FliY is a bifunctional flagellar motor switch protein that acts as a CheY-P phosphatase localized at the flagellar C-ring. It contains three functional domains: an N-terminal CheY-binding region homologous to FliM residues 6-15, a middle CheC-like phosphatase domain with conserved Glu/Asn catalytic residues, and a C-terminal FliN-like domain that integrates it into the flagellar switch complex. FliY catalyzes the dephosphorylation of the phosphorylated response regulator CheY-P at the site of its action (the flagellar motor), thereby contributing to signal termination in the chemotaxis pathway. B. subtilis uses a three-phosphatase system (CheC, FliY, CheX) instead of the CheZ found in E. coli. FliY functions constitutively to maintain optimal CheY-P levels near the motor, while CheC responds to attractant stimuli. FliY can complement Salmonella fliN mutants, confirming its structural role in the C-ring.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004721 phosphoprotein phosphatase activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation for phosphoprotein phosphatase activity is strongly supported by phylogenetic analysis and experimental evidence. FliY contains CheC-like phosphatase domains with conserved catalytic residues and has been directly demonstrated to catalyze CheY-P dephosphorylation [PMID:12920116]. Reason: This IBA annotation is well-supported by the phylogenetic tree inference and is consistent with extensive experimental evidence. PMID:12920116 directly demonstrates that FliY increases the rate of CheY-P hydrolysis in vitro. The domain architecture (CheC-like phosphatase domain) and structural studies further support this function. Supporting Evidence: PMID:12920116 We showed that FliY increases the rate of CheY-P hydrolysis in vitro. PMID:12920116 In particular we have identified the phosphatase as FliY file:BACSU/fliY/fliY-deep-research-falcon.md See deep research file for comprehensive analysis |
| GO:1902021 regulation of bacterial-type flagellum-dependent cell motility | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation for regulation of flagellum-dependent motility is well-supported. FliY regulates motility by controlling CheY-P levels at the flagellar motor, which determines flagellar rotation direction [PMID:12920116]. Reason: This annotation accurately captures FliY's regulatory role in motility. By dephosphorylating CheY-P at the flagellar switch, FliY modulates the duration of enhanced CheY-P levels and thereby regulates switching between clockwise and counterclockwise flagellar rotation. Supporting Evidence: PMID:12920116 Consequently, we imagine that the duration of enhanced CheY-P levels caused by activation of the CheA kinase upon attractant binding to receptors, is brief due both to adaptational processes and to phosphatase activity of FliY. |
| GO:0003774 cytoskeletal motor activity | IEA GO_REF:0000002 | REMOVE | Summary: IEA annotation based on InterPro domain IPR001172 (FliN_T3SS_HrcQb). This annotation is problematic because FliY is not itself a motor protein - it is a component of the motor switch that regulates motor activity, but does not generate force or movement directly. Reason: This is an over-annotation. FliY is a component of the flagellar motor switch complex (C-ring) but it does not have motor activity itself. The motor activity is generated by the stator units (MotA/MotB) interacting with FliG. FliY's role is to modulate CheY-P levels, not to generate rotational force. The InterPro domain hit is based on FliN homology, but FliN is a switch component, not a motor per se. The term "cytoskeletal motor activity" implies direct force generation, which FliY does not perform. Supporting Evidence: PMID:12920116 the flagellar switch, which controls direction of flagellar rotation based on levels of the chemotaxis primary response regulator, CheY-P |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | MODIFY | Summary: IEA annotation based on UniProt subcellular location mapping. UniProt indicates FliY is a peripheral membrane protein on the cytoplasmic side. However, more specific localization to the flagellar C-ring is available. Reason: While plasma membrane association is not incorrect, a more specific and informative CC annotation exists. FliY localizes specifically to the flagellar basal body C-ring. The UniProt entry itself states the switch is localized at the base of the flagellar motor, in or near the C-ring, attached to the MS-ring. Proposed replacements: bacterial-type flagellum basal body, C ring Supporting Evidence: PMID:12920116 the flagellar switch, which controls direction of flagellar rotation based on levels of the chemotaxis primary response regulator, CheY-P |
| GO:0006935 chemotaxis | IEA GO_REF:0000120 | ACCEPT | Summary: IEA annotation for chemotaxis from combined automated methods. FliY is clearly involved in chemotaxis as it is part of the signal termination machinery that dephosphorylates CheY-P at the motor. Reason: This is an appropriate biological process annotation. FliY's phosphatase activity directly contributes to chemotactic signal termination by hydrolyzing CheY-P at the flagellar switch. This role is experimentally validated in PMID:12920116. Supporting Evidence: PMID:12920116 This task is performed in Escherichia coli by CheZ, which interestingly enough is primarily located at the receptors, not at the switch. |
| GO:0009288 bacterial-type flagellum | IEA GO_REF:0000002 | MODIFY | Summary: IEA annotation based on InterPro domain IPR012826 (FliN). This is a valid but less specific localization compared to what is known about FliY. Reason: FliY localizes specifically to the C-ring of the flagellar basal body, not throughout the flagellum generally. A more specific annotation to the C-ring would be more informative and accurate. Proposed replacements: bacterial-type flagellum basal body, C ring Supporting Evidence: PMID:12920116 In this report we show that in Bacillus subtilis the flagellar switch, which controls direction of flagellar rotation |
| GO:0009425 bacterial-type flagellum basal body | IEA GO_REF:0000002 | MODIFY | Summary: IEA annotation based on InterPro domain IPR001172. This is a reasonable localization but can be made more specific to the C-ring. Reason: FliY is specifically a component of the C-ring within the basal body. While GO:0009425 is not incorrect, GO:0009433 (C-ring) would be more precise and informative. Proposed replacements: bacterial-type flagellum basal body, C ring Supporting Evidence: PMID:12920116 In particular we have identified the phosphatase as FliY, which resembles E. coli switch protein FliN only in its C-terminal part |
| GO:0016020 membrane | IEA GO_REF:0000002 | MODIFY | Summary: Very generic membrane annotation based on InterPro domain IPR012826. This is far too general given the specific localization known for FliY. Reason: This annotation is not wrong but is uninformative. FliY is a peripheral membrane protein that specifically localizes to the flagellar C-ring. The more specific term should be used. Proposed replacements: bacterial-type flagellum basal body, C ring Supporting Evidence: PMID:12920116 In this report we show that in Bacillus subtilis the flagellar switch, which controls direction of flagellar rotation |
| GO:0016787 hydrolase activity | IEA GO_REF:0000120 | MARK AS OVER ANNOTATED | Summary: Generic hydrolase activity annotation from combined automated methods. FliY does have hydrolase activity (it hydrolyzes the phosphate from CheY-P), but this term is too general. Reason: While technically correct (FliY catalyzes hydrolysis of a phosphoester bond), this annotation is too general to be informative. The more specific term GO:0004721 (phosphoprotein phosphatase activity) already captures this function with appropriate specificity. Retaining both creates redundancy without adding information. Supporting Evidence: PMID:12920116 We showed that FliY increases the rate of CheY-P hydrolysis in vitro. |
| GO:0071973 bacterial-type flagellum-dependent cell motility | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: IEA annotation for flagellum-dependent motility based on InterPro domains. FliY contributes to motility through its role in the motor switch complex. Reason: FliY is involved in flagellum-dependent motility as a component of the motor switch, but this is not its primary molecular function. Its core function is as a CheY-P phosphatase that regulates motility. This annotation reflects a downstream consequence of its biochemical activity rather than a direct role. Supporting Evidence: PMID:12920116 the flagellar switch, which controls direction of flagellar rotation |
