PFRA (and its identical tandem copy PFRB) encodes the 69 kDa paraflagellar rod protein, PFR2 in the consolidated kinetoplastid nomenclature, a 600-residue, highly alpha-helical, coiled-coil-rich protein. Together with its paralog PFR1 (the 73 kDa PFRC/PFRD protein) it is one of the two major structural components of the paraflagellar rod (PFR), a trilaminar paracrystalline lattice that runs alongside the axoneme inside the flagellum of kinetoplastids and euglenids. PFR1 and PFR2 coiled coils are thought to form the filamentous wires of the PFR lattice. PFR2 is assembled at the distal tip of the growing flagellum into a stable structure with low turnover. Depletion of PFRA by RNAi blocks PFR construction, prevents incorporation of other PFR components, and paralyses procyclic trypanosomes. In bloodstream forms it is essential for viability and infectivity, because cells that cannot move fail cytokinesis. The PFR proteins associate with calmodulin, which itself is required for PFR assembly.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005516 calmodulin binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO transfer from the Flagellar_rod domain (IPR007824). UniProt predicts a calmodulin-binding region (residues 335-355) by sequence analysis only. Purified PFR proteins (PFR1/PFR2) bound calmodulin-Sepharose in a calcium-dependent manner, and the binding was competed by a CaMKII calmodulin-binding peptide (Ridgley et al. 2000, as summarised in PMID:19879876). Calmodulin localises to the PFR and is required for PFR assembly (PMID:23787017). Reason: Calmodulin binding by the PFR1/PFR2 pool is plausible and has biochemical support. However, the assay did not separate PFR2 from PFR1 or from co-migrating PAR proteins, so the evidence that PFR2 itself binds calmodulin is weaker than for its structural role. The term is kept as a real but non-core property; the core function of PFR2 is structural. Supporting Evidence: PMID:19879876 PFR protein binding by calmodulin-Sepharose was sensitive to competition by the calmodulin binding peptide from calmodulin kinase II but not an irrelevant peptide, suggesting that there is a specific interaction between calmodulin and components of the PFR. PMID:19879876 although the presence in the preparations of proteins of similar molecular weight such as PAR1 and PAR4 homologues may also explain these results. PMID:23787017 Unexpectedly CaM depletion resulted in total and catastrophic failure in PFR assembly; even connections linking axoneme to PFR failed to form following CaM depletion. |
| GO:0005856 cytoskeleton | IEA GO_REF:0000044 | MODIFY | Summary: UniProt SubCell mapping (Cytoplasm, cytoskeleton). PFR2 is a structural subunit of the paraflagellar rod, which is described as a stable cytoskeletal structure inside the flagellum. Reason: The location is correct in a broad sense, since the PFR is a cytoskeletal lattice, but it is unspecific and suggests a cytoplasmic cell-body cytoskeleton. GO has a dedicated term, GO:0097740 paraflagellar rod (part_of 9+2 motile cilium). In GO it is not classified under cytoskeleton, so this is a lateral refinement to the precise structure that PFR2 builds, not a descendant swap. The same replacement is proposed for the motile cilium row. Proposed replacements: paraflagellar rod Supporting Evidence: PMID:10954429 The PFR is constructed by a polar assembly process at the distal end of the flagellum resulting in a stable cytoskeletal structure with low turn-over. PMID:2793936 Its major structural component is a single protein of 600 amino acids. |
| GO:0031514 motile cilium | IEA GO_REF:0000120 | MODIFY | Summary: Combined automated annotation (InterPro + UniProt SubCell flagellum). PFR2 is located in the flagellum, specifically in the paraflagellar rod that runs alongside the axoneme. Reason: Correct but general. GO:0097740 paraflagellar rod is a part_of descendant of motile cilium (via 9+2 motile cilium) and names the exact structure of which PFR2 is a major component. Since the PFR is only found in kinetoplastids and euglenids, this specific term is fully appropriate for a trypanosome protein. Proposed replacements: paraflagellar rod Supporting Evidence: file:TRYBB/PFRA/PFRA-uniprot.txt Major component of the paraflagellar rod (PFR). PMID:34257277 Biochemical analyses indicate that major structural components of the PFR are two proteins, PFR1 and PFR2 |
| GO:0005198 structural molecule activity | IMP PMID:10954429 Flagellum ontogeny in trypanosomes studied via an inherited ... | NEW | Summary: PFR2 is one of the two major structural proteins of the PFR lattice. RNAi depletion of PFRA blocks PFR construction, and EM shows that most of the PFR structure is missing. Cryo-ET suggests that PFR1/PFR2 coiled coils make up the lattice wires. Reason: There is no molecular-function annotation describing what PFR2 actually does. It contributes structure to the PFR lattice and is non-redundant with PFR1. GO:0005200 structural constituent of cytoskeleton was not used because that term occurs_in GO:0005856 cytoskeleton, and in GO the PFR is not classified under cytoskeleton. The broader GO:0005198 is therefore the most accurate existing term. Supporting Evidence: PMID:10954429 new PFRA RNA and PFRA protein quickly disappeared and PFR construction was affected, resulting in cell paralysis. PMID:19888464 EM analysis shows that the PFR structure is greatly reduced PMID:34257277 PFR1 and PFR2 are essential and non-redundant components of the PFR. |
| GO:0060285 cilium-dependent cell motility | IMP PMID:10954429 Flagellum ontogeny in trypanosomes studied via an inherited ... | NEW | Summary: Depletion of PFRA/PFR2 by inducible RNAi (snl-1, snl-2, snl4) paralyses procyclic T. brucei. Flagellar beat frequency and amplitude drop and forward motility is lost, while the axoneme remains intact. Reason: Participation test: PFR2 does part of the work of flagellar motility because it builds the PFR, an extra-axonemal structure that the flagellar beat depends on (the structural-contribution case). It is not merely a substrate or a remote necessity. The motility phenotype has been reproduced with several independent RNAi constructs and matches the Leishmania PFR2 null mutant. The bloodstream-form lethality and cytokinesis failure are downstream of motility loss, so no cytokinesis term is proposed. Supporting Evidence: PMID:19888464 Defective motility is clearly apparent and cells are completely paralysed. PMID:19879876 Cells showed a dramatic decrease in flagellar wave frequency and amplitude that corresponded to a loss of forward cell motility. |
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Download this section (compressed HTML)Q: Does PFR2 bind calmodulin directly, independently of PFR1 and co-purifying PAR proteins, and is the predicted 335-355 region the binding site?
Q: Should structural subunits such as PFR1/PFR2 be annotated to GO:0120268 paraflagellar rod assembly, given that their depletion abolishes PFR construction, or is their structural-constituent annotation sufficient by GO convention?
Q: Which specific domains of the PFR lattice (proximal, intermediate, distal) does PFR2 occupy relative to PFR1?
Experiment: Express recombinant PFR2 (and PFR1) separately and measure calcium-dependent calmodulin binding, for example by overlay or ITC, with and without deletion of the predicted 335-355 calmodulin-binding region.
Experiment: Localise PFR2 within the PFR lattice using in situ tagging combined with cryo-ET subtomogram averaging, to assign PFR2 to specific lattice wires or densities.
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