title: Escherichia coli membranes depleted of SecYEG elicit SecA-dependent ion-channel activity but lose signal peptide specificity.
abstract: We have developed a sensitive method to detect the opening of SecA-dependent, protein-conducting channels in Xenopus oocytes. In this study, we determined the ionic current activities of the SecA-dependent channel from membrane vesicles depleted of SecYEG. We found that these SecYEG-depleted membranes produced SecA-dependent ionic currents in the oocytes, as did membranes containing SecYEG. However, reconstituted membranes depleted of SecYEG required higher concentrations of SecA to elicit ionic currents like those in membranes containing SecYEG. In contrast to membranes containing SecYEG, the proofreading capacity of signal peptides was lost for those membranes lacking SecYEG. These findings are consistent with loss of signal peptide specificity in channel activity from membranes of SecY suppressor or SecY plug domain mutants. The signal peptide specificity of the reconstituted membranes, like SecA-liposomes, can be restored by the addition of SecYEG proteoliposomes. On the other hand, the channel activity efficiency of reconstituted membranes was fully restored, while SecA-liposomes could only be partially enhanced by the addition of SecYEG, indicating that, in addition to SecYEG, other membrane proteins contribute to the efficiency of channel activity. The SecA-dependent channels in membranes that lacked SecYEG also lost ion selectivity to monovalent cations but retained selective permeability to large anions. Thus, the electrophysiological evidence presented here indicates that SecYEG is not obligatory for the channel activity of Escherichia coli membranes, as previously shown for protein translocation, and that SecYEG is important for maintenance of the efficiency and specificity of SecA-dependent channels.
authors.affiliation: Department of Biology, Petit Science Center, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Bor-Ruei
authors.initials: BR
authors.lastname: Lin
authors.forename: Ying-Hsin
authors.initials: YH
authors.lastname: Hsieh
authors.forename: Chun
authors.initials: C
authors.lastname: Jiang
authors.forename: Phang C
authors.initials: PC
authors.lastname: Tai
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002414:Cations, Monovalent / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism*; D017136:Ion Transport; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
chemical_list: D001426:Bacterial Proteins; D002414:Cations, Monovalent; D029968:Escherichia coli Proteins; D007473:Ion Channels; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins