Electrophysiological Studies in Xenopus Oocytes for the Opening of Escherichia coli SecA-Dependent Protein-Conducting Channels |
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Authors: | Bor-Ruei Lin Lila M Gierasch Chun Jiang Phang C Tai |
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Institution: | (1) Department of Biology, Georgia State University, 24 Peachtree Center Avenue, Atlanta, GA 30303, USA;(2) Departments of Biochemistry and Molecular Biology and of Chemistry, University of Massachusetts, 710 N. Pleasant Street, Amherst, MA 01003, USA |
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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′-(β,γ-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. |
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Keywords: | Protein-conducting channel SecA E coli inverted membrane vesicle Signal peptide Voltage clamp Xenopus oocyte |
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