Targeted mutagenesis of five conserved tryptophan residues of LolB involved in membrane localization of Escherichia coli lipoproteins.
LolB, catalyzing the last step of lipoprotein transfer from the inner to the outer membrane of Escherichia coli, is itself a lipoprotein anchored to the outer membrane. Five Trp residues of LolB are conserved among LolB homologs in Gram-negative bacteria. These Trp residues were mutagenized to obtain defective LolB mutants. Mutation of Trp at position 52 to Pro impaired the receptor activity and caused accumulation of the LolA-lipoprotein complex in the periplasm. Similar mutants were obtained for Trp at position 117. A mutant with Gly instead of Trp at position 148 retained the receptor activity but inhibited growth upon its overproduction. The outer membrane sorting of this mutant seemed to be defective, lipoprotein transfer thereby being perturbed when it was overproduced. Despite the strong conservation, no defective mutant for Trp at position 183 was obtained, and only weak mutants were isolated for Trp at position 18. Based on the crystal structure of LolB, the phenotypes of these mutants are discussed.
Wada
Rieko
R
Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Matsuyama
Shin-ichi
S
Tokuda
Hajime
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008074:Lipoproteins / Q000737:chemistry* / Q000378:metabolism*; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D011487:Protein Conformation; D021381:Protein Transport / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014018:Tissue Distribution; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C087012:LolB protein, E coli; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D014364:Tryptophan