Translocation ATPase Research Articles

The PATROL1 function in roots contributes to the increase in shoot biomass

Main conclusionPATOL1 contributes to increasing biomass not only by effective stomatal movement but also by root meristematic activity.PATROL1 (PROTON ATPase TRANSLOCATION CONTROL 1), a protein with a MUN domain, is involved in the intercellular trafficking of AHA1 H+-ATPase to the plasma membrane in guard cells. This allows for larger stomatal opening and more efficient photosynthesis, leading to increased biomass. Although PATROL1 is expressed not only in stomata but also in other tissues of the shoot and root, the role in other tissues than stomata has not been determined yet. Here, we investigated PATROL1 functions in roots using a loss-of-function mutant and an overexpressor. Cytological observations revealed that root meristematic size was significantly smaller in the mutant resulting in the short primary root. Grafting experiments showed that the shoot biomass of the mutant scion was increased when it grafted onto wild-type or overexpressor rootstocks. Conversely, grafting of the overexpressor scion shoot enhanced the growth of the mutant rootstock. The leaf temperatures of the grafted plants were consistent with those of their respective genotypes, indicating cell-autonomous behavior of stomatal movement and independent roles of PATROL1 in plant growth. Moreover, plasma membrane localization of AHA1 was not altered in root epidermal cells in the patrol1 mutant implying existence of a different mode of PATROL1 action in roots. Thus PATROL1 plays a role in root meristem and contributes to increase shoot biomass.

Label-Free Quantitative Proteomic Profiling Identifies Potential Active Components to Exert Pharmacological Effects in the Fruit of Alpinia oxyphylla by Mass Spectrometry

To explore the relationship between fruit developmental periods and the protein datasets of Alpinia oxyphylla, a label-free quantitative proteomic profiling analysis of the A. oxyphylla fruits sampled at the Earlystage, Metaphase and Advanced stage was carried out using the liquid chromatography combined with tandem mass spectrometry. A total of 19,219 peptide fragments and 4946 quantitative proteins were obtained. Annotation and enrichment analysis of these peptides and proteins were performed using the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases with bioinformatics software. The results showed that the differentially expressed proteins were mainly involved in metabolic and cellular processes, cellular component organization or biogenesis and response to the stimulus. Three significantly enriched metabolic pathways were non-alcoholic fatty liver disease, vibrio cholera infection and valine, leucine and isoleucine degradation. The significantly enriched proteins were NADH dehydrogenase, putative vacuolar proton translocation ATPase and putative acyltransferase component. The proteomic profiles of the fruit samples from the Advanced and the Metaphase stages differed little, while the difference in proteomic components between the Earlystage and the Advanced groups was significant. This study should lay the theoretical foundation for the effective utilization of A. oxyphylla germplasm resource in the treatment of human diseases.

Improved stomatal opening enhances photosynthetic rate and biomass production in fluctuating light.

It has been reported that stomatal conductance often limits the steady-state photosynthetic rate. On the other hand, the stomatal limitation of photosynthesis in fluctuating light remains largely unknown, although in nature light fluctuates due to changes in sun position, cloud cover, and the overshadowing canopy. In this study, we analysed three mutant lines of Arabidopsis with increased stomatal conductance to examine to what extent stomatal opening limits photosynthesis in fluctuating light. The slac1 (slow anion channel-associated 1) and ost1 (open stomata 1) mutants with stay-open stomata, and the PATROL1 (proton ATPase translocation control 1) overexpression line with faster stomatal opening responses exhibited higher photosynthetic rates and plant growth in fluctuating light than the wild-type, whereas these four lines showed similar photosynthetic rates and plant growth in constant light. The slac1 and ost1 mutants tended to keep their stomata open in fluctuating light, resulting in lower water-use efficiency (WUE) than the wild-type. However, the PATROL1 overexpression line closed stomata when needed and opened stomata immediately upon irradiation, resulting in similar WUE to the wild-type. The present study clearly shows that there is room to optimize stomatal responses, leading to greater photosynthesis and biomass accumulation in fluctuating light in nature.

The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity.

In bacteria, the translocation of proteins across the cytoplasmic membrane by the Sec machinery requires the ATPase SecA. SecA binds ribosomes and recognises nascent substrate proteins, but the molecular mechanism of nascent substrate recognition is unknown. We investigated the role of the C-terminal tail (CTT) of SecA in nascent polypeptide recognition. The CTT consists of a flexible linker (FLD) and a small metal-binding domain (MBD). Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the MBD only or the entire CTT had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function, suggesting that the CTT influences the conformation of SecA. Site-specific crosslinking indicated that F399 in SecA contacts ribosomal protein uL29, and binding to nascent chains disrupts this interaction. Structural studies provided insight into the CTT-mediated conformational changes in SecA. Our results suggest a mechanism for nascent substrate protein recognition.