| GO:0097588 archaeal or bacterial-type flagellum-dependent cell motility | IEA GO_REF:0000043 | KEEP AS NON CORE | Summary: IEA annotation from UniProt keyword mapping. This is a parent term of GO:0071973 (bacterial-type flagellum-dependent cell motility). Reason: This is essentially redundant with GO:0071973 and represents a less specific grouping term. FliY is involved in motility regulation but this is downstream of its core phosphatase function. Given the more specific child term is also annotated, this parent term adds little value. Supporting Evidence: PMID:12920116 In this report we show that in Bacillus subtilis the flagellar switch, which controls direction of flagellar rotation |
| GO:0044780 bacterial-type flagellum assembly | IMP PMID:25313396 FlgM is secreted by the flagellar export apparatus in Bacill... | REMOVE | Summary: IMP annotation from PMID:25313396 (FlgM secretion paper). However, this paper defines a minimal set of flagellar proteins required for FlgM secretion and flagellum assembly - fliY is NOT in this minimal set. Reason: PMID:25313396 explicitly defines the minimal subset of flagellar proteins required for flagellum assembly and FlgM secretion as: FliF/FliG, FliO, FliP, FliQ, FliR, FlhA, FlhB, and FliK. FliY is NOT in this list. While fliY mutants may show assembly defects due to impaired chemotaxis signaling, FliY is not a core component of the assembly machinery. This annotation likely reflects indirect/pleiotropic effects rather than a direct role in assembly. Supporting Evidence: PMID:25313396 FlgM secretion is strongly enhanced by, but does not strictly require, hook-basal body completion and instead demands a minimal subset of flagellar proteins that includes the FliF/FliG basal body proteins, the flagellar type III export apparatus components FliO, FliP, FliQ, FliR, FlhA, and FlhB, and the substrate specificity switch regulator FliK. |
| GO:0071978 bacterial-type flagellum-dependent swarming motility | IMP PMID:25313396 FlgM is secreted by the flagellar export apparatus in Bacill... | KEEP AS NON CORE | Summary: IMP annotation from PMID:25313396. Swarming motility requires functional flagella and chemotaxis, so fliY mutants may show swarming defects. Reason: FliY likely contributes to swarming motility indirectly through its role in chemotaxis regulation. However, swarming is a complex phenotype that involves many genes. This annotation reflects a downstream consequence of FliY's phosphatase function rather than a direct role in swarming per se. The annotation is retained but marked as non-core since the primary function is CheY-P dephosphorylation. Supporting Evidence: PMID:12920116 Consequently, we imagine that the duration of enhanced CheY-P levels caused by activation of the CheA kinase upon attractant binding to receptors, is brief due both to adaptational processes and to phosphatase activity of FliY. |
| GO:0006935 chemotaxis | IMP PMID:12920116 Bacillus subtilis hydrolyzes CheY-P at the location of its a... | ACCEPT | Summary: IMP annotation for chemotaxis from the key primary publication. PMID:12920116 directly demonstrates FliY's role in chemotaxis through its phosphatase activity on CheY-P. Reason: This is a well-supported annotation from the primary paper characterizing FliY function. The paper shows that FliY deletion affects chemotaxis by altering CheY-P levels at the flagellar switch. Deletion of the CheY-binding region in fliY caused the opposite phenotype to a cheY mutant, demonstrating its direct role in chemotactic signal modulation. Supporting Evidence: PMID:12920116 Deletion of this binding region in B. subtilis mutant fliM caused the same phenotype as a cheY mutant (clockwise flagellar rotation), whereas deletion of it in fliY caused the opposite. |
| GO:0016311 dephosphorylation | IMP PMID:12920116 Bacillus subtilis hydrolyzes CheY-P at the location of its a... | ACCEPT | Summary: IMP annotation for dephosphorylation biological process. FliY catalyzes dephosphorylation of CheY-P as demonstrated in PMID:12920116. Reason: This annotation accurately captures FliY's role in dephosphorylation as a biological process. The paper directly demonstrates that FliY increases CheY-P hydrolysis rates in vitro, and this dephosphorylation is central to its function in chemotactic signal termination. Supporting Evidence: PMID:12920116 We showed that FliY increases the rate of CheY-P hydrolysis in vitro. PMID:12920116 also causes hydrolysis of CheY-P to form CheY and Pi |
