LPXB (At2g04560) is a nuclear-encoded, mitochondrially targeted lipid-A-disaccharide synthase of the LpxB (glycosyltransferase family 19) family, the single Arabidopsis ortholog of E. coli LpxB. By orthology it condenses UDP-2,3-diacylglucosamine with 2,3-diacylglucosamine 1-phosphate (lipid X) to form the beta-1',6-linked lipid A disaccharide 1-phosphate, the fifth step of the plant lipid A precursor pathway that mirrors the E. coli Raetz pathway (LpxA, LpxC, LpxD, LpxB, LpxK, KdtA). Null lpxb mutants accumulate both LpxB substrates, lipid X (about 40-fold) and UDP-2,3-diacylglucosamine, and lack downstream intermediates, but grow normally. An N-terminal GFP fusion is targeted to mitochondria, where the lipid X pool of lpxb mutants is concentrated. The final plant lipid A-like product and its physiological role, possibly a component of mitochondrial membranes or a signalling lipid, are unknown.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: Mitochondrion, propagated by PAINT from a plant LpxB node (PTN008624792) whose experimental grounding is AtLpxB itself (AT2G04560 in WITH/FROM). Reason: The plant LpxB clade is mitochondrially targeted, and AtLpxB carries its own experimental localisation (5'-terminal GFP fusion co-localising with MitoTracker; lipid X pool of the lpxb-1 mutant enriched in mitochondria). The target appearing in its own WITH/FROM reflects that experimental grounding. Sub-mitochondrial location is unknown, so mitochondrion is the right precision. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN008624792 SUPPORTS TRANSFER Plant-specific LpxB node seeded by the experimentally localised AtLpxB; mitochondrial targeting is consistent with the predicted 58-residue transit peptide. Supporting Evidence: PMID:21709257 However, 5β²-terminal fusions of AtLpxD1, AtLpxD2, AtLpxB, and AtLpxK to GFP showed the same mitochondrial pattern as full-length AtLpxA and AtLpxC |
| GO:0005739 mitochondrion | IDA PMID:21709257 Pathway for lipid A biosynthesis in Arabidopsis thaliana res... | ACCEPT | Summary: Mitochondrion by GFP fusion and MitoTracker co-localisation in transgenic Arabidopsis roots (Li et al. 2011). Reason: The full-length AtLpxB-GFP fusion was too dim to localise, but the 5'-terminal fusion showed the same mitochondrial pattern as AtLpxA and AtLpxC, and in mesophyll the fusion dots did not co-localise with chloroplasts. This shows that the N-terminal targeting signal is mitochondrial, which agrees with the predicted transit peptide. It is the best available evidence for the location of the enzyme. Supporting Evidence: PMID:21709257 However, 5β²-terminal fusions of AtLpxD1, AtLpxD2, AtLpxB, and AtLpxK to GFP showed the same mitochondrial pattern as full-length AtLpxA and AtLpxC PMID:21709257 In mesophyll cells, GFP signals were shown as 1 ΞΌm dots, but were not colocalized with chloroplasts (data not shown). |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: Mitochondrion from the UniProt subcellular-location mapping, which rests on Li et al. 2011 (ECO:0000269). Reason: Same claim as the IDA row; the UniProt location line is experimentally backed by the GFP fusion data. Supporting Evidence: file:ARATH/LPXB/LPXB-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:21709257}. PMID:21709257 However, 5β²-terminal fusions of AtLpxD1, AtLpxD2, AtLpxB, and AtLpxK to GFP showed the same mitochondrial pattern as full-length AtLpxA and AtLpxC |
| GO:0005739 mitochondrion | IMP PMID:21709257 Pathway for lipid A biosynthesis in Arabidopsis thaliana res... | ACCEPT | Summary: Mitochondrion by IMP, based on subcellular fractionation of atlpxb-1 plants in which the accumulated LpxB substrate lipid X was mainly in mitochondria. Reason: The location claim is correct and agrees with the GFP data. The evidence is indirect, since it locates the substrate pool that builds up in the mutant rather than the enzyme. It still means that lipid X is made and normally consumed in mitochondria, and the claim is accepted together with the IDA row. Supporting Evidence: PMID:21709257 Lipid X levels in mitochondria were 3- and 48-fold higher than in chloroplasts or whole cell homogenates, respectively. PMID:21709257 However, 5β²-terminal fusions of AtLpxD1, AtLpxD2, AtLpxB, and AtLpxK to GFP showed the same mitochondrial pattern as full-length AtLpxA and AtLpxC |
