flvcr2a is one of the two zebrafish co-orthologs of vertebrate FLVCR2/MFSD7c, a 12-transmembrane carrier of the major facilitator superfamily (SLC49 family). It is a facilitative uniporter for choline: transport is not coupled to sodium or proton gradients, it is electrogenic, and it follows the choline concentration gradient, so the protein can move choline in either direction across the membrane it sits in. The protein resides chiefly in the plasma membrane, with reported pools in mitochondrial and endoplasmic-reticulum membranes. In vertebrates the orthologous protein is expressed in the endothelium of the central-nervous-system vasculature, where it carries circulating choline across the blood-brain barrier into the brain and takes up choline released by brain phospholipid turnover; choline supplied this way feeds phosphatidylcholine, acetylcholine and betaine synthesis, and choline is a nutrient critical for brain development. The related amine ethanolamine is a secondary substrate of the family. Loss of the mammalian ortholog blocks angiogenic growth of CNS blood vessels and produces microcephaly-associated vasculopathy, the basis of human Fowler syndrome; an older proposal that the protein imports heme has not been substantiated.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0015232 heme transmembrane transporter activity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: GO:0015232 claims flvcr2a moves heme across a membrane. No zebrafish heme-transport data exist. The PAINT node PTN000858905 spans both FLVCR paralog clades, and its heme evidence comes from two different sources: FLVCR1 (mouse Flvcr1, human FLVCR1) as a heme exporter, and a single 2010 study of human FLVCR2 reporting heme import (1.8-fold higher ZnMP uptake in FLVCR2-expressing CHO cells, enhanced [55Fe]hemin uptake in oocytes, reduced uptake after FLVCR2 siRNA). Because the term is direction-neutral it is not contradicted by the FLVCR2 literature, but the later deorphanization work failed to corroborate heme handling: heme and bilirubin were unchanged in Mfsd7c-KO fetal brain, and the authors state the heme-import report has not been confirmed. Reason: Retained, but not as a core activity. The only experimental support within the FLVCR2 clade is the 2010 heterologous heme-analog uptake work on human FLVCR2 (PMID:20823265), which UniProt still carries as a by-similarity heme b transport reaction for flvcr2a. Against that, the 2024 mouse/vertebrate study found no heme or bilirubin change on loss of the gene and explicitly flags the import claim as unconfirmed (PMID:38302740), while the demonstrated activity of the zebrafish protein itself is choline transport. Non-core is the honest position: plausible ancestral capability, no zebrafish evidence, no current mechanistic support. Propagation Review Root cause: NO FAILURE NON CORE Failure modes: SOURCE EVIDENCE WEAK Sources checked: PANTHER:PTN000858905 SUPPORTS SOURCE BUT NOT TARGET Ancestral node covering both FLVCR clades; heme transport at this node rests on FLVCR1 export plus one FLVCR2 import report, so the direction-neutral term transfers but carries little weight for the zebrafish FLVCR2 co-ortholog. UniProtKB:Q9UPI3 Β· human FLVCR2 SOURCE WEAK OR INFERRED Heme import by human FLVCR2 rests on heterologous ZnMP/[55Fe]hemin uptake assays that have not been independently reproduced; the 2024 study states the observation has not been confirmed. MGI:MGI:2444881 Β· mouse Flvcr1 SUPPORTS SOURCE BUT NOT TARGET Flvcr1 is the heme-exporting paralog; its evidence supports the node, not the flvcr2a branch. Supporting Evidence: PMID:20823265 FLVCR2 binds to hemin-conjugated agarose, and binding is competed by free hemin PMID:20823265 we consistently observed a 1.8-fold increase in the uptake of ZnMP, a heme analog, by FLVCR2-expressing CHO (CHO/huFLVCR2) cells compared to uptake by control CHO and CHO/huFLVCR1 cells PMID:20823265 mammalian cells and Xenopus laevis oocytes expressing FLVCR2 display enhanced heme uptake file:DANRE/flvcr2a/flvcr2a-uniprot.txt Reaction=heme b(in) = heme b(out) PMID:38302740 However, this observation has not been confirmed in the literature PMID:38302740 heme and bilirubin levels in fetal brains were comparable between Mfsd7cβ/β and WT embryos |
