SctN2 (SsaN, STM1415) is the ATPase of the type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2 T3SS, T3SS-2), the injectisome that Salmonella uses from inside the Salmonella-containing vacuole. The purified enzyme is a Mg-ATP-dependent ATPase whose activity depends on the conserved DCCD-box arginine Arg192 and rises non-linearly with concentration, consistent with the hexameric ring that its 2.1 A crystal structure can be modelled into. SctN2 sits in the cytoplasm and in a peripheral inner-membrane pool at the cytoplasmic face of the injectisome, where it forms a sorting-platform (C ring) complex with the stator-like SsaK/SctL2 and the FliN-like SsaQ. Through a discrete C-terminal module it docks the SPI-2 chaperones SsaE, SseA, SscA, SscB and the multicargo chaperone SrcA, and it uses ATP hydrolysis to strip cargo from its chaperone, releasing the translocator SseB from SsaE. Cells lacking it fail to secrete translocators and effectors and are strongly attenuated in systemic mouse infection. SctN2 is a paralog of the F1-ATP synthase beta subunit and of the flagellar export ATPase FliI, but it neither synthesises ATP nor conducts protons: it has no Fo partner, and the proton motive force that powers type III translocation is converted to export at the membrane export gate, not by the ATPase.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:24722491 Functional characterization of the type III secretion ATPase... | REMOVE | Summary: Pull-down evidence that SsaN binds SsaK/SctL2 (P74853), the FliH/YscL-like stator component of the SPI-2 sorting platform. The interaction is real and biologically meaningful, but the term itself carries no functional content. Reason: Removal does not mean the interaction is false: the pull-down is sound and the partner is correctly identified. "Protein binding" simply carries no functional information, and everything it encodes is already stated more informatively by the complex term GO:0030257 type III protein secretion system complex and by the export ATPase molecular function. The paper supports no more specific binding term for a stator-like partner. Supporting Evidence: PMID:24722491 SsaN interacted with SsaK and SsaQ to form the C ring complex |
| GO:0005515 protein binding | IPI PMID:24722491 Functional characterization of the type III secretion ATPase... | REMOVE | Summary: The same pull-down series showed binding to SsaQ (P74860), the FliN/YscQ-like C-ring component of the SPI-2 sorting platform. Reason: As for the SsaK row, the interaction is real but the term is uninformative; the content is complex membership in the injectisome sorting platform, already recorded as GO:0030257. Removal is not a statement that the SsaN-SsaQ interaction is doubted. Supporting Evidence: PMID:24722491 SsaN interacted with SsaK and SsaQ to form the C ring complex |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: SsaN carries Walker A and Walker B motifs of a P-loop nucleoside triphosphate hydrolase and binds and hydrolyses ATP; the DCCD-box R192G substitution abolishes activity. Reason: Correct and directly demonstrated for this protein. ATP hydrolysis activity and protein-exporting ATPase activity are the more informative functions. Supporting Evidence: PMID:24722491 Purified SsaN-Myc-His6 hydrolyzed ATP in a linear, time-dependent manner with a mean ATPase activity of 0.36 |
| GO:0005737 cytoplasm | EXP PMID:24722491 Functional characterization of the type III secretion ATPase... | ACCEPT | Summary: Subcellular fractionation of Salmonella grown under SPI-2-inducing conditions detected SsaN in the soluble (cytoplasmic) fraction as well as in the membrane fraction. SsaN has no transmembrane segment. Reason: Directly demonstrated for this protein in this organism. Supporting Evidence: PMID:24722491 were detected in both the soluble and membrane fractions |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Automated counterpart of the experimental cytoplasm row, from the FliI/YscN and SctN InterPro signatures and the UniProt subcellular location. Reason: Agrees with the fractionation data for this protein. Redundant with the EXP row but not wrong. Supporting Evidence: PMID:24722491 were detected in both the soluble and membrane fractions |
