FliI (b1941) is the soluble ATPase of the flagellar type III protein export apparatus of Escherichia coli. It is a Walker-type P-loop ATPase that is structurally related to the F1-ATP synthase alpha/beta subunits. Most of what is known about its mechanism comes from the nearly identical Salmonella ortholog. In the cytoplasm FliI forms a FliH2-FliI carrier complex that binds flagellar export substrates and chaperone-substrate complexes. At the cytoplasmic face of the basal body, six FliI subunits form a ring around the gamma-like stalk protein FliJ, and FliH tethers this ring to the C-ring protein FliN and the export gate protein FlhA. ATP hydrolysis by FliI delivers the axial flagellar proteins (rod, hook, junction, cap and filament subunits) to the gate, releases the escort complex, and switches the membrane export gate (FlhA, FlhB, FliP, FliQ, FliR) into an efficient proton-motive-force-driven protein exporter. Protein translocation itself is powered mainly by proton flux through the export gate, not through FliI. FliI self-associates, and E. coli FliI-FliI interactions have been detected in yeast two-hybrid screens. FliI does not synthesize ATP or conduct protons. The UniProt name "Flagellum-specific ATP synthase" and EC 7.1.2.2 reflect its homology to F1 subunits 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. In the Salmonella ortholog, purified FliI binds ATP, and mutating residues equivalent to catalytic residues of the F1 beta subunit abolishes flagellation. Reason: Correct. The more informative MF terms are ATP hydrolysis activity and protein-exporting ATPase activity. Evidence is from the Salmonella ortholog. Supporting Evidence: PMID:8491729 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. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: FliI is a soluble protein with no transmembrane segment. In Salmonella it exists as a cytoplasmic FliH2-FliI complex and as a peripheral inner membrane pool bound to the basal body. Reason: Correct localization for the soluble pool. The apparatus-bound pool is captured by GO:0120102 bacterial-type flagellum secretion apparatus. Supporting Evidence: PMID:12054792 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. PMID:21934659 FliI ATPase forms a complex with FliH and FliJ and escorts export substrates from the cytoplasm to the export gate complex |
| GO:0006935 chemotaxis | NAS PMID:31337100 Flagella-Driven Motility of Bacteria. | MARK AS OVER ANNOTATED | Summary: This NAS row comes from a general review of flagellar motility. FliI is a flagellar export ATPase needed to build the flagellum. It has no role in chemoreception, signal transduction or motor switching. Chemotaxis fails in fliI mutants only because no flagella are made. Reason: Indirect, pleiotropic effect of loss of flagellar assembly. The cited review describes FliI only as part of the export apparatus, not as a chemotaxis component. Supporting Evidence: PMID:31337100 The FliI6 ring hydrolyzes ATP to activate the transmembrane export gate complex, thereby driving H+-coupled flagellar protein export by the export gate |
| GO:0009288 bacterial-type flagellum | NAS PMID:31337100 Flagella-Driven Motility of Bacteria. | MODIFY | Summary: FliI is part of the cytoplasmic ATPase ring complex of the flagellar type III export apparatus at the base of the flagellum. It is not part of the rod, hook or filament. Reason: Correct but general. GO:0120102 bacterial-type flagellum secretion apparatus is the precise location, and it is also annotated from the same review. Proposed replacements: bacterial-type flagellum secretion apparatus Supporting Evidence: PMID:31337100 FliH, FliI and FliJ form the cytoplasmic ATPase ring complex with a 12 FliH to 6 FliI to 1 FliJ stoichiometry |
| 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. It 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 annotation (GO:0046933) is wrong, so this inferred process is also wrong. No FliI has been shown to synthesize ATP. The Salmonella ortholog is an ATP hydrolase that 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:ECOLI/fliI/fliI-deep-research-falcon.md Its primary biological function is not to synthesize ATP and not to form the transmembrane channel. |
| 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 whose activity is cooperative and coupled to hexamer formation. Reason: Correct catalytic activity, kept alongside the more specific protein-exporting ATPase activity proposed below. Evidence is from the Salmonella ortholog, which is closely related to P52612. 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 that exports axial flagellar subunits. FliI provides ATP-dependent substrate delivery and gate activation. Reason: Correct. This is FliI's direct process. Supporting Evidence: PMID:18216858 the FliH-FliI complex facilitates only the initial entry of export substrates into the gate PMID:29946050 ATP hydrolysis by FliI can drive the protein export without bulk PMF |
