FliI (STM1972) is the soluble ATPase of the flagellar type III protein export apparatus of Salmonella Typhimurium. It is a Mg2+-dependent ATPase whose activity is insensitive to F-, V- and P-type ATPase inhibitors. In the presence of ATP it assembles into a homohexameric ring. The ring is structurally related to the alpha3beta3 core of F1-ATPase, and the gamma-like coiled-coil protein FliJ binds in its centre. FliI exists as a cytoplasmic FliH2-FliI complex that binds export substrates and their chaperones (such as FliT), and as a FliI6-FliJ ring docked in the basal-body C ring through FliH contacts with FlhA and FliN. FliH inhibits FliI ATPase activity. ATP hydrolysis by FliI delivers substrates to the FlhA-FlhB docking platform and releases the FliH-FliI escort. It also drives FliJ-FlhA binding, which turns the export gate into an efficient membrane-potential-driven protein exporter and helps enforce the switch from rod/hook to filament substrates. Protein translocation itself is powered mainly by proton flux through the membrane export gate (a proton-protein antiporter). Export can proceed, inefficiently, without FliI or with a nearly inactive catalytic mutant. Catalytic-site mutations abolish flagellation. FliI is an evolutionary paralog of the F1-ATP synthase subunits but does not synthesize ATP or conduct protons. The UniProt name "Flagellum-specific ATP synthase" and EC 7.1.2.2 reflect that homology, not its function; the export ATPase EC is 7.4.2.8.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:20421493 Structural insight into the regulatory mechanisms of interac... | REMOVE | Summary: Pull-down assays showed that His-FliI binds the flagellar export chaperone FliT. Binding is strong to FliT lacking its C-terminal helix (FliT94) and very weak to full-length FliT. FliI and FliD bind different surfaces of FliT, consistent with FliI receiving the FliT-FliD chaperone-substrate complex for export. Reason: The interaction is real and biologically meaningful, but generic "protein binding" says nothing about function. No GO MF term fits well. GO:0051087 protein-folding chaperone binding refers to folding chaperones, and FliT is an export/anti-aggregation chaperone. The role this interaction serves is captured by protein-exporting ATPase activity and by type III secretion. Removing the row does not mean the interaction is false. Supporting Evidence: PMID:20421493 His-FliI bound strongly to GST-FliT94, very weakly to GST-FliT, and not at all to GST |
| GO:0005515 protein binding | IPI PMID:21278755 Common architecture of the flagellar type III protein export... | REMOVE | Summary: FliJ, a gamma-subunit-like coiled coil, binds the centre of the FliI hexamer ring and promotes ring formation. Reason: The FliI-FliJ interaction is well established and is central to how the ATPase ring is built. Generic protein binding is still uninformative. The association is better represented by the complex/location terms (GO:0030257, GO:0120102) and by the core protein-exporting ATPase activity. Removing the row does not dispute the interaction. Supporting Evidence: PMID:21278755 FliJ promotes the formation of FliI hexamer rings by binding to the center of the ring |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: FliI is a Walker-type P-loop ATPase (UniProt ATP-binding region 182-189). Purified FliI binds ATP. Mutating residues that correspond to F1-beta catalytic residues (K188, D272, Y363) abolishes flagellation. Reason: Correct and directly supported for this protein. ATP hydrolysis activity and protein-exporting ATPase activity are the more informative MF terms. Supporting Evidence: PMID:8491729 It demonstrated ATP binding but not hydrolysis. PMID:8943245 Mutations K188I and Y363S decreased the ATPase activity about 100-fold, increased the Km about 10-fold, blocked flagellar assembly, and were dominant. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: FliI is a soluble protein with no transmembrane segment. It exists as a cytoplasmic FliH2-FliI complex and as a FliI6 ring bound within the basal-body C ring at the cytoplasmic face of the export gate. It also behaves as a peripheral membrane protein. Reason: Correct localization. The apparatus-bound pool is captured more precisely by GO:0120102 (proposed below). Supporting Evidence: PMID:26916245 FliI ATPase exists not only as the FliI6 ring stably bound within the C ring to fully exert its ATPase activity but also as the FliH2FliI complex in the cytoplasm |