| GO:1902021 regulation of bacterial-type flagellum-dependent cell motility | IMP PMID:12920116 Bacillus subtilis hydrolyzes CheY-P at the location of its a... | ACCEPT | Summary: IMP annotation for regulation of flagellum-dependent motility from PMID:12920116. FliY regulates motility by modulating CheY-P levels at the motor switch. Reason: This is a key biological process annotation. PMID:12920116 demonstrates that FliY regulates flagellar rotation direction by controlling CheY-P levels at the switch. The paper shows deletion of FliY's CheY-binding region affects flagellar rotation phenotype. Supporting Evidence: PMID:12920116 the flagellar switch, which controls direction of flagellar rotation based on levels of the chemotaxis primary response regulator, CheY-P PMID:12920116 deletion of it in fliY caused the opposite [phenotype to cheY mutant] |
| GO:0004721 phosphoprotein phosphatase activity | IDA PMID:12920116 Bacillus subtilis hydrolyzes CheY-P at the location of its a... | ACCEPT | Summary: IDA annotation for phosphoprotein phosphatase activity - the most important and well-supported annotation for FliY. PMID:12920116 directly demonstrates in vitro phosphatase activity on CheY-P. Reason: This is the core molecular function of FliY and is directly demonstrated by in vitro biochemical assays in PMID:12920116. The paper identifies FliY as the phosphatase responsible for CheY-P hydrolysis at the flagellar switch and shows it increases CheY-P hydrolysis rates. This IDA annotation is strongly supported. Supporting Evidence: PMID:12920116 We showed that FliY increases the rate of CheY-P hydrolysis in vitro. PMID:12920116 In particular we have identified the phosphatase as FliY |
| GO:0009433 bacterial-type flagellum basal body, C ring | IDA PMID:12920116 Bacillus subtilis hydrolyzes CheY-P at the location of its a... | NEW | Summary: NEW annotation for C-ring localization. FliY localizes to the flagellar switch complex, which is the C-ring. This is implied by its function at the motor switch and its homology to FliN. Reason: FliY is a component of the flagellar motor switch (C-ring). PMID:12920116 establishes that FliY performs its phosphatase function at the flagellar switch, and its C-terminal domain is homologous to FliN, a known C-ring component. B. subtilis FliY can complement Salmonella fliN mutants, confirming its structural integration into the C-ring. Supporting Evidence: PMID:12920116 In this report we show that in Bacillus subtilis the flagellar switch, which controls direction of flagellar rotation based on levels of the chemotaxis primary response regulator, CheY-P, also causes hydrolysis of CheY-P PMID:12920116 FliY, which resembles E. coli switch protein FliN only in its C-terminal part |
| GO:0070298 negative regulation of phosphorelay signal transduction system | IDA PMID:12920116 Bacillus subtilis hydrolyzes CheY-P at the location of its a... | NEW | Summary: NEW annotation for negative regulation of phosphorelay signaling. FliY dephosphorylates the response regulator CheY-P, thereby terminating the chemotaxis phosphorelay signal. Reason: FliY acts as a negative regulator of the chemotaxis phosphorelay system by dephosphorylating CheY-P. This function terminates the signal initiated by the CheA histidine kinase. The paper explicitly describes this termination function at the motor. Supporting Evidence: PMID:12920116 Consequently, we imagine that the duration of enhanced CheY-P levels caused by activation of the CheA kinase upon attractant binding to receptors, is brief due both to adaptational processes and to phosphatase activity of FliY. |
| GO:0005515 protein binding | NAS | NEW | Summary: Added to align core_functions with existing annotations. Reason: Core function term not present in existing_annotations. |
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Download this section (compressed HTML)Q: What is the relative contribution of FliY vs CheC vs CheX to total CheY-P dephosphorylation under different chemotactic stimulation conditions?
Q: Does FliY have dual active sites like T. maritima FliY, and do both contribute equally to catalysis in B. subtilis?
Experiment: Purify B. subtilis FliY and measure Km and kcat for CheY-P dephosphorylation to compare with CheC and CheX. Quantitative kinetic parameters would enable modeling of the three-phosphatase system.
Hypothesis: FliY kinetic parameters differ from CheC and CheX, reflecting distinct roles in chemotaxis signal termination
Experiment: Use cryo-EM or crosslinking mass spectrometry to determine the precise position of FliY within the B. subtilis C-ring. High-resolution structural information would reveal FliY integration into the switch.
Hypothesis: FliY occupies a specific position in the C-ring that places it in proximity to CheY-P binding sites on FliM
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