BraF is one of the two nucleotide-binding (ATPase) subunits of LIV-I, the high-affinity, Na(+)-independent branched-chain amino acid ABC importer of Pseudomonas aeruginosa, encoded by the braC-braD-braE-braF-braG operon. BraF is a LivG-type ABC ATPase, with a Walker A ATP-binding motif and a C-terminal extension characteristic of branched-chain amino acid transporters. It associates peripherally with the cytoplasmic face of the inner membrane, where it pairs with BraG and binds the BraD/BraE permeases. With the periplasmic binding protein BraC, the complex couples ATP hydrolysis to the uptake of L-leucine, L-isoleucine and L-valine and, with lower affinity, L-alanine and L-threonine. All five Bra components are required for transport in reconstituted proteoliposomes.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005304 L-valine transmembrane transporter activity | IBA GO_REF:0000033 | ACCEPT | Summary: Valine is a substrate of the BraCDEFG (LIV-I) importer, of which BraF is the ATPase subunit. Reason: The IBA is seeded by E. coli LivG at the LivG node. LIV-I in P. aeruginosa carries valine, as shown by uptake in cells and by binding to the periplasmic BraC protein. The transporter activity belongs to the whole BraCDEFG complex, to which BraF contributes the ATPase. BraF is inside the clade that inherited the function, and no evidence says the function diverged in this gene. Supporting Evidence: PMID:113383 The former system seemed to serve not only for the entry of leucine, isoleucine, and valine, but also for that of alanine and threonine, although less effectively. PMID:1400443 leucine transport was completely dependent on the presence of all five Bra components and on ATP loaded internally to the proteoliposomes |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: BraF has a canonical ABC ATPase domain with a Walker A motif. Reason: The ABC transporter domain and Walker A ATP-binding site are clear, and the reconstituted LIV-I complex consumes ATP during transport. Supporting Evidence: file:PSEAE/braF/braF-uniprot.txt Pfam; PF00005; ABC_tran; 1. PMID:2120183 both containing amino acid sequences typical of proteins with ATP-binding sites |
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: BraF works at the cytoplasmic face of the inner (plasma) membrane, bound to the BraD/BraE permeases. Reason: BraF was found among cell membrane proteins, and BraDEFG forms a membrane complex. As an ABC ATPase it is peripheral, not integral, but the plasma membrane is where it is active. Supporting Evidence: PMID:2120183 These proteins were found among cell membrane proteins PMID:1400443 The proteins were found to form a complex in the E. coli membrane |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Inner-membrane (plasma membrane) location of the ABC ATPase subunit. Reason: This row agrees with the IBA row and with membrane fractionation of BraF. Supporting Evidence: PMID:2120183 These proteins were found among cell membrane proteins |
| GO:0015188 L-isoleucine transmembrane transporter activity | IBA GO_REF:0000033 | ACCEPT | Summary: Isoleucine is a primary substrate of the LIV-I importer. Reason: The IBA is seeded by E. coli LivG. LIV-I in P. aeruginosa transports isoleucine, and BraC binds it with sub-micromolar affinity. The activity is that of the BraCDEFG complex. Supporting Evidence: PMID:113383 The former system seemed to serve not only for the entry of leucine, isoleucine, and valine PMID:6767701 The dissociation constants of this protein for leucine, isoleucine, and valine were found to be 0.4, 0.3, and 0.5 microM, respectively. |