| GO:0015986 proton motive force-driven ATP synthesis | IEA GO_REF:0000108 | REMOVE | Summary: This row is a logical inference from the IBA GO:0046933 ATP synthase row. FliI consumes ATP to support flagellar protein export and is not part of an Fo-coupled ATP synthase. In the flagellar export system, the proton motive force drives protein translocation through the membrane export gate, not ATP synthesis. Reason: The source MF annotation (GO:0046933) is wrong, so this inferred process is also wrong. No FliI has been shown to synthesize ATP. The Salmonella enzyme is an ATP hydrolase that is insensitive to F-type ATPase inhibitors, and the PMF is used by the flagellar exporter for protein translocation. Supporting Evidence: PMID:8943245 The activity was not affected by inhibitors of the F-, V- or P-type ATPases PMID:18216859 the flagellar secretion apparatus functions as a proton-driven protein exporter file:SALTY/fliI/fliI-deep-research-falcon.md FliI hydrolyzes ATP, but it does not itself transport ATP-derived phosphate energy across the membrane or constitute the translocation pore |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000002 | ACCEPT | Summary: Purified FliI is a Mg2+-dependent ATPase. Glu211 is the catalytic base: E211Q essentially abolishes hydrolysis, and E211D reduces it about 100-fold. Activity is cooperative and coupled to hexamer formation, and FliH inhibits it. Reason: This is the correct catalytic activity, directly demonstrated on the Salmonella protein. It is kept alongside the more specific protein-exporting ATPase activity proposed below. Supporting Evidence: PMID:8943245 It had an ATPase activity of 0.16 s-1 at 25 degrees C and pH 7, and a Km for ATP of 0.3 mM; Mg2+ was required. PMID:12787361 Enzymatic activity of the FliI ATPase showed positive co-operativity, establishing that oligomerization and enzyme activity are coupled. |
| GO:0030254 protein secretion by the type III secretion system | IEA GO_REF:0000002 | ACCEPT | Summary: The flagellar export apparatus is a type III secretion system. FliI escorts substrates to the export gate and uses ATP hydrolysis for substrate entry and gate activation. fliI mutants cannot export filament subunits. Reason: Correct. FliI does part of the work of flagellar type III export: ATP-driven substrate delivery, escort release and gate activation. It is not merely required for the process. Supporting Evidence: PMID:21934659 FliI ATPase forms a complex with FliH and FliJ and escorts export substrates from the cytoplasm to the export gate complex PMID:18216858 the FliH-FliI complex facilitates only the initial entry of export substrates into the gate |
| GO:0030257 type III protein secretion system complex | IEA GO_REF:0000002 | ACCEPT | Summary: FliI is the ATPase subunit of the cytoplasmic ATPase ring complex (FliH-FliI-FliJ) of the flagellar type III export apparatus. This term is written mainly for the injectisome but covers type III secretion complexes generally. Reason: Acceptable complex-level term, consistent with the sibling reviews. The more precise flagellar location is GO:0120102 bacterial-type flagellum secretion apparatus. Supporting Evidence: PMID:33846530 The flagellar export apparatus is composed of a PMF-driven transmembrane export gate complex made of FlhA, FlhB, FliP, FliQ, and FliR and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ |
| GO:0042802 identical protein binding | IPI PMID:21278755 Common architecture of the flagellar type III protein export... | ACCEPT | Summary: FliI self-associates into a homohexameric ring. Hydrolysis occurs at the subunit interfaces, and FliJ promotes ring formation. Purified FliI forms sixfold rings in the presence of ATP, and oligomerization is coupled to activity. Reason: Homo-oligomerization is intrinsic to FliI's catalytic mechanism, since the active sites lie at subunit interfaces. The annotation is well supported for this protein. Supporting Evidence: PMID:21278755 FliJ promotes the formation of FliI hexamer rings by binding to the center of the ring PMID:12787361 the FliI ring structure has sixfold symmetry and an external diameter of approximately 10 nm |
