BraD is one of the two integral-membrane permease 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. It is a LivH-type permease with ten predicted transmembrane helices in the inner membrane. With BraE it forms the translocation pathway, energised by the BraF/BraG ATPases and fed by the periplasmic binding protein BraC. The complex imports L-leucine, L-isoleucine and L-valine with high affinity and, with lower affinity, L-alanine and L-threonine.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005304 L-valine transmembrane transporter activity | IBA GO_REF:0000033 | ACCEPT | Summary: Valine is a primary substrate of the BraCDEFG (LIV-I) importer, in which BraD is a permease subunit. Reason: The IBA is seeded by E. coli LivH (P0AEX7). BraD is 67% identical to LivH, and P. aeruginosa LIV-I carries valine in cells, with BraC binding it at sub-micromolar affinity. The activity belongs to the whole complex, to which BraD contributes part of the translocation pathway. 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: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:0005886 plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: BraD is a polytopic inner (plasma) membrane permease. Reason: BraD has ten predicted TM helices, was found among membrane proteins, and forms a membrane complex with BraEFG. 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 and solubilized from the membrane with octyl glucoside. |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Inner-membrane location of the permease subunit. Reason: This row agrees with the IBA row, the TM topology and membrane fractionation. 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 LIV-I substrate. Reason: The IBA is seeded by E. coli LivH and holds for P. aeruginosa LIV-I, as shown by uptake and BraC binding. 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: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:0015190 L-leucine transmembrane transporter activity | IBA GO_REF:0000033 | ACCEPT | Summary: Leucine is the reference LIV-I substrate. Reason: The IBA is seeded by E. coli LivH. Leucine uptake by reconstituted BraCDEFG requires all five components, so the claim is directly supported for P. aeruginosa. 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: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 LivH, whose LivHMGF core works with two binding proteins (LivJ and LivK). In this system the binding protein sets substrate specificity. P. aeruginosa has the single BraC (LIVAT) binding protein. Its specificity, and the substrate range of LIV-I in cells and in proteoliposomes, are Leu/Ile/Val plus Ala/Thr, with no report of Phe. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: UniProtKB:P0AEX7 · E. coli LivH 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 LIV-I substrate. The term is correct, but a precise import term is supported. Reason: BraD is in the WITH/FROM because its own PseudoCAP IMP is the sole seed, which is expected and not circular. LIV-I imports alanine into cells and proteoliposomes, so the precise claim is L-alanine import across plasma membrane (GO:1904273, an is_a descendant). The node spreads a BraC-dependent secondary substrate to the whole LivH clade on one P. aeruginosa seed (see notes). Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: UniProtKB:P21627 · braD (self; sole IBD seed, IMP PMID:18833300) SUPPORTS TRANSFER The target's own growth-screen IMP, corroborated by reconstitution (PMID:1400443). PANTHER:PTN000765102 · PTN000765102 SUPPORTS TRANSFER LivH permease node, 18 leaves in 16 taxa. Alanine specificity is set by the partner 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, reflecting the substrate specificity of the BraC protein. 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. |
| GO:0015808 L-alanine transport | IMP PMID:18833300 High-throughput phenotypic characterization of Pseudomonas a... | MODIFY | Summary: The PA1073 Tn5 mutant fails to grow on L-alanine as carbon source. Reconstitution corroborates alanine import by LIV-I. Reason: A sole-carbon-source growth defect is an indirect read-out of transport, but here it agrees with direct transport data, so the claim is sound. The precise term is L-alanine import across plasma membrane. The insertion is probably polar on braE-braG, which does not change the conclusion, because all bra genes are needed. 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, reflecting the substrate specificity of the BraC protein. |
| GO:0015823 phenylalanine transport | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phenylalanine transport is not supported for P. aeruginosa LIV-I. Reason: This is the process counterpart of the L-phenylalanine transporter activity IBA from E. coli LivH. No P. aeruginosa study lists Phe as a LIV-I substrate. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: UniProtKB:P0AEX7 · E. coli LivH SUPPORTS SOURCE BUT NOT TARGET Substrate range set by binding proteins that differ between the two species. 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. |
| GO:0016020 membrane | IEA GO_REF:0000002 | MODIFY | Summary: The term is correct but general. BraD is an inner (plasma) membrane permease. Reason: The specific location, plasma membrane, is supported and already annotated by IBA and IEA. Proposed replacements: plasma membrane Supporting Evidence: PMID:2120183 These proteins were found among cell membrane proteins |
