Background: SecA has been viewed as ATPase helping precursors across SecYEG channels. Results: SecA alone could promote protein translocation and ion channel activity, but loses specificity and efficiency, which can be restored by SecYEG. Conclusion: SecA plays important structural roles and can function as low affinity protein conducting channels in membranes. Significance: Establishing SecA as channels is crucial for understanding diverse mechanisms and evolution of bacterial translocation pathways.

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.

Retinoic acids (RAs), which are metabolites of vitamin A, have been shown to be involved in multiple T cell effector responses through their binding to the retinoic acid receptor, a ligand-activated transcription factor. Since the molecular mechanism of regulation by RA is still not fully uncovered, we investigated the gene expression profile of all-trans retinoic acid (ATRA)–treated human CD4+ T cells. Leucine zipper transcription factor-like 1 (LZTFL1) was upregulated by ATRA in a dose- and time-dependent manner. The expression of LZTFL1 depended on both ATRA and TCR signaling. LZTFL1 accumulated in the plasma membrane compartment of human CD4+ T cells, and during immunological synapse (IS) formation, it transiently redistributed to the T cell and APC contact zone, indicating its role in T cell activation. Live cell imaging demonstrates that at the initial stage of IS formation, LZTFL1 is concentrated at the APC contact site, and during later stages, it relocates to the distal pole. Knockdown of LZTFL1 reduced the basal- and ATRA-induced levels of IL-5 in CD4+ T cells, and overexpression of LZTFL1 enhanced the TCR-mediated NFAT signaling, suggesting that LZTFL1 is an important regulator of ATRA-induced T cell response. Together, these data indicate that LZTFL1 modulates T cell activation and IL-5 levels.

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.

BackgroundIt is known that retinoid receptor function is attenuated during T cell activation, a phenomenon that involves actin remodeling, suggesting that actin modification may play a role in such inhibition. Here we have investigated the role of actin dynamics and the effect of actin cytoskeleton modifying agents on retinoid receptor-mediated transactivation.ResultsAgents that disturb the F-actin assembly or disassembly attenuated receptor-mediated transcription indicating that actin cytoskeletal homeostasis is important for retinoid receptor function. Overexpression or siRNA-induced knockdown of cofilin-1 (CFL1), a key regulator of F-actin assembly, induced the loss of receptor function. In addition, expression of either constitutively active or inactive/dominant-negative mutants of CFL1or CFL1 kinase LIMK1 induced loss of receptor function suggesting a critical role of the LIMK1-mediated CFL1 pathway in receptor-dependent transcription. Further evidence of the role of LMK1/CFL1-mediated actin dynamics, was provided by studying the effect of Nef, an actin modifying HIV-1 protein, on receptor function. Expression of Nef induced phosphorylation of CFL1 at serine 3 and LIMK1 at threonine 508, inhibited retinoid-receptor mediated reporter activity, and the expression of a number of genes that contain retinoid receptor binding sites in their promoters. The results suggest that the Nef-mediated inhibition of receptor function encompasses deregulation of actin filament dynamics by LIMK1 activation and phosphorylation of CFL1.ConclusionWe have identified a critical role of LIMK1-mediated CFL1 pathway and actin dynamics in modulating retinoid receptor mediated function and shown that LIMK1-mediated phosphocycling of CFL1 plays a crucial role in maintaining actin homeostasis and receptor activity. We suggest that T cell activation-induced repression of nuclear receptor-dependent transactivation is in part through the modification of actin dynamics.

Previous studies showed that E. coli membranes depleted of SecYEG are capable of translocating certain precursor proteins, but not other precursors such as pPhoA, indicating a differential requirement for SecYEG. In this study, we examined the role of SecYEG in pPhoA translocation using a purified reconstituted SecA-liposomes system. We found that translocation of pPhoA, in contrast to that of pOmpA, requires the presence of purified SecYEG. A differential specificity of the SecYEG was also revealed in its interaction with SecA: EcSecYEG did not enhance SecA-mediated pOmpA translocation by purified SecA either from Pseudomonas aeruginosa or Bacillus subtilis. Neither was SecYEG required for eliciting ion channel activity, which could be opened by unfolded pPhoA or unfolded PhoA. Addition of the SecYEG complex did restore the specificity of signal peptide recognition in the ion-channel activity. We concluded that SecYEG confers specificity in interacting with protein precursors and SecAs.

Recruitment of Oct-1 protein to the octamer sequence of U6 promoter is critical for optimal transcription by RNA polymerase III. Here we report that p38 kinase inhibitors, SB202190 and SB203580, stimulated U6 promoter activity and this stimulation can be observed only in the presence of octamer sequence. SB202190-treated cell nuclear extract had about 50% increase in Oct-1 binding activity suggesting that the increased U6 promoter activity by p38 kinase inhibitor is mediated through Oct-1. Mutation in octamer sequence significantly reduced the SB202190-stimulated U6 promoter transcription and the distance between octamer and proximal sequence element of U6 promoter is also critical for the p38 kinase inhibitor-stimulated activity. Exogenous Oct-1 expression showed a concentration-dependent activation of U6 promoter that was further stimulated by the p38 kinase inhibitors. When cells were treated with p38 kinase inducer, hydrogen peroxide or phorbol 12-myristate 13-acetate (PMA), U6 promoter activity was down regulated and this inhibition was reversed by p38 kinase inhibitors. Over-expression of p38α kinase down-regulated U6 promoter activity and this inhibition was further enhanced by PMA and p38 kinase inhibitors reversed this inhibition. p38 kinase inhibitor-treated cells had 50% more U6 RNA than the control cells. Taken together, our results show a negative correlation between the p38 kinase levels and Oct-1 binding on U6 promoter, suggesting that U6 promoter is negatively regulated by p38 kinase.

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