Replacement of alanine 58 by asparagine enables the melibiose carrier of Klebsiella pneumoniae to couple sugar transport to Na+.
The melibiose carrier of Klebsiella pneumoniae couples sugar transport to H+ and Li+, while that of Escherichia coli uses Na+ besides the other two cation species (Hama and Wilson, 1992). We have shown that the K. pneumoniae melibiose carrier is capable of recognizing Na+ when the amino-terminal 81 residues are replaced by the corresponding region of the E. coli melibiose carrier (Hama and Wilson, 1993). In this amino-terminal region there are 5 residues that are not conserved between the two carriers. In this study, we changed each of the 5 residues of the K. pneumoniae carrier to the one in the E. coli carrier. The substitutions are Ile-36-->Val, Val-43-->Leu, Leu-54-->Trp, Ala-58-->Asn, and Cys-68-->Ala. With four of the five mutants, Ile-36-->Val, Val-43-->Leu, Leu-54-->Trp, and Cys-68-->Ala, sugar accumulation was not affected by Na+. In striking contrast, melibiose and methyl-1-thio-beta-D-galactopyranoside accumulation was greatly stimulated by Na+ with the Ala-58-->Asn mutant. Furthermore, Na+ uptake coupled to downhill melibiose transport was observed with the Ala-58-->Asn mutant. These results indicate that the Ala-58-->Asn substitution enables the K. pneumoniae melibiose carrier to couple sugar transport to Na+. It is clear that the Asn-58 residue (Asn-54 in the E. coli carrier) is involved in Na+ recognition.
Hama
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Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115.
Wilson
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TH
D001483:Base Sequence; D001693:Biological Transport, Active / Q000187:drug effects; D007711:Klebsiella pneumoniae / Q000378:metabolism*; D008094:Lithium / Q000378:metabolism; D008553:Melibiose / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012964:Sodium / Q000378:metabolism*; D013329:Structure-Activity Relationship; D027981:Symporters
D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009838:Oligodeoxyribonucleotides; D027981:Symporters; C021548:melibiose permease; D008553:Melibiose; D008094:Lithium; D012964:Sodium