| GO:0022857 transmembrane transporter activity | IEA GO_REF:0000002 | MODIFY | Summary: The term is correct but general for a subunit of a binding protein-dependent ABC amino acid importer. Reason: Reconstitution shows BraD works within an ATP-driven importer of Leu/Ile/Val/Ala/Thr. The specific activity is ABC-type amino acid transporter activity (GO:0015424, an is_a descendant), which BraD provides together with BraC, BraE, BraF and BraG. Proposed replacements: ABC-type amino acid transporter activity 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. 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. |
| GO:0042941 D-alanine transmembrane transport | IBA GO_REF:0000033 | UNDECIDED | Summary: Propagated from two P. aeruginosa seeds (BraD and the paralog PA4912). Both rest only on partial D-alanine growth defects in one Biolog screen. Reason: No D-alanine uptake or binding has been shown for LIV-I or BraC. The characterised substrates are L-amino acids. The D-alanine growth defect seen in all bra mutants is suggestive but could be indirect. The claim is plausible, not established. Propagation Review Root cause: SOURCE WEAK OR INFERRED Failure modes: SOURCE EVIDENCE WEAK Sources checked: UniProtKB:P21627 · braD (self; IBD seed, IMP PMID:18833300) SOURCE WEAK OR INFERRED Partial D-Ala growth defect in a Biolog screen; no uptake assay. UniProtKB:Q9HUQ1 · PA4912 SOURCE WEAK OR INFERRED Same screen. A partial D-Ala/D-Val 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 PA1073 Tn5 mutant on D-alanine as carbon source. Reason: The phenotype is real but partial, and no uptake or binding assay was done. Nothing independent corroborates D-alanine as a LIV-I substrate. Supporting Evidence: PMID:18833300 In this work, mutants for each of these genes were found to be defective in Alanine metabolism |
| GO:0055085 transmembrane transport | IEA GO_REF:0000002 | MODIFY | Summary: The term is correct but general. The specific import processes are supported. Reason: BraD is a permease of an amino acid importer. The supported specific processes are L-leucine, L-isoleucine, L-valine and L-alanine import across the plasma membrane, all descendants of transmembrane transport. Proposed replacements: L-leucine import across plasma membrane L-isoleucine import across plasma membrane L-valine import across plasma membrane L-alanine import across plasma membrane 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. 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. |
| GO:1903785 L-valine transmembrane transport | IBA GO_REF:0000033 | MODIFY | Summary: The term is correct but general. LIV-I is an importer. Reason: The IBA is seeded by E. coli LivH. The precise claim for an uptake system is L-valine import across plasma membrane (GO:1903805, an is_a descendant), which BraF already carries. Propagation Review Root cause: NO FAILURE CORE Failure modes: GRANULARITY MISMATCH Sources checked: UniProtKB:P0AEX7 · E. coli LivH SUPPORTS TRANSFER Valine transport by LIV-I holds for P. aeruginosa. Only the direction (import) is under-specified. 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, but also for that of alanine and threonine, although less effectively. |
| GO:1903801 L-leucine import across plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: Leucine import is a core LIV-I function. Reason: The IBA is seeded by E. coli LivH. It is directly supported for P. aeruginosa by reconstituted leucine uptake, which requires BraD. 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:1903806 L-isoleucine import across plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: Isoleucine import is a core LIV-I function. Reason: The sole seed is BraD's own PseudoCAP IMP, which is expected and not circular. The claim is independently supported by LIV-I uptake and BraC binding data, and it agrees with the E. coli-seeded isoleucine transporter activity IBD at this node. 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: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:1903806 L-isoleucine import across plasma membrane | IMP PMID:18833300 High-throughput phenotypic characterization of Pseudomonas a... | ACCEPT | Summary: The PA1073 mutant grows less well on L-isoleucine as carbon source, which agrees with LIV-I specificity. Reason: The growth phenotype is indirect but corroborated. LIV-I imports isoleucine, and braD is required for LIV-I activity. 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:2120184 confirming that the braD, braE, braF, and braG genes are required for the LIV-I transport system |
| GO:0055052 ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing | IDA PMID:1400443 Solubilization and reconstitution of the Pseudomonas aerugin... | NEW | Summary: BraD is a subunit of the BraDEFG membrane complex, which works with the periplasmic binding protein BraC. Reason: BraDEFG form a membrane complex that can be solubilised, and transport requires the substrate-binding protein BraC. 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. |
Loading supporting content…
Download this section (compressed HTML)Q: Is D-alanine a genuine LIV-I substrate, or is the weak D-alanine growth defect of bra mutants indirect? The D-alanine IBD at PTN000765102 rests only on BraD and PA4912 rows from one screen.
Q: Should the L-alanine IBD (sole seed BraD) sit at the LivH node PTN000765102, given that the substrate range of HAAT systems is set by the partner binding protein?
Experiment: Radiolabelled D-alanine uptake in wild-type and clean, non-polar braD deletion strains, with complementation in trans. This would test D-alanine as a LIV-I substrate without the polarity confound of Tn5 insertions.
Type: transport assay
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)