| GO:0006754 ATP biosynthetic process | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO mapping of IPR013380 (Type III secretion system ATPase SctN) to ATP synthesis. SctN ATPases consume ATP to drive protein export; they do not make it. SsaN is a hydrolase with EC 7.4.2.8, and its purified form releases phosphate from ATP. Reason: Directly contradicted by the biology and by the enzymology of this protein. The mapping is an artefact of the family's paralogy with the F1-ATP synthase beta subunit and should be removed from the IPR013380 InterPro2GO record, which is specific to T3SS export ATPases. Supporting Evidence: PMID:24722491 Purified SsaN-Myc-His6 hydrolyzed ATP in a linear, time-dependent manner with a mean ATPase activity of 0.36 PMID:8943245 The activity was not affected by inhibitors of the F-, V- or P-type ATPases |
| GO:0008564 protein-exporting ATPase activity | IEA GO_REF:0000003 | ACCEPT | Summary: Mapping from EC 7.4.2.8, the protein-exporting ATPase EC class that UniProt assigns to this entry. This is the correct and most informative molecular function for a T3SS export ATPase, and it is supported for SsaN by its in vitro ATPase activity, by the loss of secretion in an ssaN deletion and by the failure of the catalytically dead R192G variant to complement. Reason: Core molecular function, correct at the right level of specificity. Supporting Evidence: PMID:24722491 SseB secretion from the ssaN mutant strain was undetectable PMID:24722491 our results indicate that SsaN releases the translocator protein SseB from the T3SS-2 specific chaperone SsaE in an ATP-dependent manner |
| GO:0008564 protein-exporting ATPase activity | ISS GO_REF:0000024 | ACCEPT | Summary: Curator transfer from the SPI-1 T3SS ATPase InvC/SctN1 (P0A1B9), the closest characterised Salmonella paralog. The transferred function is independently demonstrated for SsaN itself. Reason: Correct conclusion, and now redundant with direct evidence on this protein rather than dependent on the similarity argument. Supporting Evidence: PMID:24722491 our results indicate that SsaN releases the translocator protein SseB from the T3SS-2 specific chaperone SsaE in an ATP-dependent manner |
| GO:0015986 proton motive force-driven ATP synthesis | IEA GO_REF:0000108 | REMOVE | Summary: Logical inference (GOC inter-ontology link) from the IBA GO:0046933 ATP synthase row. The source annotation is a mis-propagated F1-beta function, so this inferred process inherits the error. SsaN has no Fo partner and no proton channel, and nothing in the T3SS synthesises ATP. Reason: The parent annotation GO:0046933 is removed, so this row has no basis. The SPI-2 apparatus consumes the proton motive force to export proteins; it does not use it to make ATP. Supporting Evidence: PMID:25701111 conversion of the pmf to protein export is the rate limiting step for protein export via the T3SS, and the pmf therefore the primary fuel for secretion via the T3SS PMID:8943245 The activity was not affected by inhibitors of the F-, V- or P-type ATPases |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the FliI/YscN T3SS ATPase signature. This is the catalytic reaction SsaN performs. Reason: Correct. Retained alongside the more specific protein-exporting ATPase activity, which states what the hydrolysis is coupled to. Supporting Evidence: PMID:24722491 Purified SsaN-Myc-His6 hydrolyzed ATP in a linear, time-dependent manner with a mean ATPase activity of 0.36 |
| GO:0016887 ATP hydrolysis activity | IMP PMID:24722491 Functional characterization of the type III secretion ATPase... | ACCEPT | Summary: Based on the R192G DCCD-box variant, which is stable and still binds the chaperone SsaE but has no detectable ATPase activity, does not release SseB from SsaE, and does not complement the secretion defect of the deletion. Reason: Mutational evidence on this protein that directly links the catalytic activity to secretion. The evidence is arguably IDA as well, since the activity was measured on purified protein. Supporting Evidence: PMID:24722491 Purified SsaN-Myc-His6 hydrolyzed ATP in a linear, time-dependent manner with a mean ATPase activity of 0.36 |
| GO:0030254 protein secretion by the type III secretion system | IEA GO_REF:0000002 | ACCEPT | Summary: SsaN is required for secretion of the SPI-2 translocators SseB, SseC and SseD and of effectors such as SseF, SseG and SseJ, and for translocation of SseJ into infected host cells. Complementation restores secretion; the catalytically dead variant does not. Reason: Correct core biological process, and independently established for this protein by deletion, complementation and point mutation. Supporting Evidence: PMID:24722491 SseB secretion from the ssaN mutant strain was undetectable PMID:25035427 As expected, SsaN was required for the secretion of needle and translocon components |
| GO:0030257 type III protein secretion system complex | IEA GO_REF:0000002 | ACCEPT | Summary: SsaN is part of the cytoplasmic sorting platform (C ring) of the SPI-2 injectisome, in a complex with SsaK/SctL2 and SsaQ, and associates with the inner membrane under secretion-inducing conditions. Reason: Correct complex assignment, supported by pull-downs and co-fractionation of this protein with its C-ring partners. Supporting Evidence: PMID:24722491 SsaN interacted with SsaK and SsaQ to form the C ring complex |
| GO:0030430 host cell cytoplasm | IMP PMID:24722491 Functional characterization of the type III secretion ATPase... | REMOVE | Summary: The cited paper localises SsaN by fractionation of the bacterium, to the soluble and membrane fractions of Salmonella. What it shows reaching the host cell is the effector SseJ-2HA, whose translocation into HeLa cells depends on SsaN. SsaN is a cytoplasmic sorting-platform ATPase, not a secreted substrate, and no experiment places it inside the host cell. Reason: The localisation of the exported cargo has been transferred to the export machine. This is a component of the bacterial injectisome; asserting host cell cytoplasm makes it look like a translocated effector. Supporting Evidence: PMID:24722491 were detected in both the soluble and membrane fractions PMID:24722491 These results indicated that SsaN could associate with the membrane regardless of the presence of the other ATPase-associated components. |
| GO:0033644 host cell membrane | IMP PMID:24722491 Functional characterization of the type III secretion ATPase... | MODIFY | Summary: Same problem as the host cell cytoplasm row: the membrane association demonstrated in this paper is association of SsaN with the *bacterial* membrane fraction under SPI-2-inducing conditions, independent of SsaK and SsaQ. The host membrane in the paper is the vacuolar membrane that the effector SseJ decorates. Reason: The underlying observation, peripheral membrane association, is sound but has been assigned to the wrong cell. GO:0009898 cytoplasmic side of plasma membrane records what the fractionation shows without implying membrane residence: SsaN has no transmembrane segment and docks at the cytoplasmic face of the bacterial inner membrane as part of the injectisome. Proposed replacements: cytoplasmic side of plasma membrane Supporting Evidence: PMID:24722491 These results indicated that SsaN could associate with the membrane regardless of the presence of the other ATPase-associated components. file:SALTY/sctN2/sctN2-deep-research-falcon.md SsaN lacks transmembrane helices and is not classified as an integral membrane protein |
| GO:0045259 proton-transporting ATP synthase complex | IBA GO_REF:0000033 | REMOVE | Summary: IBA from the PAINT IBD placed at PANTHER node PTN008558586. In the cached PTHR15184 PAINT table that node carries both this complex term and GO:0046933, seeded entirely by F1-beta subunits (E. coli AtpD P0ABB4, human ATP5F1B P06576, yeast ATP2, S. pombe atp2, plant and rat beta subunits). The cached tree topology shows PTN008558586 is a duplication node whose two children are PTN008558588, holding the ATP synthase beta subfamilies, and PTN000390097, holding the bacterial export ATPases including PTHR15184:SF62, the subfamily of this protein. The argument here is about node placement, not donor count: the IBD sits on the duplication node that separated F1-beta from the export ATPases, so every seed lies in the sister clade. SsaN belongs to the SPI-2 injectisome sorting platform, a complex with no Fo sector and no c-ring. Reason: Paralog over-propagation across a duplication node. SsaN is part of the type III protein secretion system complex (GO:0030257), not of an ATP synthase. The IBD should be moved to PTN008558588, or an IRD placed at PTN000390097. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN008558586 · IBD node for GO:0045259 and GO:0046933 SUPPORTS SOURCE BUT NOT TARGET Duplication node that is the parent of both PTN008558588 (F1-beta subfamilies) and PTN000390097 (bacterial export ATPases, incl. PTHR15184:SF62). Sound for the F1-beta child; should not reach the export-ATPase child. UniProtKB:P0ABB4 · E. coli AtpD (F1 beta) SUPPORTS SOURCE BUT NOT TARGET Seed lies in the sister clade PTN008558588. UniProtKB:P06576 · human ATP5F1B SUPPORTS SOURCE BUT NOT TARGET Mitochondrial F1 beta; no Fo-type partner exists for SctN2. Supporting Evidence: PMID:24722491 SsaN interacted with SsaK and SsaQ to form the C ring complex file:SALTY/sctN2/sctN2-notes.md the IBD sits *on* the duplication node that separated F1-beta from the export ATPases |
