Phosphate-specific transport system accessory regulatory protein PhoU (VPARA_10440, A0A0H2MBK2) in Variovorax paradoxus strain TBEA6 is a 233-amino-acid cytoplasmic homodimeric signaling protein (InterPro IPR028366, NCBIfam TIGR02135, PIRSF PIRSF003107, PANTHER PTHR42930:SF3) encoded on genomic contig JZWI01000005 (EMBL KLN58032.1) between pstB (VPARA_10450) and phoB1 (VPARA_10430) within the phoR1-phoB1-phoU-pstB-pstA-pstC-pstS-ppx cluster (VPARA_10420-VPARA_10490). PhoU comprises two tandem symmetric three-alpha-helix bundle domains (Pfam PF01895 at residues 22-108 and 129-210) that conserve the divalent metal-coordinating acidic/arginine pockets in each bundle. Under phosphate-replete conditions, PhoU binds divalent cations (Mn(2+)/Mg(2+)), which may be important for membrane interactions, and physically bridges the PstB ATPase subunit of the PstSCAB importer to the PAS domain of the PhoR1 sensor histidine kinase, shifting PhoR1 toward its phospho-PhoB1 phosphatase state to repress the Pho regulon and maintain intracellular phosphate homeostasis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Core subcellular localization of the soluble PhoU homodimer, which associates reversibly with the cytoplasmic face of the inner membrane via PstB and PhoR1. Reason: PhoU is a soluble cytoplasmic homodimer (PIRNR003107) that interacts with PstB and the cytoplasmic PAS domain of PhoR at the inner membrane surface to form the phosphate-signaling complex (PMID:24563032). Supporting Evidence: file:VARPD/phoU/phoU-uniprot.txt CC -!- SUBCELLULAR LOCATION: Cytoplasm {ECO:0000256|PIRNR:PIRNR003107}. PMID:24563032 We have also shown that the soluble form of E. coli PhoU is a dimer that binds manganese and magnesium at conserved sites and that metal binding may be important for localizing PhoU to the membrane. |
| GO:0030643 intracellular phosphate ion homeostasis | IEA GO_REF:0000002 | ACCEPT | Summary: Core biological process; PhoU couples the transport activity of PstSCAB to PhoR1-PhoB1 signaling and modulates Pi uptake to maintain intracellular orthophosphate homeostasis. Reason: VPARA_10440 belongs to the full-length bacterial PhoU family (InterPro IPR028366, NCBIfam TIGR02135, PANTHER PTHR42930:SF3), which senses extracellular Pi sufficiency through PstSCAB and modulates both Pi import through PstSCAB and PhoR-PhoB regulon expression to maintain intracellular phosphate homeostasis (PMID:24563032). Supporting Evidence: file:VARPD/phoU/phoU-uniprot.txt CC -!- FUNCTION: Plays a role in the regulation of phosphate uptake. CC {ECO:0000256|PIRNR:PIRNR003107}. PMID:24563032 It has been reported that PhoU is a peripheral membrane protein that is a negative regulator of the signaling pathway and that it modulates P i transport through the PstSCAB proteins ( 22 – 24 ). |
| GO:0045936 negative regulation of phosphate metabolic process | IEA GO_REF:0000002 | ACCEPT | Summary: Core biological process; PhoU acts as the essential negative regulator of the PhoR1-PhoB1 phosphate starvation response and PstSCAB phosphate import under phosphate-replete conditions. Reason: When extracellular phosphate is abundant, PhoU interacts with PstB and the PAS domain of PhoR to promote PhoR phosphatase activity toward phospho-PhoB, negatively regulating expression of the Pho regulon and preventing unregulated phosphate uptake (PMID:24563032). Supporting Evidence: file:VARPD/phoU/phoU-uniprot.txt CC -!- FUNCTION: Plays a role in the regulation of phosphate uptake. CC {ECO:0000256|PIRNR:PIRNR003107}. PMID:24563032 When phoU is mutated or deleted, PhoR is a constitutive PhoB kinase leading to high expression of the Pho regulon genes. |
