SctN1 (InvC, STM2894) is the ATPase of the Salmonella pathogenicity island 1 (SPI-1) type III secretion system, the injectisome that delivers effector proteins into intestinal epithelial cells. It is a soluble, Walker-type P-loop ATPase of the F1 alpha/beta-related export ATPase family that sits in the cytoplasmic sorting platform at the base of the injectisome: SpaO pods are linked by OrgB (SctL) spokes to a hexameric InvC ring, whose C-terminal face points at the cytoplasmic domain of the export gate protein InvA, with InvI (SctO) as the central stalk. InvC recognizes chaperone-bound secretion substrates, and uses ATP hydrolysis to release the chaperone and unfold the effector so it can enter the narrow export channel; pore-lining loops and a two-helix-finger motif of the ring, features shared with ATP-driven protein translocases and unfoldases, are essential for this. Loss of InvC, or of its ATPase activity, abolishes SPI-1 secretion and epithelial-cell invasion while leaving attachment intact. InvC is peripherally associated with the inner membrane through protein-protein contacts rather than a transmembrane segment. Despite its ancestry as a paralog of the F1-ATP synthase beta subunit, it neither synthesizes ATP nor conducts protons: the injectisome has no Fo sector, and proton motive force used by type III systems is transduced by the membrane export gate, not by the ATPase.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:20185511 Salmonella pathogenicity island 1 (SPI-1) type III secretion... | MODIFY | Summary: Records a direct interaction between InvC and the SPI-1 effector SopD, detected in protein-protein interaction assays. This reflects InvC's role as the docking site for secretion substrates at the sorting platform. Reason: The interaction is real and experimentally supported, but "protein binding" carries no functional information on its own. Effector binding by the export ATPase is substrate recognition, so the informative molecular function is GO:0008564 protein-exporting ATPase activity, which covers substrate engagement and ATP-driven handling. This matches the treatment of the parallel YscN-YopR interaction in the YEREN sctN review. Proposed replacements: protein-exporting ATPase activity Supporting Evidence: PMID:20185511 we have identified an association between SopD and the SPI-1 T3S system ATPase, InvC |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: InvC has a Walker-A/P-loop (GCGKT, residues 162-166; UniProt BINDING 164..167) and ATPgammaS- and ADP-bound crystal structures define the nucleotide site. Mutation of the P-loop lysine K165 destroys activity and function. Reason: Directly supported for this protein by mutagenesis and by ligand-bound crystal structures. Correct, though ATP hydrolysis and protein-exporting ATPase activity are the more informative molecular functions. Supporting Evidence: PMID:8045880 Site-directed mutagenesis of a residue essential for the catalytical function of this family of proteins resulted in a protein devoid of ATPase activity and unable to complement an invC mutant of S. typhimurium. PMID:31393998 we validate the InvC ATP-binding site by co-crystallization of InvCΔ79 with ATPγS (2.65 å) and ADP (2.80 å) |
| GO:0005737 cytoplasm | EXP PMID:28283062 In Situ Molecular Architecture of the Salmonella Type III Se... | ACCEPT | Summary: In situ cryo-electron tomography of Salmonella minicells localized InvC to the cytoplasmic sorting platform beneath the SPI-1 basal body, in the hexameric nave of the six-pod wheel. It is a cytoplasmic protein that is peripherally attached to the inner membrane through the injectisome. Reason: Correct and experimentally established in this organism. The more precise location is the cytoplasmic face of the type III secretion complex, which is captured by the GO:0030257 annotation below. Supporting Evidence: PMID:28283062 InvC was located within the hexameric nave of the wheel with its carboxy-terminus facing the toroidal-shape structure formed by the cytoplasmic domain of InvA PMID:15060043 characteristic of a peripherally associated membrane protein |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic duplicate of the experimental cytoplasm annotation above, derived from the FliI/YscN export ATPase domain and the UniProt subcellular location. It agrees with the experimental data. Reason: Correct, though redundant with the EXP row from PMID:28283062. InvC has no transmembrane segment and is a soluble protein recruited to the cytoplasmic face of the injectisome. Supporting Evidence: PMID:15060043 InvC was localized almost exclusively in the membrane fraction |
| GO:0008564 protein-exporting ATPase activity | IEA GO_REF:0000003 | ACCEPT | Summary: This is the correct, informative molecular function. InvC hydrolyses ATP and couples that hydrolysis to the recognition, chaperone release and unfolding of secretion substrates entering the SPI-1 export channel. The GO definition covers ATP-hydrolysing enzymes that drive protein transfer across a membrane, and EC 7.4.2.8 is the export ATPase EC that UniProt assigns to this protein. Reason: Strongly supported by direct biochemistry and genetics on this protein: purified InvC is an ATPase, catalytic-site mutants are dead and non-functional, and ATP-dependent chaperone release and substrate unfolding by InvC have been shown directly. Retained as the core molecular function. Supporting Evidence: PMID:8045880 purified preparations of InvC showed significant ATPase activity PMID:16208377 InvC induces chaperone release from and unfolding of the cognate secreted protein in an ATP-dependent manner PMID:26170413 a two-helix-finger motif and a conserved loop located at the entrance of and within the predicted pore formed by the hexameric ATPase are essential for InvC function |
| GO:0015986 proton motive force-driven ATP synthesis | IEA GO_REF:0000108 | REMOVE | Summary: A logical inference (GO_REF:0000108) from the GO:0046933 ATP synthase molecular function that the IBA row asserts. InvC consumes ATP to power protein export; it does not make ATP, and the injectisome contains no Fo-like membrane sector that could couple proton flux to phosphorylation. Reason: The source molecular function is itself wrong for this protein, so the inferred process is wrong too. No type III secretion ATPase has ever been shown to synthesize ATP; the family's ATPase activity is insensitive to F-, V- and P-type ATPase inhibitors, and where a type III system uses the proton motive force, the protons pass through the membrane export gate, not through the ATPase. Supporting Evidence: PMID:8045880 purified preparations of InvC showed significant ATPase activity 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 |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000002 | ACCEPT | Summary: ATP hydrolysis is the catalytic reaction of InvC, demonstrated with purified protein and abolished by P-loop and active-site substitutions (K165E, G164C, R189G, R191H, R223H), all of which also destroy secretion and invasion. Reason: Correct and well supported for this protein. It is the chemistry underlying the more specific protein-exporting ATPase activity and is kept alongside it. Supporting Evidence: PMID:8045880 purified preparations of InvC showed significant ATPase activity PMID:15060043 Proteins with mutations in residues predicted to be located within the predicted active site (G164C, R189G, R191H, and R223H) were devoid of any detectable ATPase activity |
| GO:0030254 protein secretion by the type III secretion system | IEA GO_REF:0000002 | ACCEPT | Summary: InvC is the energizing and substrate-processing component of the SPI-1 injectisome. Depletion of InvC decreases type III secretion, and ATPase-dead InvC alleles act as dominant negatives that block secretion of SipB, SipC, SptP and InvJ. Reason: Correct and the central biological process for this protein, supported by gene-specific genetics in Salmonella as well as by the domain-based mapping. Supporting Evidence: PMID:14762212 expression in Salmonella results in invC mRNA and InvC protein depletion, decreased type III secretion and interference with host cell invasion PMID:16208377 InvC induces chaperone release from and unfolding of the cognate secreted protein in an ATP-dependent manner |
| GO:0030257 type III protein secretion system complex | IEA GO_REF:0000002 | ACCEPT | Summary: InvC is a subunit of the SPI-1 injectisome, specifically of its cytoplasmic sorting platform, where it forms the hexameric nave connected to SpaO pods through OrgB spokes and to the InvA export gate through the InvI stalk. Deleting invC destabilizes the sorting platform. Reason: Correct complex assignment, and independently established in this organism by in situ cryo-electron tomography and by reconstitution of soluble SpaO/SpaOC/OrgB/InvC complexes. Supporting Evidence: PMID:28283062 InvC affected the overall stability of the sorting platform PMID:31288030 Here we reconstitute soluble complexes of the Salmonella Typhimurium sorting platform proteins including the ATPase InvC, the regulator OrgB, the protein SpaO and a recently identified subunit SpaOC |