| GO:0015192 L-phenylalanine transmembrane transporter activity | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phenylalanine has not been reported as a substrate of P. aeruginosa LIV-I. Reason: The IBA comes from E. coli LivG, whose LivHMGF core works with two binding proteins (LivJ and LivK). Substrate specificity in this system is set by the binding protein. P. aeruginosa has the single BraC (LIVAT) binding protein. Its binding specificity, and the substrate range of LIV-I in cells and in proteoliposomes, have been characterised as Leu/Ile/Val plus Ala/Thr, with no report of Phe. The phenylalanine function probably reflects the E. coli binding-protein repertoire rather than the ATPase lineage. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: UniProtKB:P0A9S7 · E. coli LivG SUPPORTS SOURCE BUT NOT TARGET The E. coli phenylalanine function depends on the E. coli binding-protein repertoire. P. aeruginosa LIV-I specificity is characterised as LIVAT. Supporting Evidence: PMID:1400443 Alanine and threonine in addition to branched-chain amino acids were transported by the proteoliposomes, reflecting the substrate specificity of the BraC protein. PMID:6767701 The binding activity of the protein was specific for the branched-chain amino acids. |
| GO:0015808 L-alanine transport | IBA GO_REF:0000033 | MODIFY | Summary: L-alanine is a secondary substrate of LIV-I. The term is correct, but a more precise import term is supported. Reason: BraF appears in the WITH/FROM of this IBA because its own PseudoCAP IMP row is the seed, which is expected and not circular. LIV-I transports alanine into P. aeruginosa cells and into proteoliposomes, so the precise claim is L-alanine import across plasma membrane (GO:1904273, an is_a descendant through L-alanine transmembrane transport). Note that the node propagates a secondary substrate to the whole LivG clade on a single P. aeruginosa seed (see notes). Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: UniProtKB:P21629 · braF (self; sole IBD seed, IMP PMID:18833300) SUPPORTS TRANSFER The target's own PseudoCAP growth-screen IMP is the seed. It is corroborated by reconstitution (PMID:1400443). PANTHER:PTN000656921 · PTN000656921 SUPPORTS TRANSFER LivG ATPase node, 25 leaves in 15 taxa, including several paralogous HAAT ATPases per genome. Alanine is a secondary substrate set by the binding protein, so its clade-wide validity is uncertain. Proposed replacements: L-alanine import across plasma membrane Supporting Evidence: PMID:1400443 Alanine and threonine in addition to branched-chain amino acids were transported by the proteoliposomes PMID:113383 but also for that of alanine and threonine, although less effectively |
| GO:0015808 L-alanine transport | IMP PMID:18833300 High-throughput phenotypic characterization of Pseudomonas a... | MODIFY | Summary: The Biolog/plate-growth screen shows reduced growth of the PA1071 mutant on L-alanine. Reconstitution data corroborate L-alanine uptake by LIV-I. Reason: A growth defect on a sole carbon source is an indirect read-out of transport. Here it agrees with direct evidence that BraCDEFG imports alanine, so the claim is sound. The precise term is L-alanine import across plasma membrane. The Tn5 insertion in braF may also be polar on braG, but both genes are needed for LIV-I anyway. Proposed replacements: L-alanine import across plasma membrane Supporting Evidence: PMID:18833300 In this work, mutants for each of these genes were found to be defective in Alanine metabolism, while utilization of L-Valine and L-Isoleucine was also altered in some cases PMID:1400443 Alanine and threonine in addition to branched-chain amino acids were transported by the proteoliposomes |
| GO:0015823 phenylalanine transport | IEA GO_REF:0000108 | MARK AS OVER ANNOTATED | Summary: Inferred from the L-phenylalanine transporter activity IBA, which is itself an over-annotation for P. aeruginosa. Reason: This row follows from GO:0015192 and inherits its weakness. No P. aeruginosa study lists phenylalanine as a LIV-I substrate. Supporting Evidence: PMID:1400443 reflecting the substrate specificity of the BraC protein |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000002 | ACCEPT | Summary: BraF is an ABC ATPase subunit. The LIV-I complex hydrolyses ATP in a substrate- and BraC-dependent way. Reason: This is BraF's own catalytic activity, which drives the transport cycle. The reconstituted complex shows ATP consumption coupled to leucine translocation. The BraF/BraG contribution has not been separated, but both carry ABC ATPase domains. Supporting Evidence: PMID:1400443 indicating that the LIV-I transport system has an ATPase activity coupled to translocation of branched-chain amino acids across the membrane file:PSEAE/braF/braF-uniprot.txt PROSITE; PS50893; ABC_TRANSPORTER_2; 1. |
