CheY (PP_4340) is a chemotaxis response regulator of Pseudomonas putida KT2440. It is a small (124 aa) single-domain receiver protein of the CheY-like superfamily, consisting of one REC (response regulatory) domain with the conserved phosphoaccepting aspartate (Asp52) and a Mg2+ cofactor site. The gene lies next to cheZ and cheA2 (PP_4338) in a cheYZA chemotaxis cluster. In the chemotaxis pathway, chemoreceptors control the histidine kinase CheA, which autophosphorylates and transfers the phosphoryl group to CheY; purified P. putida CheA phosphorylates CheY in vitro, and CheY is found associated with CheA in KT2440 cell extracts. Phosphorylated CheY diffuses through the cytoplasm to the flagellar motor, where it binds the C-ring switch proteins (FliM/FliN) and changes the direction of flagellar rotation, so that receptor and kinase activity is converted into changes in swimming behaviour. The phosphatase CheZ terminates the signal by accelerating CheY-P dephosphorylation.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000160 phosphorelay signal transduction system | IEA GO_REF:0000002 | ACCEPT | Summary: CheY is the response regulator of the chemotaxis phosphorelay: it accepts the phosphoryl group from the histidine kinase CheA on its conserved aspartate. P. putida CheA transfers phosphate to CheY in vitro, and the protein carries the REC domain with the phosphoaccepting Asp52. Reason: The GO:0000160 definition (histidine kinase autophosphorylation and transfer of the phosphate to an aspartate) describes the CheA-to-CheY step directly. The InterPro-based IEA is consistent with the domain architecture and with P. putida phosphotransfer data. Supporting Evidence: PMID:40197389 The addition of CheY to phosphorylated CheA led to a labeling of CheY and a reduction in the phospholabeling of CheA (Figure 2—figure supplement 2a), indicating a phosphate transfer happened between CheA and CheY. file:PSEPK/cheY/cheY-uniprot.txt MOD_RES 52 /note="4-aspartylphosphate" /evidence="ECO:0000256|PROSITE-ProRule:PRU00169" file:PSEPK/cheY/cheY-deep-research-falcon.md A 2022 KT2440 synthetic biology study (genetic code expansion) uses CheY as an exemplar “chemosensory response regulator in chemotaxis” and explicitly refers to **CheY (PP_4340)** as activated by phosphorylation by **CheA (PP_4338)**. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: CheY is a soluble, diffusible response regulator that carries the signal from the receptor-associated CheA kinase to the cytoplasmic C-ring of the flagellar motor. The single REC domain has no signal peptide or transmembrane segment. Reason: Cytoplasm is correct and is the most specific location the evidence supports for the protein itself; it acts transiently at arrays and at the motor but is not a resident component of either. The UniProt subcellular location line rests on an ARBA rule and is not used as evidence here. Supporting Evidence: file:PSEPK/cheY/cheY-deep-research-falcon.md Functionally, CheY is a **diffusible cytosolic** signal carrier between the receptor-associated kinase complex and the flagellar motor, acting at the **cytoplasmic face** of the motor’s C-ring/switch (via interaction with FliM and related switch components). PMID:40197389 After CheY accepts the phosphate group from CheA, it binds FliM and FliN of the motor-switch complex, resulting in a switch in the flagellar rotational direction and bacteria tumbling |
| GO:0000156 phosphorelay response regulator activity | IC PMID:40197389 Coordinated regulation of chemotaxis and resistance to coppe... | NEW | Summary: Proposed molecular function. CheY is phosphorylated by CheA on its receiver aspartate and in that state regulates the flagellar switch, which is the definition of phosphorelay response regulator activity ("regulated by transfer of a phosphate from a histidine residue in the sensor, to an aspartate residue in the response regulator"). Reason: GOA carries no molecular-function row for this protein. The activity is inferred by the curator from the P. putida CheA-to-CheY phosphotransfer assay together with the PROSITE REC domain and phosphoaspartate site; the cached text does not give the locus tag of the CheY used, so IC rather than IDA is used. This is the module-level activity of CheY in the chemotaxis signal transduction module. Supporting Evidence: PMID:40197389 The addition of CheY to phosphorylated CheA led to a labeling of CheY and a reduction in the phospholabeling of CheA file:PSEPK/cheY/cheY-uniprot.txt DOMAIN 2..119 /note="Response regulatory" /evidence="ECO:0000259|PROSITE:PS50110" file:PSEPK/cheY/cheY-uniprot.txt MOD_RES 52 /note="4-aspartylphosphate" /evidence="ECO:0000256|PROSITE-ProRule:PRU00169" |
| GO:0006935 chemotaxis | IC PMID:30761113 Concentration Dependent Effect of Plant Root Exudates on the... | NEW | Summary: Proposed process. PP_4340 is encoded in the same cluster as cheA2 (PP_4338), the CheA paralog that is required for chemotaxis in KT2440, and it is the phosphoacceptor of CheA in the chemotaxis phosphorelay. Reason: CheY performs a step of chemotaxis itself (it is the diffusible output that switches the motor), so this is participation rather than requirement. No KT2440 cheY mutant has been characterised, so the annotation is IC, inferred from the cheA2 mutant phenotype, gene neighbourhood and phosphotransfer data. Consistent with the chemotaxis annotation on CheA (PP_4338). Supporting Evidence: PMID:30761113 mutation of cheA2 abolished chemotaxis, which is consistent with the notion the CheA2 forms part of the che chemotaxis pathway file:PSEPK/cheY/cheY-deep-research-falcon.md The same work groups PP_4340 within a proposed operon/gene block **PP4340–PP4338**, consistent with cheY being part of a chemotaxis/motility module under flagellar regulon control. |
Loading supporting content…
Download this section (compressed HTML)Q: Does deletion of PP_4340 (cheY) in P. putida KT2440 produce the expected smooth-swimming (non-tumbling) chemotaxis-defective phenotype, and which of the multiple KT2440 chemosensory pathways does it serve?
Q: Which flagellar switch components (FliM/FliN/FliG) does P. putida CheY-P engage, and is the binding interface conserved relative to the E. coli paradigm?
Experiment: Construct a PP_4340 deletion mutant and assess swimming/tumbling behavior and chemotaxis toward defined attractants to confirm the predicted role in motor switching.
Experiment: Reconstitute CheA-CheY phosphotransfer in vitro with KT2440 proteins and measure phosphorylation kinetics and Mg2+ dependence at the active-site aspartate (Asp52).
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)