DNA Topology and the Initiation of Virus DNA Packaging.

During progeny assembly, viruses selectively package virion genomes from a nucleic acid pool that includes host nucleic acids. For large dsDNA viruses, including tailed bacteriophages and herpesviruses, immature viral DNA is recognized and translocated into a preformed icosahedral shell, the prohead. Recognition involves specific interactions between the viral packaging enzyme, terminase, and viral DNA recognition sites. Generally, viral DNA is recognized by terminase’s small subunit (TerS). The large terminase subunit (TerL) contains translocation ATPase and endonuclease domains. In phage lambda, TerS binds a sequence repeated three times in cosB, the recognition site. TerS binding to cosB positions TerL to cut the concatemeric DNA at the adjacent nicking site, cosN. TerL introduces staggered nicks in cosN, generating twelve bp cohesive ends. Terminase separates the cohesive ends and remains bound to the cosB-containing end, in a nucleoprotein structure called Complex I. Complex I docks on the prohead’s portal vertex and translocation ensues. DNA topology plays a role in the TerSλ-cosBλ interaction. Here we show that a site, I2, located between cosN and cosB, is critically important for an early DNA packaging step. I2 contains a complex static bend. I2 mutations block DNA packaging. I2 mutant DNA is cut by terminase at cosN in vitro, but in vivo, no cos cleavage is detected, nor is there evidence for Complex I. Models for what packaging step might be blocked by I2 mutations are presented.

Effects of the protonophore carbonyl-cyanide m-chlorophenylhydrazone on intracytoplasmic membrane assembly in Rhodobacter sphaeroides

The effect of carbonyl-cyanide m-chlorophenyl-hydrazone (CCCP) on intracytoplasmic membrane (ICM) assembly was examined in the purple bacterium Rhodobacter sphaeroides. CCCP blocks generation of the electrochemical proton gradient required for integral membrane protein insertion. ICM formation was induced for 8h, followed by a 4-h exposure to CCCP. Measurements of fluorescence induction/relaxation kinetics showed that CCCP caused a diminished quantum yield, a cessation in expansion of the functional absorption cross-section and a 4- to 10-fold slowing in the electron transfer turnover rate. ICM vesicles (chromatophores) and an upper-pigmented band (UPB) containing ICM growth initiation sites, were isolated and subjected to clear-native electrophoresis. Proteomic analysis of the chromatophore gel bands indicated that CCCP produced a 2.7-fold reduction in spectral counts in the preferentially assembled light-harvesting 2 (LH2) antenna, while the RC-LH1 complex, F1FO-ATPase and pyridine nucleotide transhydrogenase decreased by 1.7–1.9-fold. For 35 soluble enzymes, the ratio of 0.99 for treated/control proteins demonstrated that protein synthesis was unaffected by CCCP, suggesting that the membrane complex decline arose from the turnover of unassembled apoproteins. In the UPB fraction, an ~2-fold accumulation was observed for the preprotein translocase SecY, the SecA translocation ATPase, SecD and SecF insertion components, and chaperonins DnaJ and DnaK, consistent with the possibility that these factors, which act early in the assembly process, have accumulated in association with nascent polypeptides as stabilized assembly intermediates.

Identification of small-molecule inhibitors against SecA by structure-based virtual ligand screening.

The rapid rise of antibiotic-resistant bacteria is one of the major concerns in modern medicine. Therefore, to treat bacterial infections, there is an urgent need for new antibacterials-preferably directed against alternative bacterial targets. One such potential target is the preprotein translocation motor SecA. SecA is a peripheral membrane ATPase and a key component of the Sec secretion pathway, the major route for bacterial protein export across or into the cytoplasmic membrane. As SecA is essential for bacterial viability, ubiquitous and highly conserved in bacteria, but not present in eukaryotic cells, it represents an attractive antibacterial target. Using an in silico approach, we have defined several potentially druggable and conserved pockets on the surface of SecA. We show that three of these potentially druggable sites are important for SecA function. A starting collection of ~500 000 commercially available small-molecules was virtually screened against a predicted druggable pocket in the preprotein-binding domain of Escherichia coli SecA using a multi-step virtual ligand screening protocol. The 1040 top-scoring molecules were tested in vitro for inhibition of the translocation ATPase activity of E. coli SecA. Five inhibitors of the translocation ATPase, and not of basal or membrane ATPase, were identified with IC50 values <65 μm. The most potent inhibitor showed an IC50 of 24 μm. The antimicrobial activity was determined for the five most potent SecA inhibitors. Two compounds were found to possess weak antibacterial activity (IC50 ~198 μm) against E. coli, whereas some compounds showed moderate antibacterial activity (IC50 ~100 μm) against Staphylococcus aureus.

Conformational Changes of the Clamp of the Protein Translocation ATPase SecA

Post-translational protein translocation across the bacterial plasma membrane is mediated by the interplay of the SecA ATPase and the protein-conducting SecY channel. SecA consists of several domains, including two nucleotide-binding domains (NBD1 and NBD2), a polypeptide cross-linking domain (PPXD), a helical scaffold domain (HSD), and a helical wing domain (HWD). PPXD, HSD, and NBD2 form a clamp that positions the polypeptide substrate above the channel so that it can be pushed into the channel by a two-helix finger of the HSD. How the substrate is accommodated in the clamp during translocation is unclear. Here, we report a crystal structure of Thermotoga maritima SecA at 1.9Å resolution. Structural analysis and free-energy calculations indicate that the new structure represents an intermediate state during the transition of the clamp from an open to a closed conformation. Molecular dynamics simulations show that closure of the clamp occurs in two phases, an initial movement of PPXD, HSD, and HWD as a unit, followed by a movement of PPXD alone toward NBD2. Simulations in the presence of a polypeptide chain show that the substrate associates with the back of the clamp by dynamic hydrogen bonding and that the clamp is laterally closed by a conserved loop of the PPXD. Mutational disruption of clamp opening or closure abolishes protein translocation. These results suggest how conformational changes of SecA allow substrate binding and movement during protein translocation.

Dopamine D1-like receptors regulate the α1A-adrenergic receptor in human renal proximal tubule cells and D1-like dopamine receptor knockout mice

The homeostatic control of blood pressure hinges upon the delicate balance between prohypertensinogenic and antihypertensinogenic systems. D₁-like dopamine receptors [dopamine D₁ and D₅ receptors (D₁Rs and D₅Rs, respectively)] and the α₁A-adrenergic receptor (α₁A-AR) are expressed in the renal proximal tubule and engender opposing effects on Na(+) transport, i.e., natriuresis (via D₁Rs and D5Rs) or antinatriuresis (via α₁A-ARs). We tested the hypothesis that the D₁R/D₅R regulates the α₁A-AR. D₁-like dopamine receptors coimmunoprecipitated, colocalized, and cofractionated with α₁A-ARs in lipid rafts in immortalized human renal proximal tubule cells. Long-term treatment with the D₁R/D₅R agonist fenoldopam resulted in decreased D₁R and D₅R expression but increased α₁A-AR abundance in the plasma membrane. Short-term fenoldopam treatment stimulated the translocation of Na(+)-K(+)-ATPase from the plasma membrane to the cytosol that was partially reversed by an α₁A-AR agonist, which by itself induced Na(+)-K(+)-ATPase translocation from the cytosol to the plasma membrane. The α₁A-AR-specific agonist A610603 also minimized the ability of fenoldopam to inhibit Na(+)-K(+)-ATPase activity. To determine the interaction among D₁Rs, D₅Rs, and α₁A-ARs in vivo, we used phenylephrine and A610603 to decrease Na(+) excretion in several D1-like dopamine receptor knockout mouse strains. Phenylephrine and A61603 treatment resulted in a partial reduction of urinary Na(+) excretion in wild-type mice and its abolition in D1R knockout, D₅R knockout, and D₁R-D₅R double-knockout mice. Our results demonstrate the ability of the D₁-like dopamine receptors to regulate the expression and activity of α₁A-AR. Elucidating the intricacies of the interaction among these receptors is crucial for a better understanding of the crosstalk between anti- and pro-hypertensive systems.

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

SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG–SecDF·YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg2+, mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes.

Nonpigmented Ciliary Epithelial Cells Respond to Acetazolamide by a Soluble Adenylyl Cyclase Mechanism

The nonpigmented ciliary epithelium (NPE) is rich in soluble adenylyl cyclase (sAC), a proposed cytoplasmic bicarbonate sensor. Here, we examine the contribution of sAC to an increase in cyclic AMP (cAMP) and changes in a key ion transporter, H(+)-ATPase, in NPE exposed to acetazolamide, a carbonic anhydrase inhibitor (CAI). Cyclic AMP was measured by radioimmunoassay in primary cultured porcine NPE. The pH-sensitive dye BCECF was used to examine cytoplasmic pH regulation. Subcellular protein translocation was examined by Western blot. A transient cAMP increase, detectable within minutes of acetazolamide treatment, was prevented by KH7, a specific sAC inhibitor. Following 10-minute exposure to acetazolamide, the abundance of H(+)-ATPase B1 subunit and sAC was doubled in a plasma membrane-rich fraction, suggesting subcellular translocation. Similar evidence of H(+)-ATPase translocation was observed in NPE exposed to 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Consistent with increased capacity for proton export, acetazolamide increased the rate of pH recovery from acidification. KH7 and bafilomycin A1, an inhibitor of H(+)-ATPase, both prevented the stimulatory effect of acetazolamide on pH recovery. In a parallel study, H(+)-ATPase abundance was found to be higher in the plasma membrane of HEK293 cells that overexpress sAC compared to the normal HEK293 cells. HEK cells that overexpress sAC and had higher H(+)-ATPase abundance displayed a faster rate of pH recovery and greater sensitivity to KH7. Acetazolamide increases cAMP in a response that involves activation of sAC. Subcellular translocation of H(+)-ATPase and an increase in the capacity for proton export by acetazolamide-treated NPE cells is a cAMP-dependent response.

Abstract 468: The Dopamine D 1 -like Receptors Negatively Regulate the Expression and Function of the a 1 A Adrenergic Receptor

The homeostatic control of blood pressure hinges upon the delicate balance between pro-hypertensinogenic (e.g., sympathetic nervous system) and anti-hypertensinogenic systems (e.g., dopamine system). The D 1 -like dopamine receptors (D 1 R and D 5 R) and the α 1A adrenergic receptor (ARα 1A ) are endogenously expressed in the renal proximal tubules and engender opposing effects on sodium transport, i.e., natriuresis (D 1 R and D 5 R) or anti-natriuresis (ARα 1A ). We tested the hypothesis that the D 1 R and D 5 R interact with and regulate the ARα 1A in human renal proximal tubule cells (hRPTCs) and in mice. We found that the D 1 R and D 5 R colocalized with the ARα 1A in hRPTCs and in proximal tubules in human kidney sections. Both receptors immunoprecipitated, pulled-down, and co-fractionated with ARα 1A in lipid rafts. Short-term co-treatment with fenoldopam (1 μM, 15 min) reversed the ARα 1 agonist phenylephrine (10 μM, 15 min)-induced Na + ,K + -ATPase (NKA) translocation from the cytosol to the plasma membrane in hRPTCs (plasma membrane NKA: vehicle=100±5% vs. fenoldopam=65±3% vs. phenylephrine=177±5% vs. co-treatment=115±7%; P&lt;0.05, n=3-4). Long-term fenoldopam (1 μM, 24 hr) treatment resulted in decreased D 1 R (70.0±5.9%, P&lt;0.05, n=3) and D 5 R (50.1±10.7%, P&lt;0.05, n=3), consistent with D 1 -like receptor desensitization, but increased ARα 1A abundance (142.6±4.3%, P&lt;0.05, n=3) in hRPTCs. RNAi silencing of ARα 1A (48 hr) increased the expression of D 1 R and D 5 R. To determine the extent of regulation of each D 1 -like receptor on ARα 1A , we used the subclass-selective ARα 1 agonist phenylephrine (5 μg/kg body weight, i.p.) and the receptor-specific ARα 1A agonist A610603 (25 ng/kg body weight, i.p.) to inhibit Na + excretion in three mouse strains. Phenylephrine treatment resulted in 59.6%, 84.2%, 99.3%, and 99.5% reduction from basal level of 24-hr Na + excretion_while A61603 treatment resulted in 42.4%, 67.1%, 99.9%, and 100% reduction_in wild-type controls, D 1 R -/- , and D 5 R -/- knockout mice, and D 1 R/D 5 R -/- double knockout mice, respectively, suggesting a stronger regulatory effect of D 5 R on ARα 1A . Elucidating the intricacies of the interaction among these receptors is crucial for a better understanding of the crosstalk between anti- and pro-hypertensive systems.

A Munc13-like protein in Arabidopsis mediates H+-ATPase translocation that is essential for stomatal responses

Plants control CO2 uptake and water loss by modulating the aperture of stomata located in the epidermis. Stomatal opening is initiated by the activation of H+-ATPases in the guard-cell plasma membrane. In contrast to regulation of H+-ATPase activity, little is known about the translocation of the guard cell H+-ATPase to the plasma membrane. Here we describe the isolation of an Arabidopsis gene, PATROL1, that controls the translocation of a major H+-ATPase, AHA1, to the plasma membrane. PATROL1 encodes a protein with a MUN domain, known to mediate synaptic priming in neuronal exocytosis in animals. Environmental stimuli change the localization of plasma membrane-associated PATROL1 to an intracellular compartment. Plasma membrane localization of AHA1 and stomatal opening require the association of PATROL1 with AHA1. Increased stomatal opening responses in plants overexpressing PATROL1 enhance the CO2 assimilation rate, promoting plant growth.

Glycolipozyme MPIase is essential for topology inversion of SecG during preprotein translocation

Presecretory proteins are translocated across biological membranes through protein-conducting channels such as Sec61 (eukaryotes) and SecYEG (bacteria). SecA, a translocation ATPase, pushes preproteins out with dynamic structural changes through SecYEG. SecG, a subunit of the SecYEG channel possessing two transmembrane stretches (TMs), undergoes topology inversion coupled with SecA-dependent translocation. Recently, we characterized membrane protein integrase (MPIase), a glycolipozyme involved in not only protein integration into membranes but also preprotein translocation. We report here that SecG inversion occurs only when MPIase associates with SecYEG. We also found that MPIase modulates the dimer orientation of SecYEG. Cysteine-scanning mutagenesis mapped SecG TM 2 to a relatively hydrophilic environment. The dimer formation of SecG, crosslinked at TM 2, was not observed on SecG inversion, indicating that SecYEG undergoes a dynamic structural change during preprotein translocation.

Structure of an actin-related subcomplex of the SWI/SNF chromatin remodeler

The packaging of DNA into nucleosomal structures limits access for templated processes such as transcription and DNA repair. The repositioning or ejection of nucleosomes is therefore critically important for regulated events, including gene expression. This activity is provided by chromatin remodeling complexes, or remodelers, which are typically large, multisubunit complexes that use an ATPase subunit to translocate the DNA. Many remodelers contain pairs or multimers of actin-related proteins (ARPs) that contact the helicase-SANT-associated (HSA) domain within the catalytic ATPase subunit and are thought to regulate ATPase activity. Here, we determined the structure of a four-protein subcomplex within the SWI/SNF remodeler that comprises the Snf2 HSA domain, Arp7, Arp9, and repressor of Ty1 transposition, gene 102 (Rtt102). Surprisingly, unlike characterized actin-actin associations, the two ARPs pack like spoons and straddle the HSA domain, which forms a 92-Å-long helix. The ARP-HSA interactions are reminiscent of contacts between actin and many binding partners and are quite different from those in the Arp2/3 complex. Rtt102 wraps around one side of the complex in a highly extended conformation that contacts both ARPs and therefore stabilizes the complex, yet functions to reduce by ∼2.4-fold the remodeling and ATPase activity of complexes containing the Snf2 ATPase domain. Thus, our structure provides a foundation for developing models of remodeler function, including mechanisms of coupling between ARPs and the ATPase translocation activity.

Detailed kinetic analysis of the φ29 DNA packaging motor providing evidence for coordinated intersubunit ATPase activity of gp16

Presented is a detailed kinetic evaluation of the motor component interactions of the DNA translocation ATPase of Bacillus subtilis bacteriophage φ29. The components of the φ29 DNA packaging motor, comprised of both protein and non-protein parts, act in a coordinated manner to translocate DNA into a viral capsid, despite entropically unfavorable conditions. The precise nature of this coordination remains under investigation but recent results have shown that the gp16 pentamer acts to propel the genomic DNA in 10 base pair bursts, implying inter-subunit synchronization. We observe an emergent tandem coordination behavior in the ATPase activity of gp16 as demonstrated by a Hill coefficient of 2.4±0.2, as differentiated from its activity in DNA packaging which has been shown to have a unity Hill coefficient. Due to its relative strength and DNA packaging efficiency, understanding the molecular mechanism of force generation may prove useful to various nanotechnology applications including gene therapy, control of biological ATPases, and the powering of nanoscale mechanical devices.

Two copies of the SecY channel and acidic lipids are necessary to activate the SecA translocation ATPase

The SecA ATPase associates with the SecY complex to push preproteins across the bacterial membrane. Because a single SecY is sufficient to create the conducting channel, the function of SecY oligomerization remains unclear. Here, we have analyzed the translocation reaction using nanodiscs. We show that one SecY copy is sufficient to bind SecA and the preprotein, but only the SecY dimer together with acidic lipids supports the activation of the SecA translocation ATPase. In discs, the dimer is predominantly arranged in a back-to-back manner and remains active even if a constituent SecY copy is defective for SecA binding. In membrane vesicles and in intact cells, the coproduction of two inactive SecYs, one for channel gating and the other for SecA binding, recreates a functional translocation unit. These results indisputably argue that the SecY dimer is crucial for the activation of SecA, which is necessary for preprotein transport.

Fluorescein Analogues Inhibit SecA ATPase: The First Sub‐micromolar Inhibitor of Bacterial Protein Translocation

SecA is a central component of the general secretion system that is essential for bacterial growth and thus an ideal target for antimicrobial agents. A series of fluorescein analogues were first screened against the ATPase activity using the truncated unregulated SecA catalytic domain. Rose bengal (RB) and erythrosin B (EB) were found to be potent inhibitors SecA with IC(50) values of 0.5 μM and 2 μM, respectively. RB and EB inhibit the catalytic SecA ATPase more effectively than the F(1) F(0) -proton ATPase. We used three assays to test the effect of these compounds on full-length SecA ATPase: in solution (intrinsic ATPase), in membrane preparation, and translocation ATPase. RB and EB show the following trend in terms of IC(50) values: translocation ATPase<membrane ATPase<intrinsic ATPase. Very importantly, the potency of these fluorescein analogues in inhibiting the truncated SecA ATPase correlates with their ability to inhibit the biologically relevant protein translocation activity of SecA. The in vitro translocation of proOmpA precursors into membrane vesicles is strongly inhibited by RB with IC(50) values of approximately 0.25 μM, making RB the most potent inhibitor of SecA ATPase and SecA-dependent protein translocation reported thus far. The ability of these compounds to inhibit SecA also directly translates into antibacterial effects. Our findings show the value of fluorescein analogues as probes for mechanistic studies of SecA functions and for the potential development of new antimicrobial agents with SecA as the target.

Multiple SecA Molecules Drive Protein Translocation across a Single Translocon with SecG Inversion

SecA is a translocation ATPase that drives protein translocation. D209N SecA, a dominant-negative mutant, binds ATP but is unable to hydrolyze it. This mutant was inactive to proOmpA translocation. However, it generated a translocation intermediate of 18 kDa. Further addition of wild-type SecA caused its translocation into either mature OmpA or another intermediate of 28 kDa that can be translocated into mature by a proton motive force. The addition of excess D209N SecA during translocation caused a topology inversion of SecG. Moreover, an intermediate of SecG inversion was identified when wild-type and D209N SecA were used in the same amounts. These results indicate that multiple SecA molecules drive translocation across a single translocon with SecG inversion. Here, we propose a revised model of proOmpA translocation in which a single catalytic cycle of SecA causes translocation of 10-13 kDa with ATP binding and hydrolysis, and SecG inversion is required when the next SecA cycle begins with additional ATP hydrolysis.

Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors

A major pathway for bacterial preprotein translocation is provided by the Sec-dependent preprotein translocation pathway. Proteins destined for Sec-dependent translocation are synthesized as preproteins with an N-terminal signal peptide, which targets them to the SecYEG translocase channel. The driving force for the translocation reaction is provided by the peripheral membrane ATPase SecA, which couples the hydrolysis of ATP to the stepwise transport of unfolded preproteins across the bacterial membrane. Since SecA is essential, highly conserved among bacterial species, and has no close human homologues, it represents a promising target for antibacterial chemotherapy. However, high-throughput screening (HTS) campaigns to identify SecA inhibitors are hampered by the low intrinsic ATPase activity of SecA and the requirement of hydrophobic membranes for measuring the membrane or translocation ATPase activity of SecA. To address this issue, we have developed a colorimetric high-throughput screening assay in a 384-well format, employing an Escherichia coli ( E. coli) SecA mutant with elevated intrinsic ATPase activity. The assay was applied for screening of a chemical library consisting of ∼27,000 compounds and proved to be highly reliable (average Z′ factor of 0.89). In conclusion, a robust HTS assay has been established that will facilitate the search for novel SecA inhibitors.