| GO:0046933 proton-transporting ATP synthase activity, rotational mechanism | IBA GO_REF:0000033 | REMOVE | Summary: Same PAINT node as the complex row, and the same placement problem: the four seeds are F1-beta subunits from the sister child PTN008558588, while this protein descends through PTN000390097 into PTHR15184:SF62. Structural similarity to F1 is real and explains the family name, and a gamma-like central stalk protein exists in the flagellar homolog system (FliJ), but SsaN has no Fo channel, and the flagellar family enzyme is insensitive to F-, V- and P-type ATPase inhibitors. The purified Salmonella enzyme hydrolyses ATP; no T3SS ATPase has been shown to synthesise it. Reason: Contradicted by the enzymology and the architecture of the SPI-2 apparatus. The correct molecular function is GO:0008564 protein-exporting ATPase activity, already present from the EC mapping. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN008558586 · IBD node for GO:0046933 SUPPORTS SOURCE BUT NOT TARGET IBD placed on the duplication node separating F1-beta from the bacterial export ATPases; the target descends through PTN000390097 into PTHR15184:SF62. UniProtKB:P0ABB4 · E. coli AtpD (F1 beta) SUPPORTS SOURCE BUT NOT TARGET Rotary synthase activity requires the Fo sector, which this clade lacks. SGD:S000003882 · yeast ATP2 SUPPORTS SOURCE BUT NOT TARGET Mitochondrial F1 beta seed; not informative for a T3SS export ATPase. Supporting Evidence: PMID:24722491 Purified SsaN-Myc-His6 hydrolyzed ATP in a linear, time-dependent manner with a mean ATPase activity of 0.36 PMID:8943245 The activity was not affected by inhibitors of the F-, V- or P-type ATPases PMID:25701111 conversion of the pmf to protein export is the rate limiting step for protein export via the T3SS, and the pmf therefore the primary fuel for secretion via the T3SS |
| GO:0046961 proton-transporting ATPase activity, rotational mechanism | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO mapping of the SctN signature IPR013380 to a rotary proton pump. The term's definition requires transfer of protons across a membrane coupled to ATP hydrolysis. SsaN is a soluble, peripherally membrane-associated protein with no transmembrane segment and no membrane-embedded partner that it energises as a proton channel; the proton motive force used by type III secretion is converted to protein export at the membrane export gate. Reason: Wrong function for a T3SS export ATPase, and the source signature IPR013380 is specific to that family, so the mapping should be corrected at source. It also seeds the GO:1902600 GO_REF:0000108 inference below. Supporting Evidence: PMID:25701111 Collapsing the pmf abolishes protein export via T3SS. PMID:8943245 The activity was not affected by inhibitors of the F-, V- or P-type ATPases |
| GO:0050714 positive regulation of protein secretion | IMP PMID:24722491 Functional characterization of the type III secretion ATPase... | MODIFY | Summary: Derived from the loss of SPI-2 secretion in an ssaN deletion. SsaN is not a regulator of secretion; it is a core catalytic component of the secretion machine, and its deletion abolishes secretion rather than lowering it. Reason: The essence, that SsaN is needed for protein secretion, is right, but the regulatory framing misrepresents a machine subunit as a regulator. The direct term for the same evidence is GO:0030254 protein secretion by the type III secretion system, which this entry already carries by IEA. Proposed replacements: protein secretion by the type III secretion system Supporting Evidence: PMID:24722491 SseB secretion from the ssaN mutant strain was undetectable |
| GO:0051087 protein-folding chaperone binding | IPI PMID:24722491 Functional characterization of the type III secretion ATPase... | ACCEPT | Summary: Pull-down interaction with the SPI-2 chaperone SscA (H9L426), one of the four T3SS-2 chaperones tested. Chaperone docking is the substrate-selection step of this ATPase and maps to a discrete C-terminal module of the protein. Reason: Supported by direct interaction data on this protein, and informative: it names the cargo-selection activity rather than generic protein binding. Supporting Evidence: PMID:24722491 Therefore, we next examined for interactions between SsaN and the T3SS-2 specific chaperones SsaE, SseA, SscA, and SscB. |
| GO:0051087 protein-folding chaperone binding | IPI PMID:24722491 Functional characterization of the type III secretion ATPase... | ACCEPT | Summary: Pull-down interaction with the SPI-2 chaperone SscB (H9L491). Reason: Same experiment and same conclusion as the other chaperone rows; correct and informative. Supporting Evidence: PMID:24722491 Therefore, we next examined for interactions between SsaN and the T3SS-2 specific chaperones SsaE, SseA, SscA, and SscB. |
