FliI (CC_3040) is the soluble ATPase of the flagellar type III protein export apparatus of Caulobacter crescentus CB15. It is encoded with fliJ in a class II flagellar operon, and fliI mutants block assembly of the polar flagellum at an early stage and are non-motile. FliI is found both in the cytoplasm and associated with the membrane, and conserved residues of its Walker-type ATP binding motif are required for flagellar assembly. By homology to the well-characterized Salmonella protein, FliI forms a hexameric ring around the gamma-like stalk FliJ and, with the regulator FliH, docks at the cytoplasmic face of the export gate (FlhA, FlhB, FliP, FliQ, FliR). It hydrolyzes ATP to deliver axial flagellar proteins (rod, hook, junction, cap and filament subunits) and their chaperones to the gate, release the FliH-FliI escort, and switch the gate into an efficient proton-motive-force-driven protein exporter. Protein translocation itself is powered mainly by proton flux through the membrane export gate. FliI is an evolutionary paralog of the F1-ATP synthase alpha/beta subunits, but it does not synthesize ATP or conduct protons; the UniProt name "Flagellum-specific ATP synthase" and EC 7.1.2.2 reflect that homology rather than its function.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: FliI is a Walker-type P-loop ATPase (ATP-binding region 164-171). In Caulobacter, conserved residues of its bipartite ATP-binding motif are required for flagellar assembly. Reason: Correct and directly supported for this protein by mutational analysis of the ATP-binding motif. ATP hydrolysis activity and protein-exporting ATPase activity are the more informative molecular-function terms. Supporting Evidence: PMID:9286988 Mutational analysis of FliI showed that two highly conserved amino acid residues in a bipartite ATP binding motif are necessary for flagellar assembly. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Subcellular fractionation of Caulobacter found FliI in the cytoplasm and in a membrane-associated pool. This matches the soluble FliH-FliI pool and the apparatus-bound ring described for Salmonella. Reason: Correct localization, supported by fractionation of the Caulobacter protein itself. Supporting Evidence: PMID:9286988 Subcellular fractionation showed that FliI is present both in the cytoplasm and in association with the membrane. |
| GO:0009288 bacterial-type flagellum | IEA GO_REF:0000002 | MODIFY | Summary: FliI is a peripheral component of the flagellar type III export apparatus at the cytoplasmic face of the basal body. It is not part of the extracellular filament or hook. Reason: The annotation is correct but general. GO:0120102 bacterial-type flagellum secretion apparatus names FliI (with FliH and FliJ) as a soluble component in its definition, and is the precise location. Proposed replacements: bacterial-type flagellum secretion apparatus 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 |
| GO:0015986 proton motive force-driven ATP synthesis | IEA GO_REF:0000108 | REMOVE | Summary: This row is a logical inference from the TreeGrafter 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 ortholog has been shown to synthesize ATP, and the Salmonella enzyme is an ATP hydrolase whose activity is insensitive to F-type ATPase inhibitors. 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:CAUVC/fliI/fliI-deep-research-falcon.md FliI does not synthesize ATP physiologically |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000002 | ACCEPT | Summary: ATP hydrolysis is FliI's catalytic reaction. Purified Salmonella FliI is a Mg2+-dependent ATPase, and in Caulobacter the ATP-binding motif residues are required for function. Reason: This is the correct catalytic activity. It is retained 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:9286988 Mutational analysis of FliI showed that two highly conserved amino acid residues in a bipartite ATP binding motif are necessary for flagellar assembly. |
| GO:0030254 protein secretion by the type III secretion system | IEA GO_REF:0000002 | ACCEPT | Summary: Caulobacter FliI was identified as a component of the flagellar type III protein secretion system. The flagellar export apparatus is a type III secretion system that exports axial flagellar subunits. Reason: Correct. FliI supplies ATP-dependent substrate delivery and gate activation for flagellar type III export. Supporting Evidence: PMID:9286988 Identification of the fliI and fliJ components of the Caulobacter flagellar type III protein secretion system. 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 flagellar type III export complex. This term is written mainly for the injectisome, but it covers type III secretion complexes generally. Reason: Acceptable complex-level term for the export ATPase. The more precise flagellar location is GO:0120102 bacterial-type flagellum secretion apparatus. Supporting Evidence: PMID:9286988 Identification of the fliI and fliJ components of the Caulobacter flagellar type III protein secretion system. |
| GO:0044781 bacterial-type flagellum organization | IEA GO_REF:0000002 | MODIFY | Summary: Caulobacter fliI mutants block flagellar assembly at an early stage. FliI's role is in building the flagellum by exporting its axial subunits. Reason: Correct but general. The child term GO:0044780 bacterial-type flagellum assembly states the specific role shown by the Caulobacter mutants. Proposed replacements: bacterial-type flagellum assembly Supporting Evidence: PMID:9286988 Three mutant strains in which flagellar assembly was blocked at an early stage were isolated. |
| GO:0046034 ATP metabolic process | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: This InterPro2GO mapping comes from the F1/V1/A1 alpha/beta N-terminal domain (IPR004100), whose mapping reflects rotary ATP synthase biology. FliI hydrolyzes ATP only as the energy source for protein export. It does not take part in cellular ATP synthesis or ATP turnover as a metabolic pathway. Reason: The term is technically true, since FliI hydrolyzes ATP. But GO captures energy-coupled ATP hydrolysis through the MF (GO:0016887), not as ATP metabolism. The domain-based mapping carries ATP synthase meaning over to an export ATPase. It adds no information and implies involvement in ATP synthesis. Supporting Evidence: PMID:18216858 the energy of ATP hydrolysis being used to disassemble and release the FliH-FliI complex from the protein about to be exported |
