Role of glycine residues in the structure and function of lactose permease, an Escherichia coli membrane transport protein.
By using oligonucleotide-directed, site-specific mutagenesis, the role of 34 Gly residues in the lactose permease of Escherichia coli has been studied systematically. Each of 34 out of a total of 36 Gly residues was replaced with Cys in a functional permease mutant devoid of Cys residues (C-less permease), as previous experiments demonstrate that Gly-402 and Gly-404 can be deleted by truncation of the C-terminus with no loss of activity [Roepe, P. D., et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3992; McKenna, E., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2969]. Out of the 34 Cys-replacement mutants described, 15 transport lactose with high activity, 16 exhibit decreased but significant ability to catalyze lactose accumulation, and 3 (Gly-64-->Cys, Gly-115-->Cys and Gly-147-->Cys) exhibit no activity whatsoever. The inactive mutants were studied in more detail by replacement of Gly with Ala, Val, or Pro. C-less permease with Gly-115-->Ala or Gly-147-->Ala transports lactose almost as well as the control, while mutants with Val or Pro in place of Gly have little or no capacity to accumulate the disaccharide. In contrast, mutants with Ala, Val, or Pro in place of Gly-64 are inactive. Strikingly, however, when the mutations are placed in the wild-type background, Gly-64-->Ala permease transports lactose, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside, and methyl 1-thio-beta,D-galactopyranoside 40-60% as well as wild-type permease, while Gly-64-->Val or Gly-64-->Pro permease is inactive toward all of these substrates. The results indicate that although none of the Gly residues in lactose permease is mandatory for activity, the bulk of the side chain at positions 64, 115, and 147, rather than conformational flexibility at these positions, is particularly important.
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Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1662.
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Colacurcio
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Kaback
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D001692:Biological Transport; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005998:Glycine / Q000737:chemistry*; D007785:Lactose / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D027981:Symporters
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D007785:Lactose; D005998:Glycine