Identification and initial topological analysis of the Rickettsia prowazekii ATP/ADP translocase

Gregory V Plano, H. H. Winkler

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

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°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.

Original languageEnglish
Pages (from-to)3389-3396
Number of pages8
JournalJournal of Bacteriology
Volume173
Issue number11
StatePublished - Jun 24 1991
Externally publishedYes

Fingerprint

Rickettsia prowazekii
ATP Translocases Mitochondrial ADP
Sodium Dodecyl Sulfate
Disulfides
Reducing Agents
Escherichia coli
Polyacrylamide Gel Electrophoresis
Spheroplasts
Membranes
Adenine Nucleotides
Antibodies
Hydrophobic and Hydrophilic Interactions
Heating
Genes
Peptide Hydrolases
Cell Membrane
Amino Acids
Peptides

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

Cite this

Identification and initial topological analysis of the Rickettsia prowazekii ATP/ADP translocase. / Plano, Gregory V; Winkler, H. H.

In: Journal of Bacteriology, Vol. 173, No. 11, 24.06.1991, p. 3389-3396.

Research output: Contribution to journalArticle

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