SctN (YscN) is the ATPase of the plasmid-encoded Ysc-Yop type III secretion system (injectisome) of Yersinia enterocolitica. It is a soluble, cytoplasmic protein that assembles into a hexameric ring at the cytoplasmic face of the injectisome basal body, at the centre of the sorting platform formed with YscK/SctK, YscQ/SctQ and YscL/SctL; YscO/SctO is a central-stalk (gamma-like) analogue that stimulates its activity, while YscL/SctL binds it directly and inhibits ATP hydrolysis allosterically. SctN hydrolyses ATP to recognize secretion substrates through their N-terminal secretion signals, to strip cognate Syc chaperones and unfold effectors before they enter the export channel, and it is required for assembly of the cytosolic sorting platform (the latter role being structural, since a Walker-box mutant still supports C-ring formation but not secretion). Loss of sctN abolishes Yop secretion, host-cell injection and virulence. SctN is an evolutionary paralog of the F1-ATP synthase alpha/beta subunits and shares their fold and a rotary-like architecture, but it has no Fo-type proton channel partner, does not conduct protons and does not synthesize ATP; protein translocation by the injectisome additionally depends on the proton motive force acting at the membrane export gate rather than on SctN itself.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:17050689 Secretion signal recognition by YscN, the Yersinia type III ... | MODIFY | Summary: The interactor Q01249 is YopR/YscH, an early secretion substrate. Purified YscN binds YopR-GST, its in vivo substrate, but not GST-YopR or a hybrid lacking the N-terminal secretion signal, and impassable YopR hybrids capture YscN in the bacterial cytoplasm. This is secretion-signal recognition by the export ATPase, not a generic interaction. Reason: The experiment is sound but "protein binding" conveys nothing about the function. The informative molecular function it demonstrates is protein-exporting ATPase activity: substrate capture through the type III secretion signal is the first step of that activity. Proposed replacements: protein-exporting ATPase activity Supporting Evidence: PMID:17050689 we observed His YscN binding to YopR-GST, its in vivo substrate PMID:17050689 Eleven N-terminal residues function as the YopR secretion signal, which is required for both binding to YscN and blocking the type III pathway. |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: YscN carries the two Walker nucleotide-binding motifs, and mutation of box A or of the Walker-A lysine (K175E) abolishes Yop secretion. Purified YscN hydrolyses ATP and forms higher-order complexes on addition of the non-hydrolysable analogue AMP-PNP. Reason: Correct and directly supported for this protein by mutagenesis and enzymology. ATP hydrolysis activity and protein-exporting ATPase activity are the more informative molecular functions. Supporting Evidence: PMID:8132449 YscN contains the two consensus nucleotide-binding motifs (boxes A and B) described by Walker et al. PMID:20453832 Secretion could be complemented in trans by a wild-type yscN allele, but not by an yscN allele encoding YscN K175E altered in the Walker box |
| GO:0005737 cytoplasm | EXP PMID:16672607 Characterization of the Yersinia enterocolitica type III sec... | ACCEPT | Summary: YscN is a soluble protein that interacts with YscL in the cytoplasm of Y. enterocolitica. Fluorescent YscN fusions form injectisome-associated foci but also a substantial cytosolic pool, and YscN is entirely cytosolic when the C-ring component YscQ-C is missing. Reason: Experimentally established localization for this protein. The injectisome-associated pool is captured separately by the type III protein secretion system complex term. Supporting Evidence: PMID:16672607 YscN and YscL interact with one another in the cytoplasm PMID:25591178 EGFP-YscN remained completely cytosolic |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic duplicate of the experimental cytoplasm annotation above, derived from the FliI/YscN and SctN InterPro signatures and the UniProt subcellular location keyword. Reason: Correct, and consistent with the experimental row. It adds no information beyond the EXP annotation but is not wrong. Supporting Evidence: PMID:16672607 YscN and YscL interact with one another in the cytoplasm |
