Characterization of F-pilin as an inner membrane component of Escherichia coli K12.
Antipeptide antibodies were used to detect, purify, and characterize nonfilament F-pilin in the cell envelope of an F'tra+ strain of Escherichia coli. Affinity-purified goat antibodies raised against a peptide corresponding to the amino-terminal 14 amino acids of F-pilin detected F-pilin in immuno-overlay ("Western") blots of electrophoretically separated inner and outer membrane proteins. As expected, the molecule was absent from inner membrane preparations of F- or F'traA[Am] strains. Immunoreactive material was purified from inner membrane fractions and shown to be F-pilin by amino acid analysis. The anti-peptide antibodies also detected membrane forms of F-pilin produced by cells containing plasmid pTG801 (Grossman, T. & Silverman, P. (1989) J. Bacteriol. 171, 650-656). Most cell envelope pilin was in the inner membrane fraction, but a significant quantity fractionated with the outer membrane as well. The hydropathy profile of F-pilin suggested that the molecule is an integral membrane protein with two membrane-spanning domains. In confirmation, F-pilin and pTG801 pilins in inner membrane preparations were solubilized by a single extraction with the nonionic detergents Nonidet P-40 (2%) or Triton X-100 (2%), but not by 2 M KCl or 0.1 M NaOH. Moreover, analysis of traA'-'phoA constructs indicated that both the amino and carboxyl termini of F-pilin face the periplasm. The periplasmic location of the amino terminus was confirmed by immunoelectron microscopy of spheroplasts from F' and pTG801 strains, using a monoclonal antibody that recognizes an amino-terminal epitope. These data suggest a specific structure for membrane F-pilin. We discuss that structure in relation to the probable structure of filament F-pilin.
Paiva
W D
WD
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Grossman
T
T
Silverman
P M
PM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D002462:Cell Membrane / Q000737:chemistry* / Q000648:ultrastructure; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D036781:Fimbriae Proteins; D005798:Genes, Bacterial; D015151:Immunoblotting; D016253:Microscopy, Immunoelectron; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017830:Octoxynol; D010957:Plasmids; D011092:Polyethylene Glycols; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000032:analysis
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; C444040:F pilin, E coli; D011993:Recombinant Fusion Proteins; D036781:Fimbriae Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40