wzx (PA3153) encodes the O-antigen flippase of the B-band (O-specific antigen) lipopolysaccharide of Pseudomonas aeruginosa PAO1, a member of the polysaccharide transporter (PST/MOP) family. Wzx is a polytopic inner-membrane protein with 12 transmembrane segments that translocates undecaprenyl pyrophosphate-linked O-antigen repeat units from the cytoplasmic to the periplasmic leaflet of the inner membrane, where Wzy polymerises them and WaaL ligates the polymer to lipid A-core. Charged and aromatic residues in transmembrane segments 2 and 8 line a cationic central lumen needed for function, and the purified protein reconstituted in proteoliposomes mediates electrogenic, pH-modulated anion flux and proton uptake, supporting a proton-coupled antiport mechanism. Chromosomal wzx mutants lack B-band LPS and show delayed A-band LPS synthesis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: Plasma membrane by IBA from PTN001250140, seeded by E. coli Wzx-family proteins (P0AAA7, P37746, P77377, Q46841) and by this gene. Reason: Wzx-family flippases are polytopic inner-membrane proteins, so the node term suits the clade and the IBD stands on its four E. coli seeds. G3XD19 in WITH/FROM reflects its own IDA rows. Supporting Evidence: PMID:20824106 requiring the integral inner membrane proteins Wzx (O-antigen [O-Ag] flippase), Wzy (O-Ag polymerase), and WaaL (O-Ag ligase) |
| GO:0005886 plasma membrane | IDA PMID:24023388 Proton-dependent gating and proton uptake by Wzx support O-a... | ACCEPT | Summary: PseudoCAP IDA, a seed of the PTN001250140 IBD. Wzx-GFP-His8 was overexpressed and recovered from the detergent-solubilised membrane fraction before reconstitution in proteoliposomes. Reason: Full text read (cached). The paper's own membrane evidence is fractionation of the overexpressed fusion; inner-membrane topology and orientation were established in P. aeruginosa-derived constructs by the same group (PMID:20824106), and the protein has 12 predicted TM segments. Seed verdict SOUND. Supporting Evidence: PMID:24023388 Samples were sedimented in an ultracentrifuge to obtain the solubilized membrane fraction. PMID:20824106 Protein orientation in the inner membrane was confirmed via C-terminal fusion to green fluorescent protein. |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location (cell membrane, multi-pass) mapping. Reason: Consistent with the topology mapping. Supporting Evidence: PMID:20824106 Protein orientation in the inner membrane was confirmed via C-terminal fusion to green fluorescent protein. |
| GO:0009243 O antigen biosynthetic process | IDA PMID:9922263 Effect of wzx (rfbX) mutations on A-band and B-band lipopoly... | ACCEPT | Summary: PseudoCAP row from the wzx insertion-mutant paper; both insertions abolished B-band O antigen. Not an IBD seed. Reason: Abstract only. O-unit translocation is a required step of Wzy-dependent O-antigen assembly, and the mutants lack B-band LPS. The evidence described is a mutant phenotype (IMP, not IDA), but the process is correct and core. Supporting Evidence: PMID:9922263 Insertion of a Gmr cassette at two separate sites within the putative wzx gene led in both cases to the loss of B-band lipopolysaccharide (LPS) O-antigen production. |
| GO:0009276 Gram-negative-bacterium-type cell wall | IDA PMID:9922263 Effect of wzx (rfbX) mutations on A-band and B-band lipopoly... | REMOVE | Summary: PseudoCAP IDA for location in the Gram-negative cell wall, from the wzx mutant paper. Not an IBD seed. Reason: Wzx is an integral inner (plasma) membrane protein, and GO:0005886 is not a part of GO:0009276 in GO, so the term places the protein in the wrong compartment. The abstract describes LPS phenotypes of mutants and a hydrophobicity prediction for Wzx, not a localisation of Wzx; the LPS product, not the flippase, ends up in the outer membrane. Supporting Evidence: PMID:9922263 is predicted to encode a highly hydrophobic protein with multiple membrane-spanning domains PMID:20824106 requiring the integral inner membrane proteins Wzx (O-antigen [O-Ag] flippase), Wzy (O-Ag polymerase), and WaaL (O-Ag ligase) |
| GO:0015297 antiporter activity | EXP PMID:24023388 Proton-dependent gating and proton uptake by Wzx support O-a... | ACCEPT | Summary: PseudoCAP EXP. Purified Wzx in proteoliposomes mediated electrogenic, pH-modulated anion flux and proton uptake, leading to a proposed H+/O-unit antiport mechanism. Reason: Full text read (cached). The coupling of anion flux to proton movement was shown with purified protein, and conserved acidic residues are needed for it. The natural substrate (Und-PP-O unit) was not itself transported in the assay, so the antiport of the lipid-linked substrate is inferred; GO has no term for a proton-coupled lipid-linked oligosaccharide flippase (the floppase terms are defined as ATP-driven), so antiporter activity is as precise as GO allows. Supporting Evidence: PMID:24023388 These results indicate H(+)-dependent gating and H(+) uptake by WzxPa and allow for the first H(+)-dependent antiport mechanism to be proposed for lipid-linked oligosaccharide translocation across the bacterial IM. |