| GO:0045259 proton-transporting ATP synthase complex | IBA GO_REF:0000033 | REMOVE | Summary: The IBD behind this row sits at PANTHER node PTN008558586 in PTHR15184. That node is a duplication node: one child, PTN008558588, is the genuine F1-beta clade (subfamilies SF51/74/75/76/80/82/83/85) from which every seed comes (E. coli AtpD, human ATP5F1B, yeast ATP2, S. pombe atp2, plant and rat F1 subunits); the other child, PTN000390097, is the bacterial export-ATPase clade containing SF9 (SPI-1 T3SS ATPase, this protein), SF62 (SPI-2 ATPase) and SF81 (flagellar FliI). Asserting the F1 complex at the duplication node makes it leak across the paralogy boundary into export ATPases. InvC is a member of an injectisome, which has no Fo sector, no c-ring and no proton half-channels; it is in the cytoplasmic sorting platform, not in a two-sector ATP synthase. Reason: The node placement is the problem, not the number of donors: the IBD should sit on the F1-beta child PTN008558588, whose members supply all the experimental evidence, rather than on the duplication node that also subtends the T3SS/flagellar export ATPases. The complex InvC actually belongs to is the type III secretion system complex (GO:0030257), which is separately annotated and experimentally established here. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN008558586 · PTHR15184 duplication node above the F1-beta / export-ATPase split SUPPORTS SOURCE BUT NOT TARGET IBD node for GO:0045259. Its children are PTN008558588 (F1-beta clade) and PTN000390097 (export ATPases, SF9/SF62/SF81); the term should sit on the former. UniProtKB:P0ABB4 · E. coli atpD (F1 beta subunit) SUPPORTS SOURCE BUT NOT TARGET Genuine F1-beta seed; its complex is the Fo-F1 ATP synthase, absent from the injectisome. UniProtKB:P06576 · human ATP5F1B SUPPORTS SOURCE BUT NOT TARGET Mitochondrial F1-beta seed, outside the export-ATPase clade. SGD:S000003882 · yeast ATP2 SUPPORTS SOURCE BUT NOT TARGET F1-beta seed. Supporting Evidence: PMID:28283062 InvC was located within the hexameric nave of the wheel with its carboxy-terminus facing the toroidal-shape structure formed by the cytoplasmic domain of InvA file:SALTY/sctN1/sctN1-notes.md PTN008558586 is a **DUPLICATION** node |
| GO:0046034 ATP metabolic process | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: An InterPro2GO mapping from IPR004100, the F1/V1/A1 alpha/beta N-terminal domain, whose GO mapping reflects rotary ATP synthase biology. InvC does hydrolyse ATP, but only as the energy source for substrate unfolding and export; it plays no part in cellular ATP synthesis or ATP turnover as a metabolic pathway. Reason: Technically true but uninformative and misleading in context. GO captures energy-coupled ATP hydrolysis through the molecular function (GO:0016887, GO:0008564), not as participation in ATP metabolism, and the domain-based mapping carries ATP synthase meaning over to an export ATPase. Supporting Evidence: PMID:16208377 InvC induces chaperone release from and unfolding of the cognate secreted protein in an ATP-dependent manner |
| GO:0046933 proton-transporting ATP synthase activity, rotational mechanism | IBA GO_REF:0000033 | REMOVE | Summary: Same source as the GO:0045259 row: an IBD at the PTHR15184 duplication node PTN008558586, seeded exclusively by F1-beta subunits. P0A1B9 is classified in PTHR15184:SF9, the SPI-1 T3SS ATPase subfamily, which descends from the export-ATPase child PTN000390097, not from the F1-beta child PTN008558588. The export ATPases diverged from F1 alpha/beta before the synthase function of the F1-beta clade and have no Fo partner. InvC hydrolyses ATP; ATP synthesis by it has never been observed, and the family's ATPase activity is insensitive to F-, V- and P-type ATPase inhibitors. Reason: Paralog over-propagation caused by placing the F1-beta IBD at a duplication node. The assertion is also contradicted biologically: InvC neither synthesizes ATP nor transports protons, and it has no transmembrane segment. Even under the rotary models proposed for the homologous FliI6-FliJ ring, the ring hydrolyses ATP to drive protein export, and proton flux in type III systems runs through the membrane export gate. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN008558586 · PTHR15184 duplication node above the F1-beta / export-ATPase split SUPPORTS SOURCE BUT NOT TARGET IBD node for GO:0046933, seeded only by F1-beta subunits; P0A1B9 descends from the sister export-ATPase child PTN000390097 (PTHR15184:SF9). UniProtKB:P0ABB4 · E. coli atpD (F1 beta subunit) SUPPORTS SOURCE BUT NOT TARGET Rotational ATP synthase activity is established for this seed and its clade, not for export ATPases. UniProtKB:P06576 · human ATP5F1B SUPPORTS SOURCE BUT NOT TARGET F1-beta seed. PomBase:SPAC222.12c · S. pombe atp2 SUPPORTS SOURCE BUT NOT TARGET F1-beta seed. Supporting Evidence: PMID:8045880 InvC showed significant similarity to a protein family which shares sequence homology with the catalytic beta subunit of the F0F1 ATPase from a number of microorganisms 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 |