| GO:0006754 ATP biosynthetic process | IEA GO_REF:0000002 | REMOVE | Summary: This row comes from InterPro IPR013380, which is the T3SS ATPase SctN signature itself, mapped to ATP-synthase biology because of the F1 alpha/beta fold. SctN hydrolyses ATP to energize protein export; it does not make ATP. It has no Fo-type partner, and the homologous export ATPase FliI is insensitive to F-, V- and P-type ATPase inhibitors. Reason: Directly contradicted by the biology: the enzyme is an ATP consumer, and the process term asserts the opposite direction of the reaction. The InterPro2GO mapping on a T3SS-specific signature is wrong at source and affects every SctN protein. Supporting Evidence: PMID:16672607 The bacterial energy source for secretion is ATP, which is consumed by an ATPase that couples ATP hydrolysis to the unfolding of secreted proteins and the dissociation of their chaperones just prior to secretion. 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: The GO definition of this term explicitly covers the ATP-hydrolysing enzymes of the type III (virulence-related) secretion pathway. YscN is exactly that enzyme in the Yersinia Ysc system: it is a cooperative ATPase, it recognizes substrate secretion signals, and its Walker-box mutants abolish Yop secretion. UniProt assigns EC 7.4.2.8 (and not an ATP-synthase EC) to this entry, so the EC2GO mapping is correct here. Reason: This is the most informative and correct molecular function for SctN, and it is well supported by organism-specific genetics and enzymology. Supporting Evidence: PMID:16672607 A biochemical analysis of YscN reveals it to be a highly cooperative ATPase whose activity is inhibited by the addition of YscL. PMID:8132449 This mutant, impaired in Yop secretion, can be complemented in trans by a cloned yscN gene. |
| GO:0015986 proton motive force-driven ATP synthesis | IEA GO_REF:0000108 | REMOVE | Summary: A logical inference from the TreeGrafter GO:0046933 row, which is itself an F1-beta function propagated across a duplication node onto an export ATPase. Yersinia type III secretion does depend on the proton motive force, but the PMF is consumed by the machine to translocate protein, and SctN neither couples to a proton gradient nor synthesizes ATP. Reason: The source molecular-function annotation is wrong, so the inferred process is wrong. Confusing the machine's PMF requirement with ATP synthesis by SctN inverts the energetics. Supporting Evidence: PMID:15213145 Motility as well as type III-dependent secretion of Yop proteins was inhibited by CCCP. PMID:16672607 The bacterial energy source for secretion is ATP, which is consumed by an ATPase that couples ATP hydrolysis to the unfolding of secreted proteins and the dissociation of their chaperones just prior to secretion. |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000002 | ACCEPT | Summary: ATP hydrolysis is the catalytic reaction of YscN. Purified His-YscN shows cooperative ATPase activity in a malachite green assay and is inhibited non-competitively by its partner YscL. Reason: Correct catalytic activity, demonstrated for this protein. Retained alongside the more specific protein-exporting ATPase activity. Supporting Evidence: PMID:16672607 A biochemical analysis of YscN reveals it to be a highly cooperative ATPase whose activity is inhibited by the addition of YscL. PMID:28653671 An oligomerization-activated ATPase, SctN (YscN in Yersinia, see Supplementary Table 1), is thought to detach T3SS effector chaperones |
| GO:0030254 protein secretion by the type III secretion system | IEA GO_REF:0000002 | ACCEPT | Summary: YscN is essential for Yop secretion in Y. enterocolitica: a box A deletion and the Walker-A K175E substitution both abolish secretion, and a yscN deletion secretes neither effectors nor early substrates. It supplies the ATP-dependent substrate recognition, chaperone release and unfolding steps of the Ysc pathway. Reason: Correct and strongly supported by organism-specific genetics. This is the core biological process for the gene. Supporting Evidence: PMID:8132449 This mutant, impaired in Yop secretion, can be complemented in trans by a cloned yscN gene. PMID:20453832 Secretion could be complemented in trans by a wild-type yscN allele, but not by an yscN allele encoding YscN K175E altered in the Walker box |