| GO:0044780 bacterial-type flagellum assembly | IEA GO_REF:0000002 | ACCEPT | Summary: FliI exports the axial building blocks of the flagellum (rod, hook, junction, cap and filament proteins) and helps enforce the rod/hook to filament export order. Catalytic mutants block flagellar assembly. Reason: Correct core process, directly supported by mutants of the Salmonella protein. Supporting Evidence: PMID:8943245 Mutations K188I and Y363S decreased the ATPase activity about 100-fold, increased the Km about 10-fold, blocked flagellar assembly, and were dominant. PMID:38531947 we propose that FliH and FliI also support FlhA in taking appropriate conformations at different steps of flagellar protein export to bring strict order in the export substrates for efficient assembly of the flagellum |
| GO:0045259 proton-transporting ATP synthase complex | IBA GO_REF:0000033 | REMOVE | Summary: This IBD is placed at PANTHER:PTN008558586. In the PTHR15184 tree that node is a duplication, with two children: PTN000390097, the FliI/SctN type III ATPase clade (SF9, SF62, SF81), and PTN008558588, the F1-beta clade. Every seed (E. coli AtpD, human ATP5F1B, yeast ATP2, S. pombe atp2, Arabidopsis, rat, bovine and chloroplast beta subunits) lies in the F1-beta branch. P26465 (SF81) inherits the assertion only because the node sits above the duplication that created the export-ATPase paralog. FliI is part of the FliH-FliI-FliJ ring of the flagellar export apparatus, which has no Fo sector and no a/b/c subunits. Reason: The node is placed too deep. Membership of the Fo-F1 complex is a property of the F1-beta branch only. The FliI/SctN branch diverged at the duplication and forms a different complex: the cytoplasmic ATPase ring of a type III exporter. The FliJ-FliI6 ring is architecturally related to the F1 gamma-alpha3beta3 core, but it is not an ATP synthase complex. The IBD should be moved to PTN008558588, or an IRD/NOT placed at PTN000390097. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN008558586 · PTN008558586 SUPPORTS SOURCE BUT NOT TARGET Duplication node above both the F1-beta clade (PTN008558588, which holds all seeds) and the FliI/SctN clade (PTN000390097). The complex membership is valid for F1-beta descendants only; FliI is in the FliH-FliI-FliJ export ATPase ring. Supporting Evidence: PMID:33846530 The flagellar export apparatus is composed of a PMF-driven transmembrane export gate complex made of FlhA, FlhB, FliP, FliQ, and FliR and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ PMID:17202259 These results imply an evolutionary relation between the flagellum and F0F1-ATPsynthase and a similarity in the mechanism between FliI and F1-ATPase despite the apparently different functions of these proteins. |
| GO:0046933 proton-transporting ATP synthase activity, rotational mechanism | IBA GO_REF:0000033 | REMOVE | Summary: This IBD comes from the same PTN008558586 duplication node as the complex row, and its seeds are all F1-beta subunits. The row uses contributes_to, so the claim is that FliI is a subunit of a complex with rotary proton-transporting ATP synthase activity. The complex FliI actually forms (FliH12-FliI6-FliJ at the export gate) hydrolyzes ATP to drive protein export. FliJ is structurally gamma-like and a rotary mechanism for the FliI6-FliJ ring has been proposed, but no coupled proton channel or ATP synthesis exists. Reason: Contradicted by the Salmonella biochemistry. FliI's ATPase is insensitive to F-, V- and P-type inhibitors. Protons move through the export gate (FlhA has ion-channel activity and the gate alone is a proton-protein antiporter), not through FliI or its ring, and the PMF powers protein translocation, not ATP synthesis. A rotary-like architecture does not make the complex an ATP synthase, so even the weaker contributes_to claim fails. The target lies in the paralogous FliI/SctN branch below a duplication node, and there is target-specific evidence of functional divergence. This is paralog over-propagation from an IBD placed above the duplication. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN008558586 · PTN008558586 SUPPORTS SOURCE BUT NOT TARGET IBD placed at a duplication node; seeds (AtpD, ATP5F1B, ATP2, atp2) are all in the F1-beta child PTN008558588. P26465 descends via the FliI/SctN paralog branch PTN000390097, whose members hydrolyse ATP for protein export and lack an Fo partner. Supporting Evidence: PMID:8943245 The activity was not affected by inhibitors of the F-, V- or P-type ATPases PMID:21934659 the export gate complex by itself is a proton-protein antiporter PMID:29946050 FlhA has an ion channel activity, and the FlhA-FliJ interaction enables effective utilization of PMF for protein export PMID:21278755 FliJ promotes the formation of FliI hexamer rings by binding to the center of the ring |
