title: Electrophysiological studies in Xenopus oocytes for the opening of Escherichia coli SecA-dependent protein-conducting channels.
abstract: Protein translocation in Escherichia coli requires protein-conducting channels in cytoplasmic membranes to allow precursor peptides to pass through with adenosine triphosphate (ATP) hydrolysis. Here, we report a novel, sensitive method that detects the opening of the SecA-dependent protein-conducting channels at the nanogram level. E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. Observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein proOmpA or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5'-(beta,gamma-methylene)-diphosphonate, a nonhydrolyzable ATP analogue, both of which are known to inhibit SecA protein activity. Endogenous oocyte precursor proteins also stimulated ion current activity and can be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides continued to enhance ionic currents. Thus, the requirement of signal peptides and ATP hydrolysis for the SecA-dependent currents resembles biochemical protein translocation assay with E. coli membrane vesicles, indicating that the Xenopus oocyte system provides a sensitive assay to study the role of Sec and precursor proteins in the formation of protein-conducting channels using electrophysiological methods.
authors.affiliation: Department of Biology, Georgia State University, 24 Peachtree Center Avenue, Atlanta, GA 30303, USA.
authors.forename: Bor-Ruei
authors.initials: BR
authors.lastname: Lin
authors.forename: Lila M
authors.initials: LM
authors.lastname: Gierasch
authors.forename: Chun
authors.initials: C
authors.lastname: Jiang
authors.forename: Phang C
authors.initials: PC
authors.lastname: Tai
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000166:cytology; D011189:Potassium Chloride / Q000494:pharmacology; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D019810:Sodium Azide / Q000494:pharmacology; D014982:Xenopus laevis
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D007473:Ion Channels; C085044:LamB signal peptide, E coli; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; C078167:OMPA outer membrane proteins; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D019810:Sodium Azide; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins