Intrinsic membrane targeting of the flagellar export ATPase FliI: interaction with acidic phospholipids and FliH

The specialised ATPase FliI is central to export of flagellar axial protein subunits during flagellum assembly. We establish the normal cellular location of FliI and its regulatory accessory protein FliH in motile Salmonella typhimurium, and ascertain the regions involved in FliH(2)/FliI heterotrimerisation. Both FliI and FliH localised to the cytoplasmic membrane in the presence and in the absence of proteins making up the flagellar export machinery and basal body. Membrane association was tight, and FliI and FliH interacted with Escherichia coli phospholipids in vitro, both separately and as the preformed FliH(2)/FliI complex, in the presence or in the absence of ATP. Yeast two-hybrid analysis and pull-down assays revealed that the C-terminal half of FliH (H105-235) directs FliH homodimerisation, and interacts with the N-terminal region of FliI (I1-155), which in turn has an intra-molecular interaction with the remainder of the protein (I156-456) containing the ATPase domain. The FliH105-235 interaction with FliI was sufficient to exert the FliH-mediated down-regulation of ATPase activity. The basal ATPase activity of isolated FliI was stimulated tenfold by bacterial (acidic) phospholipids, such that activity was 100-fold higher than when bound by FliH in the absence of phospholipids. The results indicate similarities between FliI and the well-characterised SecA ATPase that energises general protein secretion. They suggest that FliI and FliH are intrinsically targeted to the inner membrane before contacting the flagellar secretion machinery, with both FliH105-235 and membrane phospholipids interacting with FliI to couple ATP hydrolysis to flagellum assembly.     

Genetic and biochemical analysis of Salmonella typhimurium FliI, a flagellar protein related to the catalytic subunit of the F0F1 ATPase and to virulence proteins of mammalian and plant pathogens.

FliI is a Salmonella typhimurium protein that is needed for flagellar assembly and may be involved in a specialized protein export pathway that proceeds without signal peptide cleavage. FliI shows extensive sequence similarity to the catalytic beta subunit of the F0F1 ATPase (A. P. Volger, M. Homma, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 173:3564-3572, 1991). It is even more similar to the Spa47 protein of Shigella flexneri (M. M. Venkatesan, J. M. Buysse, and E. V. Oaks, J. Bacteriol. 174:1990-2001, 1992) and the HrpB6 protein of Xanthomonas campestris (S. Fenselau, I. Balbo, and U. Bonas, Mol. Plant-Microbe Interact. 5:390-396, 1992), which are believed to play a role in the export of virulence proteins. Site-directed mutagenesis of residues in FliI that correspond to catalytically important residues in the F1 beta subunit resulted in loss of flagellation, supporting the hypothesis that FliI is an ATPase. FliI was overproduced and purified almost to homogeneity. It demonstrated ATP binding but not hydrolysis. An antibody raised against FliI permitted detection of the protein in wild-type cells and an estimate of about 1,500 subunits per cell. An antibody directed against the F1 beta subunit of Escherichia coli cross-reacted with FliI, confirming that the proteins are structurally related. The relationship between three proteins involved in flagellar assembly (FliI, FlhA, and FliP) and homologs in a variety of virulence systems is discussed.     

Selective binding of virulence type III export chaperones by FliJ escort orthologues InvI and YscO

Bacteria secrete flagella subunits and deliver virulence effectors via type III export systems. During flagellar filament assembly, a chaperone escort mechanism has been proposed to enhance the export of early, minor flagellar filament components by selectively binding and cycling their chaperones. Here we identify virulence orthologues of the flagellar chaperone escort FliJ and show that the orthologues Salmonella InvI and Yersinia YscO are, like FliJ, essential for their type III export pathway and similarly, do not bind export substrates. Like FliJ, they recognize a subset of export chaperones, in particular those of the host membrane translocon components required for subsequent effector delivery.     

Oligomerization and activation of the FliI ATPase central to bacterial flagellum assembly

FliI is the peripheral membrane ATPase pivotal to the type III protein export mechanism underlying the assembly of the bacterial flagellum. Gel filtration and multiangle light scattering showed that purified soluble native FliI protein was in a monomeric state but, in the presence of ATP, FliI showed a propensity to oligomerize. Electron microscopy revealed that FliI assembles to a ring structure, the yield of which was increased by the presence of a non-hydrolysable ATP analogue. Single particle analysis of the resulting electron micrograph images, to which no symmetry was applied, showed that the FliI ring structure has sixfold symmetry and an external diameter of approximately 10 nm. The oligomeric ring has a central cavity of 2.5-3.0 nm, which is comparable to the known diameter of the flagellar export channel into which export substrates feed. Enzymatic activity of the FliI ATPase showed positive co-operativity, establishing that oligomerization and enzyme activity are coupled. Escherichia coli phospholipids increased enzyme co-operativity, and in vitro cross-linking demonstrated that they promoted FliI multimerization. The data reveal central facets of the structure and action of the flagellar assembly ATPase and, by extension, the homologous ATPases of virulence-related type III export systems.     

Soluble components of the flagellar export apparatus,FliI, FliJ,and FliH,do not deliver flagellin,the major filament protein,from the cytosol to the export gate

Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook–filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear.     

Protein export through the bacterial flagellar type III export pathway

For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly–disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

ATP-induced FliI hexamerization facilitates bacterial flagellar protein export

FliI ATPase forms a homo-hexamer to fully exert its ATPase activity, facilitating bacterial flagellar protein export. However, it remains unknown how FliI hexamerization is linked to protein export. Here, we analyzed the capability of ring formation by FliI and its catalytic mutant variants. Compared to ATP a non-hydrolysable ATP analog increased the probability of FliI hexamerization. In contrast, FliI(E221Q), which retained the affinity for ATP but has lost ATPase activity, efficiently formed the hexamer even in the presence of ATP. The mutations, which reduced the binding affinity for ATP, significantly abolished the ring formation. These results indicate that ATP-binding induces FliI hexamerization and that the release of ADP and Pi destabilizes the ring structure. FliI(E221Q) facilitated flagellar protein export in the absence of the FliH regulator of the export apparatus although not at the wild-type FliI level while the other did not. We propose that FliI couples ATP binding and hydrolysis to its assembly-disassembly cycle to efficiently initiate the flagellar protein export cycle.     

Salmonella typhimurium mutants defective in flagellar filament regrowth and sequence similarity of FliI to F0F1, vacuolar, and archaebacterial ATPase subunits.

Many flagellar proteins are exported by a flagellum-specific export pathway. In an initial attempt to characterize the apparatus responsible for the process, we designed a simple assay to screen for mutants with export defects. Temperature-sensitive flagellar mutants of Salmonella typhimurium were grown at the permissive temperature (30 degrees C), shifted to the restrictive temperature (42 degrees C), and inspected in a light microscope. With the exception of switch mutants, they were fully motile. Next, cells grown at the permissive temperature had their flagellar filaments removed by shearing before the cells were shifted to the restrictive temperature. Most mutants were able to regrow filaments. However, flhA, fliH, fliI, and fliN mutants showed no or greatly reduced regrowth, suggesting that the corresponding gene products are involved in the process of flagellum-specific export. We describe here the sequences of fliH, fliI, and the adjacent gene, fliJ; they encode proteins with deduced molecular masses of 25,782, 49,208, and 17,302 Da, respectively. The deduced sequence of FliI shows significant similarity to the catalytic beta subunit of the bacterial F0F1 ATPase and to the catalytic subunits of vacuolar and archaebacterial ATPases; except for limited similarity in the motifs that constitute the nucleotide-binding or catalytic site, it appears unrelated to the E1E2 class of ATPases, to other proteins that mediate protein export, or to a variety of other ATP-utilizing enzymes. We hypothesize that FliI is either the catalytic subunit of a protein translocase for flagellum-specific export or a proton translocase involved in local circuits at the flagellum.     

Oligomerization of the bacterial flagellar ATPase FliI is controlled by its extreme N-terminal region

Salmonella FliI is the flagellar ATPase which converts the energy of ATP hydrolysis into the export of flagellar proteins. It forms a ring-shaped oligomer in the presence of ATP, its analogs, or phospholipids. The extreme N-terminal region of FliI has an unstable conformation and is responsible for the interaction with other components of the export apparatus and for regulation of the catalytic mechanism. To understand the role of this N-terminal region in more detail, we used multi-angle light-scattering, analytical ultracentrifugation, far-UV CD and biochemical methods to characterize a partially functional variant of FliI, missing its first seven amino acid residues (His-FliI(Delta1-7)), whose ATPase activity is about ten times lower than that of wild-type FliI. His-FliI(Delta1-7) is monomeric in solution. The deletion increased the content of alpha-helix, suggesting that the deletion stabilizes the unstable N-terminal region into an alpha-helical conformation. The deletion did not influence the K(m) value for ATP. However, unlike the wild-type, ATP and acidic phospholipids did not induce oligomerization of His-FliI(Delta1-7) or increase its ATPase activity. These results suggest that the deletion suppresses the oligomerization of FliI, and that a conformational change in the unstable N-terminal region is required for FliI oligomerization to effectively couple the energy of ATP hydrolysis to the translocation of flagellar proteins.     

Interaction of the Extreme N-Terminal Region of FliH with FlhA Is Required for Efficient Bacterial Flagellar Protein Export

The flagellar type III protein export apparatus plays an essential role in the formation of the bacterial flagellum. FliH forms a complex along with FliI ATPase and is postulated to provide a link between FliI ring formation and flagellar protein export. Two tryptophan residues of FliH, Trp7 and Trp10, are required for the effective docking of the FliH-FliI complex to the export gate made of six membrane proteins. However, it remains unknown which export gate component interacts with these two tryptophan residues. Here, we performed targeted photo-cross-linking of the extreme N-terminal region of FliH (FliH(EN)) with its binding partners. We replaced Trp7 and Trp10 of FliH with p-benzoyl-phenylalanine (pBPA), a photo-cross-linkable unnatural amino acid, to produce FliH(W7pBPA) and FliH(W10pBPA). They were both functional and were photo-cross-linked with one of the export gate proteins, FlhA, but not with the other gate proteins, indicating that these two tryptophan residues are in close proximity to FlhA. Mutant FlhA proteins that are functional in the presence of FliH and FliI but not in their absence showed a significantly reduced function also by N-terminal FliH mutations even in the presence of FliI. We suggest that the interaction of FliH(EN) with FlhA is required for anchoring the FliI hexamer ring to the export gate for efficient flagellar protein export.     

Role of the N-terminal domain of FliI ATPase in bacterial flagellar protein export

FliI, the ATPase involved in bacterial flagellar protein export, forms a complex with its regulator FliH in the cytoplasm and hexamerizes upon docking to the export gate composed of integral membrane proteins. The extreme N-terminal region of FliI is involved not only in its interaction with FliH but also in its oligomerization, but the regulatory mechanism of oligomerization remains unclear. Using in-frame 10-residue deletions within the 100 residues of the N-terminal domain, we demonstrate that the first 20 residues are required for FliH binding and that the conformation of the N-terminal domain is sensitive to the export function, even though the oligomerization and FliH-binding ability are retained and the ATPase activity is maintained in most of the deletion variants.     

Self-assembly and type III protein export of the bacterial flagellum

The bacterial flagellum is a supramolecular structure consisting of a basal body, a hook and a filament. Most of the flagellar components are translocated across the cytoplasmic membrane by the flagellar type III protein export apparatus in the vicinity of the flagellar base, diffuse down the narrow channel through the nascent structure and self-assemble at its distal end with the help of a cap structure. Flagellar proteins synthesized in the cytoplasm are targeted to the export apparatus with the help of flagellum-specific chaperones and pushed into the channel by an ATPase, whose activity is controlled by its regulator to enable the energy of ATP hydrolysis to be efficiently coupled to the translocation reaction. The export apparatus switches its substrate specificity by monitoring the state of flagellar assembly in the cell exterior, allowing this huge and complex macromolecular assembly to be built efficiently by a highly ordered and well-regulated assembly process.     

Roles of the extreme N‐terminal region of FliH for efficient localization of the FliH–FliI complex to the bacterial flagellar type III export apparatus

Most bacterial flagellar proteins are exported by the flagellar type III protein export apparatus for their self‐assembly. FliI ATPase forms a complex with its regulator FliH and facilitates initial entry of export substrates to the export gate composed of six integral membrane proteins. The FliH–FliI complex also binds to the C ring of the basal body through a FliH–FliN interaction for efficient export. However, it remains unclear how these reactions proceed within the cell. Here, we analysed subcellular localization of FliI–YFP by fluorescence microscopy. FliI–YFP was localized to the flagellar base, and its localization required both FliH and the C ring. The ATPase activity of FliI was not required for its localization. FliI–YFP formed a complex with FliHΔ1 (missing residues 2–10) but the complex did not show any localization. FliHΔ1 did not interact with FliN, and alanine‐scanning mutagenesis revealed that only Trp‐7 and Trp‐10 of FliH are essential for the interaction with FliN. Overproduction of the FliH–FliI complex improved the export activity of the fliN mutant whereas neither of the FliH(W7A)‐FliI nor FliH(W10A)‐FliI complexes did, suggesting that Trp‐7 and Trp‐10 of FliH are also required for efficient localization of the FliH–FliI complex to the export gate.     

Early steps in assembly of the yeast vacuolar H+-ATPase.

