PilN (PA5043) is a bitopic inner-membrane protein of the type IVa pilus alignment subcomplex of Pseudomonas aeruginosa, encoded second in the pilMNOPQ operon. It has a short cytoplasmic N-terminal tail, a single transmembrane helix and a periplasmic domain of coiled coils and a ferredoxin-like fold, related to the GspL protein of type II secretion systems. The periplasmic domain heterodimerises with PilO, and PilN and PilO also form homodimers in vivo; the PilNO pair binds the periplasmic lipoprotein PilP, which in turn binds the secretin PilQ. The conserved N-terminal tail of PilN binds the cytoplasmic actin-like protein PilM, monomerising PilM and closing its domains around the ATP site, and so links the cytoplasmic motor side of the machine to the periplasmic and outer membrane components. Loss of PilN, or of its conserved N-terminal residues, destabilises the subcomplex, abolishes surface pili and abolishes twitching motility.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005886 plasma membrane | EXP PMID:19857645 PilM/N/O/P proteins form an inner membrane complex that affe... | ACCEPT | Summary: UniProt experimental inner-membrane location from Ayers et al. 2009 fractionation of P. aeruginosa. Reason: PilN has a single predicted transmembrane helix and co-fractionates with the inner membrane. In a Gram-negative bacterium the inner membrane is GO:0005886 plasma membrane; this is where PilN acts as the anchor of the alignment subcomplex. Supporting Evidence: PMID:19857645 all four proteins, including the nominally cytoplasmic PilM, colocalized to the inner membrane file:PSEAE/pilN/pilN-uniprot.txt Cell inner membrane {ECO:0000269|PubMed:19857645} |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (cell inner membrane) to plasma membrane. Reason: Same claim as the EXP row; the UniProt location it maps from is experimentally supported, and the TM segment agrees. Supporting Evidence: file:PSEAE/pilN/pilN-uniprot.txt Single-pass type II membrane protein PMID:7565110 PilN and PilO have long internal hydrophobic domains which may serve to localize them to the cytoplasmic membrane |
| GO:0043107 type IV pilus-dependent motility | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA from PTN002220336, whose sole seed is the PseudoCAP IMP on this protein. PilN in its own WITH/FROM is expected for a seed. Reason: The seed is sound (see the IMP rows) and the node is well scoped: its seven leaves in six taxa (Neisseria, Geobacter x2, Shewanella, Xanthomonas, Vibrio, Pseudomonas) are all PilN subfamily members (PTHR40278:SF2), so the inherited claim is reasonable for the clade. Supporting Evidence: PMID:23457250 Constructs expressing the PilN_N5A variant or deletion constructs lacking residues 2 to 9 or 4 to 18 did not restore twitching motility, phage sensitivity, or surface PilA in a pilN mutant |
| GO:0043107 type IV pilus-dependent motility | IMP PMID:19857645 PilM/N/O/P proteins form an inner membrane complex that affe... | ACCEPT | Summary: PseudoCAP IMP from Ayers et al. 2009 on pilM/N/O/P mutants and the PilMNOP complex. Reason: PilN is a structural component of the pilus machine (the inner-membrane anchor of the alignment subcomplex, binding PilM, PilO and PilP), so it does part of the work of pilus-driven twitching rather than merely being required for it. Abstract-only here; deferred to the curator for the motility assay itself. Supporting Evidence: PMID:19857645 The highly conserved pilM/N/O/P/Q gene cluster is among the core set of genes required for cell surface expression of type IV pili and associated twitching motility. |
| GO:0043107 type IV pilus-dependent motility | IMP PMID:7565110 Characterization of a five-gene cluster required for the bio... | ACCEPT | Summary: PseudoCAP IMP from Martin et al. 1995: pilM-P transposon mutants lack twitching and are complemented. Reason: Direct mutant and complementation data in a twitching assay for a core machine component. Seed of the PTN002220336 motility IBD. Supporting Evidence: PMID:7565110 PilM-P were found to be required for fimbrial biogenesis by complementation studies using twitching motility and sensitivity to fimbrial-specific phage as indicators of the presence of functional fimbriae. |
| GO:0043683 type IV pilus assembly | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA from PTN002220336, sole seed the PseudoCAP IMP on this protein. Reason: Core process for PilN; the node contains only PilN orthologues, and the PilN/GspL-like alignment role in pilus assembly is conserved in Neisseria and other members. Supporting Evidence: PMID:19857645 We provide evidence that an intact inner membrane complex is required for optimal formation of the outer membrane complex of the type IVa pilus system in P. aeruginosa |
| GO:0043683 type IV pilus assembly | IMP PMID:19857645 PilM/N/O/P proteins form an inner membrane complex that affe... | ACCEPT | Summary: PseudoCAP IMP: pilN deletion mutants lack surface pili and destabilise the PilMNOP subcomplex. Reason: Core process. PilN anchors the alignment subcomplex of the assembly machine; its loss abolishes surface pili. Supporting Evidence: file:PSEAE/pilN/pilN-uniprot.txt Deletion mutants lack surface pili. PMID:19857645 Stability studies via Western blot analyses revealed that loss of one component has a negative impact on the levels of other members of the putative complex. |
| GO:0043683 type IV pilus assembly | IMP PMID:7565110 Characterization of a five-gene cluster required for the bio... | ACCEPT | Summary: PseudoCAP IMP from Martin et al. 1995; pilM-P mutants lack fimbriae by phage sensitivity, twitching and electron microscopy. Reason: Mutant evidence for fimbrial biogenesis confirmed by EM. Correct core process. Seed of the PTN002220336 assembly IBD. Supporting Evidence: PMID:7565110 This was confirmed by electron microscopy. |
| GO:0044096 type IV pilus | IMP PMID:7565110 Characterization of a five-gene cluster required for the bio... | REMOVE | Summary: PseudoCAP located_in type IV pilus, IMP, from the Martin et al. 1995 mutant screen; the same paper and code were used for pilM, pilO and pilP. Reason: The IMP code and the abstract both point to a mutant phenotype: pilM-P mutants lack functional fimbriae; the full text (not available here) was not read. Loss of pili does not place the PilN protein in the pilus, and an IMP cannot support this located_in claim. PilN is a bitopic inner-membrane protein (PMID:19857645) of the alignment subcomplex, not part of the surface fibre that GO:0044096 defines. The phenotype is already captured by the assembly and motility rows from the same paper. This row is the sole seed of the PTN002220336 type IV pilus IBD. Supporting Evidence: PMID:7565110 PilM-P were found to be required for fimbrial biogenesis by complementation studies using twitching motility and sensitivity to fimbrial-specific phage as indicators of the presence of functional fimbriae. PMID:19857645 all four proteins, including the nominally cytoplasmic PilM, colocalized to the inner membrane |
Loading supporting content…
Download this section (compressed HTML)Q: GO has no cellular component for the type IV pilus assembly machine distinct from GO:0044096, the surface fibre. Should a "type IV pilus machine" (or alignment subcomplex) complex term be added so that PilM, PilN, PilO and PilP can be annotated to the machine rather than to the fibre?
Experiment: Use in vivo crosslinking of chromosomal Cys variants under extension- and retraction-locked conditions (pilT or pilB backgrounds with ATPase-dead alleles) to test whether PilN switches between homodimer and PilNO heterodimer states during the extension/retraction cycle.
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)