| GO:0030254 protein secretion by the type III secretion system | NAS PMID:31337100 Flagella-Driven Motility of Bacteria. | ACCEPT | Summary: Same term as the InterPro-based row, here stated by the curator from a review of the flagellar export apparatus. Reason: Correct and consistent with the ortholog experimental literature. Supporting Evidence: PMID:31337100 The FliI6 ring hydrolyzes ATP to activate the transmembrane export gate complex, thereby driving H+-coupled flagellar protein export by the export 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. The term is written mainly for the injectisome but covers type III secretion complexes in general. Reason: Acceptable complex-level term. The more precise flagellar location is GO:0120102 bacterial-type flagellum secretion apparatus. Supporting Evidence: PMID:31337100 FliH, FliI and FliJ form the cytoplasmic ATPase ring complex with a 12 FliH to 6 FliI to 1 FliJ stoichiometry |
| GO:0030257 type III protein secretion system complex | NAS PMID:31337100 Flagella-Driven Motility of Bacteria. | ACCEPT | Summary: Same term as the InterPro-based row, stated from a review of the flagellar type III export apparatus. Reason: Correct. See the IEA row. Supporting Evidence: PMID:31337100 The type III protein export apparatus consists of an export gate complex made of five transmembrane proteins, 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:19834901 Benchmarking yeast two-hybrid systems using the interactions... | KEEP AS NON CORE | Summary: E. coli FliI-FliI self-interaction was detected in a yeast two-hybrid screen of E. coli motility proteins. This fits the ATP-dependent homohexameric ring formed by the ortholog. Reason: The self-association is real biology, since FliI works as a hexamer. But "identical protein binding" does not describe FliI's function. The ATPase and protein-export terms capture that. Supporting Evidence: PMID:19834901 90 motility-related proteins from Escherichia coli were tested in all pairwise combinations PMID:12787361 the FliI ring structure has sixfold symmetry and an external diameter of approximately 10 nm |
| GO:0042802 identical protein binding | IPI PMID:24561554 The binary protein-protein interaction landscape of Escheric... | KEEP AS NON CORE | Summary: FliI-FliI self-interaction from the E. coli genome-wide binary (yeast two-hybrid) interactome. It is consistent with FliI hexamer formation. Reason: Real self-association, but an uninformative MF term. It is kept as non-core. Supporting Evidence: PMID:12787361 Enzymatic activity of the FliI ATPase showed positive co-operativity, establishing that oligomerization and enzyme activity are coupled |
| GO:0042802 identical protein binding | IPI PMID:8604139 Interacting components of the flagellar motor of Escherichia... | KEEP AS NON CORE | Summary: Yeast two-hybrid study of E. coli flagellar motor proteins. The cached abstract names FliG, FliM, FliN, FliF and H-NS interactions only. The FliI self-interaction was presumably reported in the full text, which is not cached. Ortholog work independently shows FliI homo-oligomerization. Reason: The curator read the full text, and the self-interaction agrees with the known hexamer. It is not overruled, but the term is uninformative and non-core. Supporting Evidence: PMID:21278755 FliJ promotes the formation of FliI hexamer rings by binding to the center of the ring |
| GO:0044780 bacterial-type flagellum assembly | IEA GO_REF:0000002 | ACCEPT | Summary: FliI exports the axial structural subunits needed to build the flagellum. In the Salmonella ortholog, catalytic-residue mutants lose flagellation. Reason: Correct and core. FliI directly takes part in flagellar assembly by exporting its axial components. Supporting Evidence: PMID:8491729 Site-directed mutagenesis of residues in FliI that correspond to catalytically important residues in the F1 beta subunit resulted in loss of flagellation |
| GO:0045259 proton-transporting ATP synthase complex | IBA GO_REF:0000033 | REMOVE | Summary: PAINT placed the IBD for this term at PTN008558586. Every seed is an F1-ATP synthase beta subunit (E. coli AtpD, human ATP5F1B, yeast ATP2, S. pombe atp2, and plant, rat and bovine F1 subunits). PTN008558586 is a duplication node. One child (PTN008558588) is the F1-beta clade. The other child (PTN000390097) is the bacterial FliI/SctN type III ATPase clade (PTHR15184 subfamilies SF9, SF62 and SF81), which includes P52612 (SF81). The IBD therefore sits one node too deep and covers a paralog clade in which no member has shown ATP synthase activity or membership of an Fo-F1 complex. FliI forms a separate FliH12-FliI6-FliJ1 ATPase ring on the flagellar export apparatus. Reason: Paralog over-propagation from a misplaced IBD. P52612 is outside the F1-beta clade that carries the function. The Salmonella ortholog is structurally related to F1 alpha/beta but is part of the flagellar export apparatus, not an ATP synthase. The IBD should move to PTN008558588, or a NOT/IRD should be 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 spanning the F1-beta clade (PTN008558588) and the FliI/SctN type III ATPase clade (PTN000390097); all seeds are F1-beta subunits. UniProtKB:P0ABB4 · E. coli AtpD (F1 beta) SUPPORTS SOURCE BUT NOT TARGET Genuine F1-ATP synthase subunit, but a paralog of FliI. Supporting Evidence: PMID:17202259 FliI consists of three domains, and the whole structure shows extensive similarities to the alpha and beta subunits of F0F1-ATPsynthase PMID:31337100 FliH, FliI and FliJ form the cytoplasmic ATPase ring complex with a 12 FliH to 6 FliI to 1 FliJ stoichiometry |