| GO:0030257 type III protein secretion system complex | IEA GO_REF:0000002 | ACCEPT | Summary: YscN forms a hexameric ring at the cytoplasmic side of the injectisome and assembles there together with YscK, YscL and YscQ; its recruitment requires the secretin YscC, the MS-ring protein YscJ, YscK, YscL and YscQ. The GO definition explicitly includes the cytoplasmic, membrane-associated ATPase of the type III system. Reason: Correct complex assignment, supported by imaging and assembly genetics in this organism. Supporting Evidence: PMID:20453832 At the cytosolic side of the injectisome, an ATPase of the AAA + family (YscN) forms a hexameric ring that is activated by oligomerization PMID:20453832 After completion of the membrane rings, an ATPase–C ring complex formed by YscK, YscL, YscN, and YscQ joins the machinery. |
| GO:0046034 ATP metabolic process | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Mapped from IPR004100, the F1/V1/A1 alpha/beta N-terminal domain, whose GO mapping reflects rotary ATP synthase biology. SctN does hydrolyse ATP, so the term is not false, but the hydrolysis is an energy-coupling step of protein export rather than participation in cellular ATP metabolism. Reason: Energy-coupled ATP hydrolysis is captured by the molecular function (GO:0016887) and by the export process terms. As a process annotation this adds no information and, arriving from an ATP-synthase domain mapping alongside GO:0006754, it implies a role in ATP production that SctN does not have. Supporting Evidence: PMID:16672607 The bacterial energy source for secretion is ATP, which is consumed by an ATPase that couples ATP hydrolysis to the unfolding of secreted proteins and the dissociation of their chaperones just prior to secretion. |
| GO:0046933 proton-transporting ATP synthase activity, rotational mechanism | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter grafted P40290 onto PANTHER node PTN001807733. In the cached PTHR15184 PAINT table the GO:0046933 IBD sits at PTN008558586, seeded by four genuine F1-beta subunits (E. coli AtpD P0ABB4, human ATP5F1B P06576, S. cerevisiae ATP2, S. pombe atp2). PTN001807733 lies in clade PTN000390097, the eubacterial sister clade of the F1-beta clade PTN008558588 under that duplication node, and PTN000390097 contains precisely the export-ATPase subfamilies SF9, SF62 and SF81. P40290 is in SF9. So the function asserted at the IBD node arose in, and is grounded only in, the sister clade; the export-ATPase lineage has no Fo partner, no proton channel and no ATP-synthesis activity. The rotary-like architecture is real — YscO/SctO is a central-stalk analogue that stimulates the ATPase — but architecture is not synthase activity. Reason: Contradicted by the biology of this protein: YscN consumes ATP to drive Yop export and transports no protons. The graft node sits outside the clade whose descendants carry the experimental evidence for the IBD, so this is a paralog over-propagation across a duplication node rather than an inherited function. Supporting Evidence: PMID:16672607 The bacterial energy source for secretion is ATP, which is consumed by an ATPase that couples ATP hydrolysis to the unfolding of secreted proteins and the dissociation of their chaperones just prior to secretion. PMID:8943245 The activity was not affected by inhibitors of the F-, V- or P-type ATPases file:YEREN/sctN/sctN-deep-research-falcon.md It is a cytoplasmic, peripheral component of the injectisome PMID:25591178 a protein with homology to the central stalk of the FoF1-ATPase that stimulates ATPase activity (SctO; YscO) |