| GO:0046933 proton-transporting ATP synthase activity, rotational mechanism | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter transferred the F1-beta ATP synthase MF to FliI. PAINT asserts GO:0046933 at the F1-beta node PTN008558586, seeded by E. coli AtpD, human ATP5F1B, yeast ATP2 and S. pombe atp2. P0CAT8 is classified in PTHR15184:SF9 (FliI/SctN type III ATPase subfamily). In the current PANTHER tree PTN008558586 is a duplication node whose two children are the F1-beta clade (PTN008558588, holding every seed) and the FliI/SctN export ATPase clade (PTN000390097, SF9/SF62/SF81); the graft node PTN000390110 sits inside the latter. The IBD was therefore placed one node too deep, on the paralog-creating duplication, and leaks to a lineage with no Fo partner. FliJ is structurally similar to the gamma stalk, and a rotary mechanism has been proposed for the FliI6-FliJ ring. Even so, that ring hydrolyzes ATP to drive protein export and has no coupled proton channel. Reason: The annotation is contradicted by the biology. FliI does not synthesize ATP or transport protons. Its ATPase is insensitive to F-type ATPase inhibitors, and proton flux in the flagellar export system runs through the export gate (a proton-protein antiporter involving FlhA), not through FliI. The target descends from the IBD node only through the paralogous export-ATPase branch, not the F1-beta branch whose seeds carry the function, so this is a paralog over-propagation. 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:21278755 FliJ promotes the formation of FliI hexamer rings by binding to the center of the ring |
| GO:1902600 proton transmembrane transport | IEA GO_REF:0000002 | REMOVE | Summary: This row is an InterPro2GO mapping from the F1/V1/A1 alpha/beta N-terminal domain. FliI is a soluble peripheral ATPase with no transmembrane segment. The flagellar export apparatus as a whole uses the proton motive force, but proton translocation happens in the membrane export gate, where FlhA has ion-channel activity. FliI's contribution is to promote the FliJ-FlhA interaction that lets the gate use the PMF efficiently. That is not transmembrane transport of protons by FliI. The UniProt FUNCTION text also offers an old alternative: a "proton translocase involved in local circuits at the flagellum". No evidence supports it. Reason: The domain-based mapping carries a rotary ATP synthase function over to an export ATPase. The protons are carried by the export gate (FlhA-FlhB-FliPQR), not FliI. Assigning this process to FliI confuses dependence on the PMF with participation in proton transport. Supporting Evidence: PMID:29946050 FlhA has an ion channel activity, and the FlhA-FliJ interaction enables effective utilization of PMF for protein export PMID:21934659 the export gate complex by itself is a proton-protein antiporter |
| GO:0008564 protein-exporting ATPase activity | ISS PMID:8943245 Enzymatic characterization of FliI. An ATPase involved in fl... | NEW | Summary: FliI hydrolyzes ATP to support export of flagellar proteins through the type III export apparatus. The GO definition covers ATP-hydrolyzing enzymes of type III secretion pathways. Caulobacter FliI is a component of the flagellar type III secretion system, and its ATP-binding motif is required for flagellar assembly. Enzymology and the export role are established for the Salmonella ortholog. Reason: This is the most specific and informative MF for FliI, and it is missing from GOA for P0CAT8. Other FliI entries carry it, for example through the EC 7.4.2.8 mapping. It is based on the Caulobacter genetics and fractionation together with ortholog biochemistry; the ISS source is the purified Salmonella FliI (PMID:8943245). Supporting Evidence: PMID:8943245 It had an ATPase activity of 0.16 s-1 at 25 degrees C and pH 7 PMID:9286988 fliI encodes a 50-kDa polypeptide whose sequence is closely related to that of the Salmonella typhimurium FliI protein, an ATPase thought to energize the export of flagellar subunits across the cytoplasmic membrane through a type III protein secretion system. PMID:29946050 ATP hydrolysis by FliI can drive the protein export without PMF. |
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Download this section (compressed HTML)Q: The GO:0046933 and GO:0045259 IBDs sit on PTN008558586, a duplication node whose children are the F1-beta clade (PTN008558588) and the FliI/SctN export ATPase clade (PTN000390097), which contains the TreeGrafter graft node PTN000390110 used for P0CAT8. Should the IBDs move down to PTN008558588, or should an IRD/NOT be placed at PTN000390097, so that ATP synthase terms stop propagating to type III export ATPases?
Q: Should UniProt rename P0CAT8 from "Flagellum-specific ATP synthase" (EC 7.1.2.2, H+-transporting) to "Flagellum-specific ATPase / flagellar export ATPase FliI" with EC 7.4.2.8? Should it also drop the "proton translocase" clause from the FUNCTION comment, which drives the ATP-synthesis and proton-transport keywords?
Q: Does Caulobacter FliI, like the Salmonella protein, form a FliH12-FliI6-FliJ1 complex that is recruited to the new pole when the flagellum is assembled in the predivisional cell?
Experiment: Purify P0CAT8 and measure Mg2+-dependent ATPase kinetics, sensitivity to F-type ATPase inhibitors, and hexamer formation with Caulobacter FliJ. Then test whether Walker-A/catalytic-glutamate mutants complement a clean fliI deletion.
Type: biochemistry and genetic complementation
Experiment: Image a functional fluorescent FliI fusion through the cell cycle to test for cell-cycle-dependent polar recruitment to the nascent flagellar basal body, and whether that recruitment depends on FliH and the FlhA export gate.
Type: live-cell fluorescence microscopy
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