Cation-coupling in chimeric melibiose carriers derived from Escherichia coli and Klebsiella pneumoniae. The amino-terminal portion is crucial for Na+ recognition in melibiose transport.
The melibiose carrier of Escherichia coli couples sugar transport to H+, Na+, and Li+, while that of Klebsiella pneumoniae utilizes only H+ and Li+. We made five chimeric carriers derived from the two carriers to identify the region(s) involved in Na+ recognition. The chimeric carriers E2K10, E4K8, E6K6, E8K4, and E10K2 have the amino-terminal 77, 144, 197, 298, and 349 amino acid residues derived from E. coli and the rest derived from K. pneumoniae, respectively. Melibiose accumulation through the chimeric carriers E2K10, E4K8, and E6K6 was strongly stimulated by Na+ and Li+ as is the case with the E. coli carrier. On the other hand, there was very little stimulation with the carriers E8K4 and E10K2. These results suggest that, 1) the amino-terminal 77 amino acids of the E. coli carrier, which has 5 different and 4 fewer amino acids than the K. pneumoniae carrier, have a crucial role in Na+ recognition in melibiose transport and 2) the carboxyl-terminal half of the carrier also forms a part of the Na+ recognition site which may be distorted in chimeric structures. In contrast with melibiose accumulation, there was very little Na+ stimulation of TMG (methyl-1-thio-beta-D-galactopyranoside) transport and no Na+ stimulation was observed in lactose transport with any of the chimeric carriers, whereas in E. coli Na+ stimulates TMG and lactose transport. These results suggest that there is no universal Na+ recognition site for all the sugar substrates. Instead different parts of the carrier seem to participate in cation recognition for different sugar substrates.
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Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115.
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D001483:Base Sequence; D002712:Chlorides / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D008094:Lithium / Q000494:pharmacology; D018021:Lithium Chloride; D008553:Melibiose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D027981:Symporters
D002712:Chlorides; D026901:Membrane Transport Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D027981:Symporters; D012965:Sodium Chloride; C021548:melibiose permease; D008553:Melibiose; D008094:Lithium; D012964:Sodium; D018021:Lithium Chloride