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.

Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes

“…The other membrane SecDF•YajC complex stabilizes the SecYEG complex during protein translocation [16][17][18][19][20][21]. On the other hand, SecA can integrate into lipids and form a ring-like pore structures [22] that alone could serve as protein-conducting channels for protein translocation and ion channel activity in the absence of SecYEG [23,24]. The SecA-mediated channel activity in oocytes requires the presence of functional SecA and precursors, as well as bacterial membranes [4].…”

Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

“…Therefore, SecA is considered an excellent target for the development of broad-spectrum antimicrobials. [3] Prior efforts identified inorganic azide as a SecA inhibitor in the mM range. [2a, 2g, 4] We have recently developed three structural classes of SecA inhibitors (Figure 1) and started examining this issue.…”

“…Since the publication of our earlier work, there have been additional reports of SecA inhibitors with potencies in the high μM to low mM range. [3a, 5] A recent paper reported a low μM inhibitor against citrus pathogen , Candidatus Liberibacter asiaticus. [6] The three structural classes of small molecule SecA inhibitors (Figure 1) that we developed include Rose Bengal and its Class A analogues, [7] Class B pyrimidine analogues, [8] and Class C triazole-pyrimidine analogues [9] with the best having IC 50 and/or MIC values in the high nM range (Tables 1 and 2).…”

Mechanisms of Rose Bengal inhibition on SecA ATPase and ion channel activities

“…The voltage clamp adapted from an electrophysiological method was used to measure the opening of protein conducting channels as described previously [15,16,32]. Briefly, the 50 nl sample mixtures were injected into dark pole site of oocytes.…”

“…Purified soluble SecA can insert into membranes, and become an integrated membrane protein [11,12,13]. In addition, there are evidences that SecA alone can integrate into lipids and form a ring-like pore structures [14] that could serve as protein-conducting channels for protein translocation and ion channel activity in the absence of SecYEG [15,16]. SecA from various bacteria with liposomes can also elicit channel activity [15,17].…”