| GO:0042941 D-alanine transmembrane transport | IBA GO_REF:0000033 | UNDECIDED | Summary: Propagated from two P. aeruginosa seeds (BraF and the paralog PA4910). Both rest only on partial D-alanine growth defects in one Biolog screen. Reason: D-alanine uptake by LIV-I has not been measured, and D-alanine binding by BraC has not been shown. The characterised substrate range is L-Leu/Ile/Val/Ala/Thr. Every bra mutant grew less well on D-Ala, which is suggestive, but a partial growth defect could also be an indirect metabolic effect of losing L-amino acid uptake. The claim is plausible but not established, so I neither accept nor reject the IBA. Propagation Review Root cause: SOURCE WEAK OR INFERRED Failure modes: SOURCE EVIDENCE WEAK Sources checked: UniProtKB:P21629 · braF (self; IBD seed, IMP PMID:18833300) SOURCE WEAK OR INFERRED Partial D-Ala growth defect in a Biolog screen; no uptake assay. UniProtKB:Q9HUQ3 · PA4910 SOURCE WEAK OR INFERRED Same screen. A partial D-Ala nitrogen-source growth defect in a paralogous HAAT system. Supporting Evidence: PMID:18833300 mutants for each of these genes were found to be defective in Alanine metabolism |
| GO:0042941 D-alanine transmembrane transport | IMP PMID:18833300 High-throughput phenotypic characterization of Pseudomonas a... | UNDECIDED | Summary: Reduced (+/-) growth of the PA1071 Tn5 mutant on D-alanine as carbon source, from a Biolog screen with plate confirmation. Reason: The phenotype is real, but it is a partial growth defect with no uptake or binding assay. Nothing independent corroborates D-alanine as a LIV-I substrate, so whether BraF takes part in D-alanine transport stays open. Supporting Evidence: PMID:18833300 In this work, mutants for each of these genes were found to be defective in Alanine metabolism |
| GO:1903714 isoleucine transmembrane transport | IEA GO_REF:0000117 | MODIFY | Summary: The term is correct but general. The import-specific term is supported and already on BraF. Reason: LIV-I is an importer. L-isoleucine import across plasma membrane (GO:1903806, an is_a descendant) is supported by the binding-protein and uptake data and is already annotated by IBA and IMP. Proposed replacements: L-isoleucine import across plasma membrane Supporting Evidence: PMID:113383 The former system seemed to serve not only for the entry of leucine, isoleucine, and valine |
| GO:1903785 L-valine transmembrane transport | IEA GO_REF:0000117 | MODIFY | Summary: The term is correct but general. The import-specific term is supported and already on BraF. Reason: L-valine import across plasma membrane (GO:1903805, an is_a descendant) is the precise claim for an importer and is already annotated by IBA and IMP. Proposed replacements: L-valine import across plasma membrane Supporting Evidence: PMID:113383 The former system seemed to serve not only for the entry of leucine, isoleucine, and valine |
| GO:1903805 L-valine import across plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: Valine import is a core LIV-I function. Reason: This IBA is seeded only by BraF's own PseudoCAP IMP, which is expected and not circular. The claim is supported independently by P. aeruginosa LIV-I uptake and binding studies, and it agrees with the E. coli-seeded L-valine transporter activity IBD at the same node. Caveat for the node (not for BraF): it also covers several uncharacterised paralogous HAAT ATPases, whose substrates are set by their own binding proteins. Supporting Evidence: PMID:113383 The former system seemed to serve not only for the entry of leucine, isoleucine, and valine PMID:6767701 The dissociation constants of this protein for leucine, isoleucine, and valine were found to be 0.4, 0.3, and 0.5 microM, respectively. |