| GO:0015774 polysaccharide transport | ISS PMID:8606190 An O-antigen processing function for Wzx (RfbX): a promising... | MODIFY | Summary: PseudoCAP ISS from S. enterica Wzx (RfbX), whose mutant accumulates undecaprenyl pyrophosphate-linked O units on the cytoplasmic side of the membrane. Reason: Abstract only. The Salmonella evidence is sound and transfers, but Wzx moves single lipid-linked repeat units (oligosaccharides) across the bilayer rather than transporting polysaccharide, which is formed afterwards by Wzy. GO:0034203 glycolipid translocation, already annotated to this gene, describes the step exactly. Proposed replacements: glycolipid translocation Supporting Evidence: PMID:8606190 We demonstrate that a wzx mutant accumulates undecaprenol pyrophosphate-linked O units which appear to be on the cytoplasmic side of the cytoplasmic membrane, suggesting that the wzx gene encodes a flippase for O-unit translocation across that membrane. |
| GO:0016020 membrane | IDA PMID:9922263 Effect of wzx (rfbX) mutations on A-band and B-band lipopoly... | MODIFY | Summary: PseudoCAP IDA for generic membrane from the wzx mutant paper. Reason: Correct but general. Wzx is an inner (plasma) membrane protein, a location already annotated to the gene. The abstract itself offers only a hydropathy prediction. Proposed replacements: plasma membrane Supporting Evidence: PMID:20824106 Protein orientation in the inner membrane was confirmed via C-terminal fusion to green fluorescent protein. |
| GO:0034203 glycolipid translocation | IDA PMID:20824106 Membrane topology mapping of the O-antigen flippase (Wzx), p... | ACCEPT | Summary: PseudoCAP IDA for glycolipid translocation (flipping of Und-PP-linked O units) from the topology mapping paper. Not an IBD seed. Reason: Full text read (cached). The topology paper does not itself assay translocation, but the claim is the core process of Wzx and is supported by the Salmonella flippase mutant (PMID:8606190), by complementation of B-band O antigen by lumen-residue mutants (PMID:22554073) and by the proteoliposome work (PMID:24023388). Supporting Evidence: PMID:22554073 This translocation is mediated by the integral IM flippase Wzx. PMID:24023388 they transport undecaprenyl pyrophosphate-linked sugar repeat units from the cytoplasmic to the periplasmic leaflets of the inner membrane (IM) for final assembly |
| GO:0043165 Gram-negative-bacterium-type cell outer membrane assembly | IDA PMID:22554073 A cationic lumen in the Wzx flippase mediates anionic O-anti... | KEEP AS NON CORE | Summary: PseudoCAP IDA for outer membrane assembly from the site-directed mutagenesis paper, which scored each Wzx variant for restoration of B-band O antigen. Not an IBD seed. Reason: Full text read (cached). The assay measures B-band LPS production, an outer-membrane component, so the term is defensible as a downstream process, but the direct role of Wzx is O-unit translocation in O-antigen synthesis, captured by the accepted GO:0034203 and GO:0009243 rows. Supporting Evidence: PMID:22554073 followed by assessment of each construct's ability to restore B-band O-Ag production, identifying eight residues important for flippase function |
| GO:0055085 transmembrane transport | IEA GO_REF:0000108 | MODIFY | Summary: Transmembrane transport inferred from the antiporter activity row. Reason: Correct but general. The transport process Wzx performs is the transbilayer flipping of lipid-linked O units, GO:0034203 glycolipid translocation, already annotated to the gene. Proposed replacements: glycolipid translocation Supporting Evidence: PMID:8606190 suggesting that the wzx gene encodes a flippase for O-unit translocation across that membrane |
| GO:0098567 periplasmic side of plasma membrane | IDA PMID:20824106 Membrane topology mapping of the O-antigen flippase (Wzx), p... | REMOVE | Summary: PseudoCAP IDA and a seed of the PTN001250140 plasma membrane IBD (via its GO:0005886 parent). The topology paper maps Wzx as a 12-TM integral inner-membrane protein with both cytoplasmic and periplasmic loops. Reason: Full text read (cached). GO:0098567 is for proteins on the periplasmic face of the membrane; Wzx spans the bilayer, and the paper's own map assigns regions to cytoplasmic, TM and periplasmic loci. Seed verdict WRONG. The plasma membrane IBD stands on the sound GO:0005886 seed and four E. coli seeds. Supporting Evidence: PMID:20824106 The results from truncation fusion expression and the corresponding differential enzyme activity ratios allowed for the assignment of specific regions of the proteins to cytoplasmic, transmembrane (TM), or periplasmic loci. |
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Download this section (compressed HTML)Q: Should GO have a proton-coupled lipid-linked oligosaccharide flippase activity term? The existing floppase terms (including GO:0015161 lipid III floppase activity, whose definition describes the enterobacterial common antigen repeat-unit flip) sit under ATP-driven floppase activity, which does not fit MOP-family Wzx proteins.
Experiment: Reconstitute purified Wzx with Und-PP-linked O units in proteoliposomes and measure their transbilayer movement as a function of a proton gradient, to show substrate translocation directly.
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