| GO:0051087 protein-folding chaperone binding | IPI PMID:24722491 Functional characterization of the type III secretion ATPase... | ACCEPT | Summary: Interaction with SsaE (H9L4A0), the chaperone of the translocator SseB. This is the pair used in the in vitro release assay: SsaN dissociates SseB from SsaE in an ATP-dependent way, and the catalytically dead R192G variant still binds SsaE but cannot release the cargo. Reason: The best-supported chaperone interaction for this protein, and mechanistically central to its function. Supporting Evidence: PMID:24722491 our results indicate that SsaN releases the translocator protein SseB from the T3SS-2 specific chaperone SsaE in an ATP-dependent manner |
| GO:0051087 protein-folding chaperone binding | IPI PMID:24722491 Functional characterization of the type III secretion ATPase... | ACCEPT | Summary: Pull-down interaction with the SPI-2 translocator chaperone SseA (O84944). Reason: Supported by the same interaction series; consistent with the C-terminal chaperone-docking module characterised structurally for this protein. Supporting Evidence: PMID:24722491 Therefore, we next examined for interactions between SsaN and the T3SS-2 specific chaperones SsaE, SseA, SscA, and SscB. PMID:25035427 The C-terminal region of T3SS ATPases mediates binding with multiple contact points along the chaperone. |
| GO:1902600 proton transmembrane transport | IEA GO_REF:0000108 | REMOVE | Summary: Logical inference from the InterPro2GO GO:0046961 row. SsaN has no transmembrane segment and transports no protons. The SPI-2 injectisome, like the flagellar export apparatus, is powered mainly by the proton motive force, but that conversion happens at the inner-membrane export gate; dependence of the system on the pmf is not participation of the ATPase in proton transport. Reason: The source annotation GO:0046961 is removed, and the process itself is carried out by other components of the apparatus. Supporting Evidence: PMID:25701111 Collapsing the pmf abolishes protein export via T3SS. PMID:25701111 Evidence from both fT3SS and vT3SS suggests that the inner membrane export gate component FlhA (YscV and homologs in vT3SS), as well as the cytoplasmic component FliJ (YscO) are involved in controlling the conversion of the pmf to protein export |
Loading supporting content…
Download this section (compressed HTML)Q: Should the PAINT IBDs for GO:0046933 and GO:0045259 be moved from PANTHER:PTN008558586 down to PTN008558588, the child that contains the ATP synthase beta subfamilies and all of the seed proteins? As placed, the assertions cross a duplication node and reach PTN000390097, the bacterial export ATPase clade (PTHR15184:SF62 SPI-2, SF9 SPI-1, SF81 flagellar), which has no Fo sector. Failing that, should an IRD or NOT be recorded at PTN000390097?
Q: Should the InterPro2GO mappings for IPR013380 (Type III secretion system ATPase SctN) drop GO:0046961 and GO:0006754? The signature is specific to export ATPases, so the two mappings mis-assign rotary proton pumping and ATP synthesis to the whole family, and they also generate the GO_REF:0000108 rows GO:1902600 and (via GO:0046933) GO:0015986.
Q: Should the AgBase rows GO:0030430 host cell cytoplasm and GO:0033644 host cell membrane on P74857 be withdrawn or re-pointed? PMID:24722491 localises SsaN to bacterial soluble and membrane fractions, and it is the effector SseJ, not the ATPase, that is shown in the host cell.
Q: Is the hexameric ring of SsaN observed directly? The crystal structure is of a monomer and the hexamer is modelled on F1; cryo-EM or analytical ultracentrifugation of full-length SsaN, ideally with SsaK/SctL2, would settle the stoichiometry of the SPI-2 sorting platform.
Experiment: Measure the contribution of the proton motive force versus SsaN ATP hydrolysis to SPI-2 secretion directly, by uncoupling the pmf in cells expressing wild-type or R192G SsaN and quantifying translocator and effector export. This would test for the injectisome the division of labour established for the flagellar system.
Type: bacterial physiology and secretion assay
Experiment: Determine a cryo-EM structure of full-length SsaN with SsaK/SctL2 and SsaQ, in situ if possible, to establish the oligomeric state and how the ATPase is held at the cytoplasmic face of the SPI-2 injectisome.
Type: structural biology
Experiment: Test whether SsaN unfolds its substrates rather than only releasing chaperones, using hydrogen-deuterium exchange or single-molecule force measurements on SseB with wild-type and R192G SsaN.
Type: 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)