A novel labeling approach supports the five-transmembrane model of subunit a of the Escherichia coli ATP synthase.
Cysteine mutagenesis and surface labeling has been used to define more precisely the transmembrane spans of subunit a of the Escherichia coli ATP synthase. Regions of subunit a that are exposed to the periplasmic space have been identified by a new procedure, in which cells are incubated with polymyxin B nonapeptide (PMBN), an antibiotic derivative that partially permeabilizes the outer membrane of E. coli, along with a sulfhydryl reagent, 3-(N-maleimidylpropionyl) biocytin (MPB). This procedure permits reaction of sulfhydryl groups in the periplasmic space with MPB, but residues in the cytoplasm are not labeled. Using this procedure, residues 8, 27, 37, 127, 131, 230, 231, and 232 were labeled and so are thought to be exposed in the periplasm. Using inside-out membrane vesicles, residues near the end of transmembrane spans 1, 64, 67, 68, 69, and 70 and residues near the end of transmembrane spans 5, 260, 263, and 265 were labeled. Residues 62 and 257 were not labeled. None of these residues were labeled in PMBN-permeabilized cells. These results provide a more detailed view of the transmembrane spans of subunit a and also provide a simple and reliable technique for detection of periplasmic regions of inner membrane proteins in E. coli.
Wada
T
T
Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275, USA.
Long
J C
JC
Zhang
D
D
Vik
S B
SB
D000595:Amino Acid Sequence; D002463:Cell Membrane Permeability; D004926:Escherichia coli / Q000201:enzymology*; D008239:Lysine / Q000031:analogs & derivatives; D008301:Maleimides; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D015335:Molecular Probes; D008969:Molecular Sequence Data; D019897:Periplasm / Q000201:enzymology; D011112:Polymyxin B / Q000031:analogs & derivatives / Q000494:pharmacology; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D013439:Sulfhydryl Reagents
D008301:Maleimides; D008565:Membrane Proteins; D015335:Molecular Probes; D013439:Sulfhydryl Reagents; C038934:polymyxin B nonapeptide; C047272:3-(N-maleimidopropionyl)biocytin; D006180:Proton-Translocating ATPases; D011112:Polymyxin B; D008239:Lysine