| GO:0016020 membrane | IBA GO_REF:0000033 | MODIFY | Summary: GO:0016020 asserts only that flvcr2a acts in a membrane. That is certainly true for a 12-transmembrane MFS carrier, but uninformative. The PAINT node PTN000858822 is broad, spanning FLVCR1/FLVCR2 and the lysosomal SLC49A4/DIRC2 members, whose membranes differ; the generic parent is what can safely be placed at such a node. For flvcr2a itself the site of action is resolved: human FLVCR2 is at the plasma membrane by confocal imaging, choline movement is measured across the plasma membrane, and the zebrafish protein's own choline-transport assay is a plasma-membrane uptake assay. Reason: The node placement is sound but the granularity is not: within the same aspect a specific child is supported for this gene product. is_active_in plasma membrane states where flvcr2a does its work, which the generic membrane term leaves open. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000858822 SUPPORTS TRANSFER Family node mixes plasma-membrane FLVCRs with lysosomal SLC49A4/DIRC2 orthologs, so the generic membrane term is the safe node-level call; it under-specifies a gene with resolved localization. UniProtKB:Q9UPI3 Β· human FLVCR2 SUPPORTS TRANSFER Donor is plasma-membrane localized by confocal imaging, supporting the more specific child term. Proposed replacements: plasma membrane Supporting Evidence: PMID:38778100 Confocal imaging shows that FLVCR1 and FLVCR2 are localized at the plasma membrane PMID:38302740 our results show that MFSD7c facilitates the transport of choline and it behaves like a channel for the movement of choline via the plasma membrane file:DANRE/flvcr2a/flvcr2a-uniprot.txt Cell membrane file:DANRE/flvcr2a/flvcr2a-uniprot.txt Multi-pass membrane protein |
| GO:0020037 heme binding | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: GO:0020037 claims flvcr2a binds heme. The proximate evidence in the FLVCR2 clade is direct but singular: human FLVCR2 co-precipitates with hemin-conjugated agarose and the binding is competed by free hemin. No zebrafish binding data exist, and the physiological relevance of the interaction is unsettled since loss of the mammalian protein does not perturb heme or bilirubin levels. Reason: The binding assay is real and was performed on the orthologous FLVCR2 protein, so the annotation should not be discarded; but heme binding is not the activity this protein is known to perform in vivo, and the choline-transport function is what the zebrafish protein was directly shown to do. Non-core. Propagation Review Root cause: NO FAILURE NON CORE Failure modes: SOURCE EVIDENCE WEAK Sources checked: UniProtKB:Q9UPI3 Β· human FLVCR2 SUPPORTS TRANSFER Hemin-agarose pulldown with free-hemin competition is a direct binding assay on the orthologous protein. PANTHER:PTN000858905 SUPPORTS TRANSFER Node placement is defensible; the binding is a family-level property with unclear physiological output. Supporting Evidence: PMID:20823265 FLVCR2 binds to hemin-conjugated agarose, and binding is competed by free hemin PMID:38302740 heme and bilirubin levels in fetal brains were comparable between Mfsd7cβ/β and WT embryos |
| GO:0097037 heme export | IBA GO_REF:0000033 | REMOVE | Summary: GO:0097037 asserts that flvcr2a exports heme out of the cell, a direction-specific process term. Its WITH/FROM is the ancestral node plus human FLVCR1 alone, i.e. the evidence is entirely from the heme-exporting paralog. Within the FLVCR2 clade the direction claim is the opposite of what was reported and was explicitly excluded: the study that proposed FLVCR2 as a heme importer notes that earlier work clearly shows FLVCR2 does not export heme. Reason: This is a paralog-specific function propagated across a node that also contains the FLVCR2 clade. Heme export is FLVCR1 biology; the FLVCR2 literature reports import, not export (PMID:20823265), and the 2024 work finds no heme phenotype at all on loss of the FLVCR2 ortholog (PMID:38302740). Removing the export term does not deny a possible heme interaction, which remains covered by the direction-neutral GO:0015232 and GO:0020037 rows kept as non-core. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: UniProtKB:Q9Y5Y0 Β· human FLVCR1 SUPPORTS SOURCE BUT NOT TARGET FLVCR1 is the characterized heme exporter; the export direction is a property of that paralog and was specifically reported not to hold for FLVCR2. PANTHER:PTN000858905 SUPPORTS SOURCE BUT NOT TARGET The node spans FLVCR1 and FLVCR2; placing a direction-specific export term here lets an FLVCR1-only function reach the FLVCR2 co-orthologs, including zebrafish flvcr2a. Supporting Evidence: PMID:20823265 a report by Quigley and colleagues ( 37 ) clearly shows that FLVCR2 does not export heme. PMID:20823265 FLVCR2 binds to hemin-conjugated agarose, and binding is competed by free hemin PMID:38302740 However, this observation has not been confirmed in the literature PMID:38302740 heme and bilirubin levels in fetal brains were comparable between Mfsd7cβ/β and WT embryos |