| GO:0008915 lipid-A-disaccharide synthase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Lipid-A-disaccharide synthase activity, propagated from PAINT node PTN000769503 seeded by E. coli LpxB (P10441) and AtLpxB itself. Reason: AtLpxB is a full-length single-copy LpxB family member (31% identity to E. coli LpxB, Pfam LpxB) inside the clade that inherited the activity. Mutant chemistry supports it in planta, because both LpxB substrates accumulate only in lpxb nulls. Core function. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000769503 SUPPORTS TRANSFER LpxB ancestral node; AtLpxB is a single-copy ortholog with no evidence of loss of function. UniProtKB:P10441 SUPPORTS TRANSFER Biochemically characterised E. coli lipid-A-disaccharide synthase. Supporting Evidence: PMID:21709257 Single copy genes encoding AtLpxB (AT2G04560), AtLpxK (AT3G20480) and AtKdtA (AT5G03770) ( Table 1 ) were also found, with 31, 26, and 34% sequence identity to the corresponding E. coli proteins, respectively PMID:21709257 The accumulation of lipid X together with UDP-2,3-diacyl-GlcN in three independently grown atlpxb-1 null mutant plants ( Figs. 3 and 4 ) demonstrates that both compounds are likely substrates for AtLpxB in vivo |
| GO:0008915 lipid-A-disaccharide synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Lipid-A-disaccharide synthase activity from combined InterPro (IPR003835), Rhea and EC 2.4.1.182 mappings. Reason: Same core claim as the IBA and IMP rows; LpxB family membership is consistent with the in planta substrate accumulation. Supporting Evidence: file:ARATH/LPXB/LPXB-uniprot.txt Belongs to the LpxB family. PMID:21709257 The accumulation of lipid X together with UDP-2,3-diacyl-GlcN in three independently grown atlpxb-1 null mutant plants ( Figs. 3 and 4 ) demonstrates that both compounds are likely substrates for AtLpxB in vivo |
| GO:0008915 lipid-A-disaccharide synthase activity | IMP PMID:21709257 Pathway for lipid A biosynthesis in Arabidopsis thaliana res... | ACCEPT | Summary: Lipid-A-disaccharide synthase activity by IMP. In two independent null alleles (atlpxb-1, atlpxb-2), both LpxB substrates accumulate (lipid X 42-fold; UDP-2,3-diacyl-GlcN detectable only in lpxb). Reason: Loss of the gene causes accumulation of both the donor and acceptor substrates of the LpxB reaction, while the LpxB product (disaccharide 1-phosphate) is found only in mutants of the next steps (lpxk, kdta). This is strong genetic evidence for the activity. No in vitro assay has been reported, but together with orthology the IMP is sound. Core function. Supporting Evidence: PMID:21709257 Lipid X accumulates 42-fold in the atlpxb-1 mutant compared to the Col-0 wild type by this criterion PMID:21709257 The accumulation of lipid X together with UDP-2,3-diacyl-GlcN in three independently grown atlpxb-1 null mutant plants ( Figs. 3 and 4 ) demonstrates that both compounds are likely substrates for AtLpxB in vivo PMID:21709257 The independently isolated atlpxb-2 , atlpxk-2 , and atlpxk-3 mutants showed similar lipid accumulation patterns as the atlpxb-1 and atlpxk-1 mutants. |
| GO:0009245 lipid A biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: Lipid A biosynthetic process from PAINT node PTN000769503 (E. coli LpxB). Wei et al. 2020 (RAR) flagged this as a probable plant misannotation, but for LPXB the flag is refuted. Reason: The node placement is sound, and gene-specific experiments support the inherited process. AtLpxB catalyses a step of the lipid A precursor pathway, so it does the work of the process. lpxb nulls accumulate the two LpxB substrates, lipid X and UDP-2,3-diacyl-GlcN. Epistasis with LpxC-RNAi and lpxd2 orders LpxB after LpxC and LpxD, as in E. coli. The LpxB product is found downstream in lpxk and kdta mutants. The plant end product is unknown, but this does not weaken involvement in the lipid A precursor pathway. The low taxon rate-ratio reflected sparse plant curation, not absence of the pathway. This row also carries the experimental (IMP) evidence, which in GOA is attached only to the obsolete lipid X term. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000769503 SUPPORTS TRANSFER LpxB ancestral node; Arabidopsis mutant lipid chemistry matches the inherited role. UniProtKB:P10441 SUPPORTS TRANSFER E. coli LpxB, an enzyme of the Raetz lipid A pathway. Supporting Evidence: PMID:21709257 our work demonstrates that plants synthesize lipid A precursors using the same enzymatic pathway present in E. coli PMID:21709257 The accumulation of lipid X together with UDP-2,3-diacyl-GlcN in three independently grown atlpxb-1 null mutant plants ( Figs. 3 and 4 ) demonstrates that both compounds are likely substrates for AtLpxB in vivo PMID:21709257 The disaccharide-1-phosphate intermediate generated by LpxB ( Fig. 1 ) accumulated in the atlpxk-1 and atkdta-1 mutants file:ARATH/LPXB/LPXB-uniprot.txt acetyl-alpha-D-glucosamine: step 5/6. {ECO:0000269|PubMed:21709257}. |