| GO:0030145 manganese ion binding | IC file:VARPD/phoU/phoU-uniprot.txt | NEW | Summary: Core molecular function; PhoU coordinates Mn(2+) cations via conserved acidic/arginine pockets in its two tandem PhoU three-helix bundle domains (Pfam PF01895), which may be important for PhoU membrane interactions. Reason: Inferred by curator (IC) from the two conserved PhoU three-helix bundle domains (Pfam PF01895 at residues 22-108 and 129-210) in file:VARPD/phoU/phoU-uniprot.txt preserving the conserved acidic/arginine pockets (including Glu96/Arg97 and Glu198/Arg199) shown by ICP-MS, fluorescence titration, and site-directed mutagenesis to bind Mn(2+), which may be important for PhoU membrane interactions in E. coli PhoU (PMID:24563032). Supporting Evidence: file:VARPD/phoU/phoU-uniprot.txt FT DOMAIN 22..108 FT /note="PhoU" FT /evidence="ECO:0000259|Pfam:PF01895" FT DOMAIN 129..210 FT /note="PhoU" FT /evidence="ECO:0000259|Pfam:PF01895" PMID:24563032 We have also shown that the soluble form of PhoU is a dimer that binds manganese and magnesium. Alteration of highly conserved residues in PhoU by site-directed mutagenesis shows that these sites play a role in binding metals. Analysis of these phoU mutants suggests that metal binding may be important for PhoU membrane interactions. |
| GO:0000287 magnesium ion binding | IC file:VARPD/phoU/phoU-uniprot.txt | NEW | Summary: PhoU coordinates Mg(2+) cations via conserved acidic/arginine pockets in its two tandem PhoU three-helix bundle domains (Pfam PF01895), which may be important for PhoU membrane interactions. Reason: Inferred by curator (IC) from the two conserved PhoU three-helix bundle domains (Pfam PF01895 at residues 22-108 and 129-210) in file:VARPD/phoU/phoU-uniprot.txt preserving the conserved acidic/arginine pockets (including Glu96/Arg97 and Glu198/Arg199) shown by ICP-MS, fluorescence titration, and site-directed mutagenesis to bind Mg(2+) as well as Mn(2+), which may be important for PhoU membrane interactions in E. coli PhoU (PMID:24563032). Supporting Evidence: file:VARPD/phoU/phoU-uniprot.txt FT DOMAIN 22..108 FT /note="PhoU" FT /evidence="ECO:0000259|Pfam:PF01895" FT DOMAIN 129..210 FT /note="PhoU" FT /evidence="ECO:0000259|Pfam:PF01895" PMID:24563032 We have also shown that the soluble form of PhoU is a dimer that binds manganese and magnesium. Alteration of highly conserved residues in PhoU by site-directed mutagenesis shows that these sites play a role in binding metals. Analysis of these phoU mutants suggests that metal binding may be important for PhoU membrane interactions. |
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Download this section (compressed HTML)Q: Does Variovorax paradoxus PhoU (VPARA_10440) also directly regulate polyphosphate kinase (Ppk, VPARA_28600) or exopolyphosphatase (Ppx, VPARA_10490, encoded at the opposite end of the same gene cluster) in response to fluctuations in PstSCAB transport flux?
Suggested experts: McCleary WR
Experiment: Perform bacterial two-hybrid (BACTH) and Ni-NTA co-elution assays with Variovorax paradoxus PhoU (VPARA_10440), PstB (VPARA_10450), and the PAS domain of PhoR1 (VPARA_10420), and measure Mn(2+)/Mg(2+) binding by ICP-MS and intrinsic fluorescence.
Hypothesis: V. paradoxus PhoU forms a divalent-cation-binding homodimer that bridges PstB and the PAS domain of PhoR1 to assemble the membrane-associated phosphate-signaling complex (PMID:24563032).
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