Identification and initial topological analysis of the Rickettsia prowazekii ATP/ADP translocase.
The Rickettsia prowazekii ATP/ADP translocase was identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis using antibodies raised against a synthetic peptide corresponding in sequence to the carboxyl-terminal 17 amino acids of the carrier. Both the translocase of R. prowazekii and that expressed by Escherichia coli transformants containing the rickettsial gene had an apparent molecular mass of 36,500 Da by SDS-PAGE analysis, a mass considerably less than that deduced from the sequence of the gene. The SDS-solubilized translocase aggregated upon heating at 100 degrees C in the presence of disulfide bond-reducing agents. Similar concentrations of disulfide bond-reducing agents inhibited the exchange transport of adenine nucleotides by both R. prowazekii and translocase-expressing E. coli. These data suggested that an intramolecular disulfide bond in the translocase was essential for transport activity. The antipeptide antibodies used for identification of the translocase bound preferentially to inside-out membrane vesicles of translocase-expressing E. coli relative to right-side-out spheroplasts, thus indicating that the carboxyl terminus of the carrier is located on the cytoplasmic side of the bacterial inner membrane. Protease studies were unable to localize the carboxyl terminus because of the resistance of this region of the native translocase to proteolytic cleavage. These data in conjunction with hydrophobicity analysis were used to construct an initial topological model of the translocase within the cell membrane.
Plano
G V
GV
Department of Microbiology and Immunology, University of South Alabama, College of Medicine, Mobile 36688.
Winkler
H H
HH
D000255:Adenosine Triphosphate / Q000493:pharmacokinetics; D000595:Amino Acid Sequence; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006358:Hot Temperature; D008623:Mercaptoethanol / Q000494:pharmacology; D000226:Mitochondrial ADP, ATP Translocases / Q000235:genetics* / Q000302:isolation & purification; D008956:Models, Chemical; D008969:Molecular Sequence Data; D012283:Rickettsia prowazekii / Q000235:genetics*
D008623:Mercaptoethanol; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; D004229:Dithiothreitol