| GO:0009245 lipid A biosynthetic process | IEA GO_REF:0000002 | ACCEPT | Summary: Lipid A biosynthetic process from the InterPro2GO mapping of IPR003835 (glycosyl transferase family 19, LpxB). Reason: Same claim as the IBA row; it is supported in Arabidopsis by the lpxb mutant lipid chemistry. Supporting Evidence: PMID:21709257 our work demonstrates that plants synthesize lipid A precursors using the same enzymatic pathway present in E. coli PMID:21709257 The accumulation of lipid X together with UDP-2,3-diacyl-GlcN in three independently grown atlpxb-1 null mutant plants ( Figs. 3 and 4 ) demonstrates that both compounds are likely substrates for AtLpxB in vivo |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | REMOVE | Summary: Chloroplast, predicted by AtSubP sequence analysis. Reason: The prediction conflicts with the experimental data. UniProt annotates a mitochondrial transit peptide, the AtLpxB 5'-GFP fusion co-localised with MitoTracker, and in mesophyll cells the Lpx fusion signals did not co-localise with chloroplasts. Some lipid X was found in chloroplast fractions of lpxb-1 plants, but this is about 3-fold less than in mitochondria. It locates a lipid that may be exported, not the enzyme. No experiment places LpxB in plastids. Supporting Evidence: PMID:21709257 In mesophyll cells, GFP signals were shown as 1 ΞΌm dots, but were not colocalized with chloroplasts (data not shown). PMID:21709257 However, 5β²-terminal fusions of AtLpxD1, AtLpxD2, AtLpxB, and AtLpxK to GFP showed the same mitochondrial pattern as full-length AtLpxA and AtLpxC PMID:21709257 Lipid X levels in mitochondria were 3- and 48-fold higher than in chloroplasts or whole cell homogenates, respectively. |
| GO:2001289 obsolete lipid X metabolic process | IMP PMID:21709257 Pathway for lipid A biosynthesis in Arabidopsis thaliana res... | MODIFY | Summary: Obsolete lipid X metabolic process, based on lipid X accumulation in atlpxb-1. Reason: GO:2001289 is obsolete (QuickGO lists it as made in error and an unnecessary grouping term, with no replacement). The experimental finding, accumulation of the LpxB substrate lipid X in lpxb nulls, is direct genetic evidence that AtLpxB acts in lipid A precursor biosynthesis. It is best captured as lipid A biosynthetic process, which the gene already carries (IBA/IEA). Proposed replacements: lipid A biosynthetic process Supporting Evidence: PMID:21709257 Lipid X accumulates 42-fold in the atlpxb-1 mutant compared to the Col-0 wild type by this criterion PMID:21709257 The accumulation of lipid X together with UDP-2,3-diacyl-GlcN in three independently grown atlpxb-1 null mutant plants ( Figs. 3 and 4 ) demonstrates that both compounds are likely substrates for AtLpxB in vivo |
| GO:2001289 obsolete lipid X metabolic process | IMP PMID:21709257 Pathway for lipid A biosynthesis in Arabidopsis thaliana res... | MODIFY | Summary: Obsolete lipid X metabolic process (involved_in), from the same lpxb mutant lipid X data. Reason: Same claim and the same obsolete term as the previous row. LpxB consumes lipid X as its acceptor substrate, so the supported process is lipid A biosynthetic process (GO:0009245). Proposed replacements: lipid A biosynthetic process Supporting Evidence: PMID:21709257 The accumulation of lipid X together with UDP-2,3-diacyl-GlcN in three independently grown atlpxb-1 null mutant plants ( Figs. 3 and 4 ) demonstrates that both compounds are likely substrates for AtLpxB in vivo PMID:21709257 our work demonstrates that plants synthesize lipid A precursors using the same enzymatic pathway present in E. coli |
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Download this section (compressed HTML)Q: Does recombinant mature AtLpxB condense UDP-2,3-diacyl-GlcN and lipid X in vitro, and does it complement an E. coli lpxB mutant?
Q: Is AtLpxB a peripheral membrane protein of the mitochondrial inner or outer membrane, as E. coli LpxB is peripheral to the inner membrane?
Q: Why does lipid X from lpxb-1 plants also appear in chloroplast fractions? Is there lipid trafficking from mitochondria to plastids?
Experiment: Express mature AtLpxB (without the 58-residue transit peptide) in E. coli, assay with UDP-2,3-diacyl-GlcN and lipid X, identify the disaccharide 1-phosphate by LC-ESI/MS, and test rescue of a conditional E. coli lpxB mutant.
Hypothesis: AtLpxB is a functional lipid-A-disaccharide synthase.
Type: in vitro enzyme assay and heterologous complementation
Experiment: Protease protection and carbonate extraction of mitochondria from lines expressing full-length tagged LPXB under its native promoter.
Hypothesis: AtLpxB is associated with a mitochondrial membrane where its lipid acceptor resides.
Type: sub-mitochondrial fractionation
Experiment: Challenge lpxb null mutants with bacterial pathogens and mitochondrial stressors, and compare mitochondrial membrane lipid profiles with wild type.
Hypothesis: The plant lipid A-like product has a conditional role in mitochondrial membrane function or defence.
Type: stress and pathogen phenotyping
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