| GO:0046933 proton-transporting ATP synthase activity, rotational mechanism | IBA GO_REF:0000033 | REMOVE | Summary: The IBD at PTN008558586 is seeded only by F1-beta subunits (E. coli AtpD, human ATP5F1B, yeast ATP2, S. pombe atp2). It is placed at a duplication node that also contains the FliI/SctN clade (PTN000390097; SF9, SF62, SF81), where P52612 sits. FliJ resembles the gamma stalk, and a rotary mechanism has been proposed for the FliI6-FliJ ring. But that ring hydrolyzes ATP to drive protein export and has no coupled proton channel. The proton flux of flagellar export runs through the export gate, a proton-protein antiporter in which FlhA has ion-channel activity. It does not run through FliI. Reason: Contradicted by the biology. FliI does not synthesize ATP or transport protons, and the ortholog ATPase is insensitive to F-type ATPase inhibitors. The target lies in a paralog clade outside the F1-beta clade whose seeds carry the function, so this is a node-placement error. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN008558586 · PTN008558586 SUPPORTS SOURCE BUT NOT TARGET Duplication node spanning the F1-beta clade (PTN008558588) and the FliI/SctN type III ATPase clade (PTN000390097); all seeds are F1-beta subunits. UniProtKB:P0ABB4 · E. coli AtpD (F1 beta) SUPPORTS SOURCE BUT NOT TARGET Genuine F1-ATP synthase subunit, but a paralog of FliI. 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 is needed to assemble the flagellum and so is needed for swimming motility. It has no role in torque generation or motor rotation. Reason: The effect on motility is downstream of FliI's direct role in flagellum assembly and protein export. Supporting Evidence: PMID:8491729 FliI is a Salmonella typhimurium protein that is needed for flagellar assembly |
| GO:0071973 bacterial-type flagellum-dependent cell motility | NAS PMID:31337100 Flagella-Driven Motility of Bacteria. | KEEP AS NON CORE | Summary: Same term as the InterPro-based row, from a review of flagellar motility. Reason: Downstream of flagellum assembly. See the IEA row. Supporting Evidence: PMID:31337100 A type III protein export apparatus transports axial component proteins from the cytoplasm to the distal end of the growing flagellar structure to construct the axial structure beyond the cellular membranes |
| GO:0120102 bacterial-type flagellum secretion apparatus | NAS PMID:31337100 Flagella-Driven Motility of Bacteria. | ACCEPT | Summary: FliI forms the cytoplasmic ATPase ring of the flagellar type III export apparatus, together with FliH and FliJ. Reason: The precise location of the apparatus-bound FliI pool. Supporting Evidence: PMID:31337100 The type III protein export apparatus consists of an export gate complex made of five transmembrane proteins, FlhA, FlhB, FliP, FliQ and FliR, and a cytoplasmic ATPase ring complex consisting of FliH, FliI and FliJ |
| GO:0008564 protein-exporting ATPase activity | ISS PMID:8943245 Enzymatic characterization of FliI. An ATPase involved in fl... | NEW | Summary: FliI hydrolyzes ATP to deliver flagellar export substrates to the type III export gate and to activate the gate. The GO definition covers ATP-hydrolyzing enzymes of type III secretion pathways. The evidence is from the Salmonella ortholog: purified-enzyme kinetics and in vitro reconstitution of ATP-driven export. Reason: This is the most specific and informative MF for FliI, and GOA lacks it for P52612. It is inferred from the closely related Salmonella ortholog and matches the sibling CAUVC fliI review. 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:29946050 ATP hydrolysis by FliI can drive the protein export without bulk PMF |
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Download this section (compressed HTML)Q: The GO:0046933 and GO:0045259 IBDs are placed at PTN008558586, a duplication node whose children are the F1-beta clade (PTN008558588) and the bacterial FliI/SctN type III ATPase clade (PTN000390097; PTHR15184:SF9, SF62, SF81). All seeds are F1-beta proteins. Should the IBDs move down to PTN008558588, or should a NOT be placed at PTN000390097, so that ATP synthase terms stop propagating to FliI and SctN?
Q: Should UniProt rename P52612 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 involved in local circuits" clause from the FUNCTION comment?
Q: Do E. coli K-12 FliI kinetics, FliH regulation and FliJ-dependent hexamerization match the Salmonella ortholog? No direct enzymology exists for P52612.
Experiment: Purify P52612 and measure Mg2+-dependent ATPase kinetics, sensitivity to F-type ATPase inhibitors (azide, DCCD, efrapeptin), stimulation by phospholipids, and hexamer formation with E. coli FliJ and FliH.
Type: biochemistry
Experiment: Complement a clean E. coli K-12 fliI deletion with Walker-A and catalytic-glutamate mutants, and measure hook and flagellin export and swimming motility.
Type: genetic complementation
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