title: Reconstitution of periplasmic transport in inside-out membrane vesicles. Energization by ATP.
abstract: The periplasmic histidine permease of Salmonella typhimurium has been reconstituted in inside-out vesicles (IOV) of Escherichia coli by disrupting the cells with a French press in the presence of a high concentration of the periplasmic histidine-binding protein, HisJ. Efflux from IOV, which is equivalent to uptake in whole cells, is induced by ATP. The reconstituted system depends on the presence of the membrane-bound permease proteins, HisQ, HisM, and HisP, and does not function if reconstitution is performed in the presence of a mutant HisJ protein, HisJ5625, that can bind histidine normally but can't interact properly with the membrane complex. Efflux is not induced by the nonhydrolyzable ATP analog, adenyl-5'-yl imidodiphosphate, supporting the contention that ATP hydrolysis is necessary. 8-Azido ATP inactivates IOV, indicating that the ATP effect occurs through the HisP protein, which has previously been shown to be modified by 8-azido ATP (Hobson, A., Weatherwax, R., and Ames, G.F.-L. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 733-7337). The estimated Km of the vesicles for ATP is about 200 microM. Vanadate, an inhibitor of phosphohydrolase enzymes, inhibits ATP-induced efflux. We conclude that ATP is likely to be the proximal energy source for periplasmic permeases.
authors.affiliation: Department of Biochemistry, University of California, Berkeley 94720.
authors.forename: G F
authors.initials: GF
authors.lastname: Ames
authors.forename: K
authors.initials: K
authors.lastname: Nikaido
authors.forename: J
authors.initials: J
authors.lastname: Groarke
authors.forename: J
authors.initials: J
authors.lastname: Petithory
mesh_terms: D000255:Adenosine Triphosphate / Q000502:physiology*; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000502:physiology; D006639:Histidine / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000502:physiology*; D012486:Salmonella typhimurium / Q000502:physiology
chemical_list: D026901:Membrane Transport Proteins; D006639:Histidine; D000255:Adenosine Triphosphate