| GO:0046961 proton-transporting ATPase activity, rotational mechanism | IEA GO_REF:0000002 | REMOVE | Summary: IPR013380 is the SctN-specific InterPro entry, so this mapping asserts that being a T3SS ATPase implies pumping protons by a rotational mechanism. The GO definition requires transfer of protons across a membrane coupled to ATP hydrolysis. SctN is a soluble cytoplasmic protein with no transmembrane segment and no proton-conducting partner; in the homologous flagellar system it is the membrane export gate, not the ATPase, that carries protons. Reason: The proton-translocation half of the term is false for this protein. The ATP-hydrolysis half is already covered correctly by GO:0016887 and GO:0008564. The mapping is an error on a T3SS-specific signature and propagates to all SctN proteins. Supporting Evidence: PMID:21934659 the export gate complex by itself is a proton-protein antiporter PMID:8943245 The activity was not affected by inhibitors of the F-, V- or P-type ATPases |
| GO:1902600 proton transmembrane transport | IEA GO_REF:0000002 | REMOVE | Summary: Mapped from the F1/V1/A1 alpha/beta N-terminal domain. Yersinia type III secretion is inhibited by the protonophore CCCP, so the injectisome as a machine uses the proton motive force, but the protons move through the membrane export apparatus; SctN is soluble and cytoplasmic and translocates none of them. Reason: Confuses the machine's dependence on the PMF with participation of this subunit in proton transport. A domain-level mapping carrying rotary ATP synthase semantics onto an export ATPase. Supporting Evidence: PMID:15213145 Motility as well as type III-dependent secretion of Yop proteins was inhibited by CCCP. PMID:29946050 FlhA has an ion channel activity, and the FlhA-FliJ interaction enables effective utilization of PMF for protein export |
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Download this section (compressed HTML)Q: TreeGrafter grafts P40290 onto PANTHER:PTN001807733 and transfers GO:0046933. The GO:0046933 IBD is at PTN008558586, whose seeds are four F1-beta subunits, while PTN001807733 sits in the sister clade PTN000390097 that contains only the type III / flagellar export-ATPase subfamilies (SF9, SF62, SF81). Should PAINT move the IBD down to the F1-beta clade PTN008558588, or place an IRD/NOT at the root of PTN000390097, so that ATP synthase terms stop propagating to every SctN and FliI protein?
Q: InterPro IPR013380 is the SctN-specific signature yet carries InterPro2GO mappings to GO:0046961 (proton-transporting ATPase activity, rotational mechanism) and GO:0006754 (ATP biosynthetic process), and IPR004100 carries GO:1902600 and GO:0046034. Should these mappings be replaced by GO:0008564, GO:0016887, GO:0030254 and GO:0030257 for the T3SS-specific entries?
Q: SctO/YscO is a gamma-stalk analogue that stimulates the SctN ATPase, and the SctN6-SctO assembly is architecturally rotary. Is there any experimental test of rotation in an injectisome ATPase, and if rotation were shown, which GO molecular function (other than the proton-coupled GO:0046961) should describe a rotary but non-proton-translocating export ATPase?
Q: YscN K175E abolishes secretion but still supports C-ring assembly. Is the structural (scaffolding) role of SctN in sorting-platform formation separable enough from its catalytic role to deserve its own annotation, and is there a GO term for a secretion-system assembly scaffold?
Experiment: Test directly whether purified YscN or a YscN6-YscO complex reconstituted into proteoliposomes conducts protons, using an ACMA or pyranine quench assay with and without ATP. A negative result would provide the direct experimental refutation of GO:0046961 and GO:1902600 that is currently only inferred from the flagellar system.
Type: reconstitution and proton flux assay
Experiment: Measure ATP synthesis (luciferase assay) by YscN in inverted membrane vesicles energized with a pH gradient, alongside an F1Fo-containing control, to confirm that SctN cannot run the reaction in the synthetic direction.
Type: biochemistry
Experiment: Reconstitute Yop export in inverted membrane vesicles with purified YscN, YscL, YscO and chaperone-effector pairs to determine how much of the translocation step is driven by ATP hydrolysis versus by the proton motive force at the SctV export gate, in the way that has been done for the flagellar system.
Type: in vitro reconstitution
Experiment: Assay chaperone release and effector unfolding by purified Y. enterocolitica YscN with SycE-YopE, which has been shown for Salmonella InvC but not for this protein, and test the dependence on YscL and YscO.
Type: biochemistry
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