the of the permease to solute from the of the in the the solute has to the The loop 2/3 motif may be important in promoting the between and may be that loop 2/3 and/or loop 8/9 may be as a for and [14C]Lactose was as described and lactose uptake was in strain the lacY plasmids described within the accumulation was in that is The are as the of activity.
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