Vacuolar proton-translocating ATPases are composed of a complex of integral membrane proteins, the Vo sector, attached to a complex of peripheral membrane proteins, the V1 sector. We have examined the early steps in biosynthesis of the yeast vacuolar ATPase by biosynthetically labeling wild-type and mutant cells for varied pulse and chase times and immunoprecipitating fully and partially assembled complexes under nondenaturing conditions. In wild-type cells, several V1 subunits and the 100-kDa Vo subunit associate within 3-5 min, followed by addition of other Vo subunits with time. Deletion mutants lacking single subunits of the enzyme show a variety of partial complexes, including both complexes that resemble intermediates in the assembly pathway of wild-type cells and independent V1 and Vo sectors that form without any apparent V1Vo subunit interaction. Two yeast sec mutants that show a temperature-conditional block in export from the endoplasmic reticulum accumulate a complex containing several V1 subunits and the 100-kDa Vo subunit during incubation at elevated temperature. This complex can assemble with the 17-kDa Vo subunit when the temperature block is reversed. We propose that assembly of the yeast V-ATPase can occur by two different pathways: a concerted assembly pathway involving early interactions between V1 and Vo subunits and an independent assembly pathway requiring full assembly of V1 and Vo sectors before combination of the two sectors. The data suggest that in wild-type cells, assembly occurs predominantly by the concerted assembly pathway, and V-ATPase complexes acquire the full complement of Vo subunits during or after exit from the endoplasmic reticulum.     

Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit

In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway.     

Genetic characterization of conserved charged residues in the bacterial flagellar type III export protein FlhA

For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ΔfliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.     

Interactions between C ring proteins and export apparatus components: a possible mechanism for facilitating type III protein export

The flagellar switch proteins of Salmonella, FliG, FliM and FliN, participate in the switching of motor rotation, torque generation and flagellar assembly/export. FliN has been implicated in the flagellar export process. To address this possibility, we constructed 10-amino-acid scanning deletions and larger truncations over the C-terminal domain of FliN. Except for the last deletion variant, all other variants were unable to complement a fliN null strain or to restore the export of flagellar proteins. Most of the deletions showed strong negative dominance effects on wild-type cells. FliN was found to associate with FliH, a flagellar export component that regulates the ATPase activity of FliI. The binding of FliM to FliN does not interfere with this FliN-FliH interaction. Furthermore, a five-protein complex consisting of FliG, His-tagged FliM, FliN, FliH and FliI was purified by nickel-affinity chromatography. FliJ, a putative general chaperone, is bound to FliM even in the absence of FliH. The importance of the C ring as a possible docking site for export substrates, chaperones and FliI through FliH for their efficient delivery to membrane components of the export apparatus is discussed.     

The ATPase FliI can interact with the type III flagellar protein export apparatus in the absence of its regulator,FliH

Salmonella FliI is the ATPase that drives flagellar protein export. It normally exists as a complex together with the regulatory protein FliH. A fliH null mutant was slightly motile, with overproduction of FliI resulting in substantial improvement of its motility. Mutations in the cytoplasmic domains of FlhA and FlhB, which are integral membrane components of the type III flagellar export apparatus, also resulted in substantially improved motility, even at normal FliI levels. Thus, FliH, though undoubtedly important, is not essential.     

The FliS chaperone selectively binds the disordered flagellin C-terminal D0 domain central to polymerisation

Assembly of each Salmonella typhimurium flagellum filament requires export and polymerisation of ca. 30000 flagellin (FliC) subunits. This is facilitated by the cytosolic chaperone FliS, which binds to the 494 residue FliC and inhibits its polymerisation. Yeast two-hybrid assays, co-purification and affinity blotting showed that FliS binds specifically to the C-terminal 40 amino acid component of the disordered D0 domain central to polymerisation. Without FliS binding, the C-terminus is degraded. Our data provide further support for the view that FliS is a domain-specific bodyguard preventing premature monomer interaction.     

The FliN-FliH interaction mediates localization of flagellar export ATPase FliI to the C ring complex

FliH regulates the flagellar export ATPase FliI, preventing nonproductive ATP hydrolysis. FliH has been shown to stably associate with the C ring protein FliN. Analysis of this complex reveals that FliH is required for FliI localization to the C ring, and thus FliH not only inhibits FliI ATPase activity but also may act to target FliI to the basal body. Quantitative binding studies revealed a KD of 110 nM for FliH binding to FliN. The KD for FliH binding of a FliN variant from a temperature-sensitive nonflagellate fliN point mutant was determined to be 270 nM, suggesting a molecular explanation for its phenotype. Another variant FliN from a temperature-sensitive mutant with a different phenotype displayed binding with an intermediate affinity. Weak export activity in a fliN null mutant was greatly increased by overproduction of FliI, mimicking a previously observed FliH bypass effect and supporting the conclusion that FliN-FliH binding is important for localization of FliI to the C ring and thus the membrane-embedded export apparatus beyond. A model incorporating the present findings is presented.