| GO:1903805 L-valine import across plasma membrane | IMP PMID:18833300 High-throughput phenotypic characterization of Pseudomonas a... | ACCEPT | Summary: The PA1071 mutant grows less well on L-valine as nitrogen source. This agrees with the known substrate range of LIV-I. Reason: The phenotype in the screen is weak (+/- on N source only) and indirect. The claim is still correct, because valine import by LIV-I is established biochemically and BraF is required for LIV-I activity. Supporting Evidence: PMID:18833300 while utilization of L-Valine and L-Isoleucine was also altered in some cases PMID:2120184 confirming that the braD, braE, braF, and braG genes are required for the LIV-I transport system |
| GO:1903806 L-isoleucine import across plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: Isoleucine import is a core LIV-I function. Reason: The node is seeded by BraF's PseudoCAP IMP and agrees with the E. coli-seeded L-isoleucine transporter activity IBD. The claim is independently supported for P. aeruginosa. Supporting Evidence: PMID:113383 The former system seemed to serve not only for the entry of leucine, isoleucine, and valine |
| GO:1903806 L-isoleucine import across plasma membrane | IMP PMID:18833300 High-throughput phenotypic characterization of Pseudomonas a... | ACCEPT | Summary: The PA1071 mutant grows less well on L-isoleucine as carbon source, which agrees with LIV-I specificity. Reason: The growth phenotype alone is indirect. It is corroborated by direct evidence that LIV-I imports isoleucine and that BraF is an essential LIV-I component. Supporting Evidence: PMID:18833300 while utilization of L-Valine and L-Isoleucine was also altered in some cases PMID:2120184 confirming that the braD, braE, braF, and braG genes are required for the LIV-I transport system |
| GO:1903801 L-leucine import across plasma membrane | IDA PMID:1400443 Solubilization and reconstitution of the Pseudomonas aerugin... | NEW | Summary: Leucine is the reference substrate of LIV-I. Its ATP-dependent uptake into proteoliposomes requires BraF together with the other four Bra proteins. Reason: BraF does part of the work, supplying the ATPase that drives translocation. In reconstituted proteoliposomes, leucine uptake needed all five Bra components and internal ATP. BraF has no leucine annotation at present, although the BraD permease receives one by IBA. Supporting Evidence: PMID:1400443 In this reconstituted system, leucine transport was completely dependent on the presence of all five Bra components and on ATP loaded internally to the proteoliposomes. |
| GO:0055052 ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing | IDA PMID:1400443 Solubilization and reconstitution of the Pseudomonas aerugin... | NEW | Summary: BraF is a subunit of the BraDEFG membrane complex, which works with the periplasmic binding protein BraC. Reason: BraDEFG form a complex in the membrane that can be solubilised, and transport requires the substrate-binding protein BraC. This is a binding protein-dependent ABC importer complex. Supporting Evidence: PMID:1400443 The proteins were found to form a complex in the E. coli membrane and solubilized from the membrane with octyl glucoside. |
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Download this section (compressed HTML)Q: Is D-alanine a genuine LIV-I substrate (bound by BraC and imported by BraCDEFG), or is the weak D-alanine growth defect of bra mutants indirect? The answer decides whether the D-alanine IBD at PTN000656921, seeded only by PA1071 and PA4910 from one screen, should stand.
Q: Should substrate-specific process IBDs (L-alanine, L-valine, L-isoleucine import) sit at PTN000656921? The node spans several paralogous HAAT ATPases per genome (seven in Bradyrhizobium japonicum), and in this family the substrate is set by the partner binding protein, not by the ATPase.
Experiment: Radiolabelled D-alanine uptake in wild-type and braC or braF deletion strains, and D-alanine competition for leucine binding to purified BraC. This would test whether D-alanine is a LIV-I substrate.
Type: transport assay
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