| GO:1902600 proton transmembrane transport | IEA GO_REF:0000002 | REMOVE | Summary: An InterPro2GO mapping from the F1/V1/A1 alpha/beta N-terminal domain. InvC is a soluble protein with no transmembrane segment; it is only peripherally attached to the inner membrane through protein-protein contacts with the injectisome. Type III export can use the proton motive force, but proton movement happens in the membrane export gate (InvA/FlhA-type components), not in the ATPase. Reason: The domain-based mapping transfers rotary ATP synthase biology to an export ATPase. Assigning proton transport to InvC confuses a system-level dependence on the proton motive force with participation of this subunit in moving protons. There is no evidence for any proton-conducting activity of InvC. Supporting Evidence: PMID:15060043 characteristic of a peripherally associated membrane protein PMID:21934659 the export gate complex by itself is a proton-protein antiporter file:SALTY/sctN1/sctN1-deep-research-falcon.md InvC is not a membrane transporter subunit and is not itself secreted. PMID:18216859 the flagellar secretion apparatus functions as a proton-driven protein exporter |
Loading supporting content…
Download this section (compressed HTML)Q: Should the PTHR15184 IBDs for GO:0046933 and GO:0045259 be moved from the duplication node PTN008558586 to its F1-beta child PTN008558588, so that ATP synthase terms stop propagating into the export-ATPase clade PTN000390097 (PTHR15184:SF9 SPI-1 T3SS ATPase, SF62 SPI-2 ATPase, SF81 flagellar FliI)? Alternatively, should an IRD or NOT annotation be placed at PTN000390097? The same question applies to the TreeGrafter graft nodes PTN000390110 and PTN001807733, which deliver the same terms as IEA (GO_REF:0000118) to other members of this clade.
Q: Should InterPro2GO mappings from IPR004100 (F1/V1/A1 alpha/beta N-terminal domain) to GO:0046034 and GO:1902600 be restricted so that they do not fire on entries that also match IPR005714 (T3SS/flagellar export ATPase FliI/YscN)? That single rule would remove the ATP-metabolism and proton-transport rows from every SctN and FliI protein at once.
Q: Is there a GO term that properly captures the ATP-dependent chaperone-release and substrate-unfolding activity of type III export ATPases? GO:0043335 protein unfolding exists but is not used for any T3SS or flagellar export ATPase in GOA, so either the convention is that GO:0008564 subsumes it, or the unfoldase step of type III secretion is currently unrepresentable.
Q: How is InvC ATPase activity regulated in vivo, given that OrgB (SctL) is the FliH-like partner and that the free protein is largely monomeric until it is assembled on the platform? Does OrgB inhibit or activate InvC, as FliH does for FliI?
Experiment: Measure the contribution of ATP hydrolysis versus proton motive force to SPI-1 secretion directly in Salmonella, by comparing secretion of SipB/SipC/ SptP in wild type, ATPase-dead invC point mutants, and cells treated with protonophores, including a test for suppressor mutations in invA/spaS that bypass the ATPase (the flagellar system has such bypass suppressors).
Type: bacterial genetics and secretion assay
Experiment: Reconstitute purified hexameric InvC (full length, on a SpaO/OrgB scaffold) with a chaperone-effector pair and measure single-turnover chaperone release and unfolding kinetics, plus the ATP dependence of the pore-loop and two-helix-finger mutants, to quantify the unfoldase step attributed to InvC.
Type: in vitro reconstitution and enzymology
Experiment: Test directly whether InvC can conduct protons or synthesize ATP: assay purified InvC (and the InvC-InvI stalk complex) reconstituted into liposomes for proton translocation and for ATP synthesis under an imposed proton gradient, as a formal falsification of the propagated ATP synthase annotations.
Type: proteoliposome biophysics
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)