| GO:0071973 bacterial-type flagellum-dependent cell motility | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: fliI mutants are non-flagellate and therefore non-motile. Swimming speed tracks export efficiency in catalytic mutants. FliI has no role in generating torque or in chemotaxis. Its effect on motility is entirely through building the flagellum. Reason: Not wrong, since FliI is a component of the flagellar machine and motility needs it. But the direct process is flagellum assembly through type III export, and motility is a downstream consequence. Supporting Evidence: PMID:25531309 In contrast, more than 80% of the fliI(E211D) mutant cells were motile, and their swimming speed was about 50% of the wild-type level |
| GO:0008564 protein-exporting ATPase activity | IDA PMID:29946050 In Vitro Reconstitution of Functional Type III Protein Expor... | NEW | Summary: FliI hydrolyzes ATP to move flagellar export substrates into and through the type III export gate. In inverted-membrane-vesicle reconstitution with Salmonella components, ATP hydrolysis by FliI drove protein export even without PMF. In vivo, FliI's ATPase escorts and releases substrates and activates the gate. Reason: This is the most specific and informative MF for FliI, and it is missing from GOA for P26465. The GO definition covers ATP-hydrolysing enzymes of type III secretion. It is consistent with the sibling FliI reviews and with the export-ATPase EC 7.4.2.8. Supporting Evidence: PMID:29946050 ATP hydrolysis by FliI can drive the protein export without PMF. PMID:10712687 A soluble complex of at least three proteins (FliH, FliI and FliJ) bind the protein to be exported and then interact with the complex at the motor to deliver the protein, which is then exported in an ATP-dependent process mediated by FliI. |
| GO:0120102 bacterial-type flagellum secretion apparatus | IDA PMID:26916245 FliH and FliI ensure efficient energy coupling of flagellar ... | NEW | Summary: The FliI6 ring is stably bound within the basal-body C ring at the cytoplasmic face of the export gate, anchored through FliH contacts with FlhA and FliN. The GO term definition names FliI, FliH and FliJ as the soluble components of this apparatus. Reason: GOA has only the generic T3SS complex and cytoplasm terms for this protein. GO:0120102 is the precise flagellar location, as applied in the sibling Caulobacter review. Supporting Evidence: PMID:26916245 FliI ATPase exists not only as the FliI6 ring stably bound within the C ring to fully exert its ATPase activity but also as the FliH2FliI complex in the cytoplasm PMID:33846530 The FliI ring structure is localized to the flagellar base through interactions of the extreme N-terminal region of FliH (FliHEN) with FlhA and a C ring protein, FliN |
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Download this section (compressed HTML)Q: PANTHER:PTN008558586, which carries the GO:0046933 and GO:0045259 IBDs, is a duplication node. Its children are the FliI/SctN type III ATPase clade (PTN000390097; SF9/SF62/SF81, including P26465 in SF81) and the F1-beta clade (PTN008558588), and all seeds are F1-beta subunits. Should the IBDs be moved down to PTN008558588, or should an IRD/NOT be placed at PTN000390097? The GO:0046933 IBA reaches 311 proteins through this node, 28 of them FliI/SctN (IPR005714) in reference genomes; TreeGrafter then extends the term to essentially every other FliI/SctN via graft nodes under PTN000390097.
Q: Should UniProt rename P26465 from "Flagellum-specific ATP synthase" (EC 7.1.2.2, H+-transporting) to "Flagellar export ATPase FliI" with EC 7.4.2.8? Should it also drop the 1991 "proton translocase involved in local circuits" hypothesis from the FUNCTION comment?
Q: Does the FliI6-FliJ ring actually rotate FliJ during ATP hydrolysis at the export gate, as the F1-like architecture suggests? And how does this couple to FlhA remodelling and substrate unfolding?
Experiment: Use single-molecule rotation assays (gold-bead or high-speed AFM) on FliI6-FliJ rings to test whether FliJ rotates during ATP hydrolysis, and whether the ring can be driven backwards (no evidence of ATP synthesis is expected).
Type: single-molecule biophysics
Experiment: Measure proton flux across the membrane in inverted vesicles containing the export gate, with and without FliH/FliI/FliJ and catalytic FliI mutants, to confirm that FliI itself makes no contribution to proton conduction.
Type: reconstituted membrane transport assay
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