| GO:0005789 endoplasmic reticulum membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: GO:0005789 places flvcr2a in the endoplasmic reticulum membrane. UniProt carries this location for the zebrafish protein only as a similarity transfer from human FLVCR2 (ECO:0000250), and it is the SubCell keyword SL-0097 that generates the IEA row. No zebrafish imaging or fractionation supports it, and the compartment where the protein's transport activity is measured is the plasma membrane. Reason: Kept, because a multi-pass MFS protein can legitimately be detected in the ER and the donor record asserts it; but this is not where the reviewed activity happens. The functional site for choline transport is the plasma membrane, where the human protein is imaged and where uptake and efflux are measured, so the ER location is retained as non-core context rather than as part of the core function. Supporting Evidence: file:DANRE/flvcr2a/flvcr2a-uniprot.txt Endoplasmic reticulum membrane file:DANRE/flvcr2a/flvcr2a-uniprot.txt Multi-pass membrane protein PMID:38778100 Confocal imaging shows that FLVCR1 and FLVCR2 are localized at the plasma membrane PMID:38302740 our results show that MFSD7c facilitates the transport of choline and it behaves like a channel for the movement of choline via the plasma membrane |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: GO:0005886 places flvcr2a in the plasma membrane. This is the best-supported location for the protein family: confocal imaging localizes human FLVCR1 and FLVCR2 to the plasma membrane, the mammalian ortholog is documented in the plasma membrane of CNS endothelium, choline flux is measured across the plasma membrane of transfected cells, and a transport-dead disease mutant still reaches the plasma membrane normally, showing that the assay depends on plasma-membrane delivery. Reason: Accepted as the functional site. The zebrafish protein's own demonstrated activity, choline uptake in transfected cells, requires it to be in the plasma membrane, and the orthologs are directly imaged there (PMID:38778100, PMID:38302740). Supporting Evidence: PMID:38778100 Confocal imaging shows that FLVCR1 and FLVCR2 are localized at the plasma membrane PMID:38302740 The reduced import of choline of the mutant was not due to reduced protein expression levels nor defective localization in the plasma membrane PMID:38302740 Besides its localization in the mitochondria, this study and our results also showed that MFSD7c was expressed in the plasma membrane PMID:38302740 our results show that MFSD7c facilitates the transport of choline and it behaves like a channel for the movement of choline via the plasma membrane file:DANRE/flvcr2a/flvcr2a-uniprot.txt Cell membrane |
| GO:0015220 choline transmembrane transporter activity | IEA GO_REF:0000117 | ACCEPT | Summary: GO:0015220 claims flvcr2a can translocate choline across a membrane. This is the activity the zebrafish protein itself was tested for: both zebrafish Mfsd7c isoforms (DaMfsd7c_a, DaMfsd7c_b), along with medaka and frog orthologs, exhibited choline transport activity in the same heterologous uptake assay used to deorphanize the human protein. Mechanistically the transport is facilitative and uncoupled: substituting lithium for sodium does not affect it, it proceeds across a broad pH range, it is electrogenic, and it reverses when the choline gradient is inverted. This row is the ARBA machine-learning prediction of the same term. Reason: Core molecular function, with species-specific experimental support rather than inference alone. UniProt records the reaction choline(out) = choline(in) with experimental evidence from the same study, and structural work on the human ortholog identifies the binding site that makes choline the substrate (a conserved tryptophan forming a cation-pi interaction with the quaternary ammonium group). The predicted term coincides with the experimentally demonstrated activity, so the prediction is correct and adds nothing to be corrected. Supporting Evidence: PMID:38302740 We tested the choline import function for several MFSD7c orthologs including the ones from zebrafish (DaMfsd7c_a and DaMfsd7c_b isoforms), medaka fish (MeMfsd7c), and frog (XeMfsd7c). Consistently, these MFSD7c orthologs also exhibited choline transport activity file:DANRE/flvcr2a/flvcr2a-uniprot.txt Reaction=choline(out) = choline(in) file:DANRE/flvcr2a/flvcr2a-uniprot.txt Choline uniporter that specifically mediates choline uptake PMID:38302740 Replacement of sodium with lithium did not affect choline import by MFSD7c PMID:38302740 These results show that the import of choline by MFSD7c is electrogenic. |
| GO:0015871 choline transport | IEA GO_REF:0000108 | ACCEPT | Summary: GO:0015871 is the process counterpart of the choline transporter activity, derived by inter-ontology inference from GO:0015220. The zebrafish protein was directly shown to transport choline in a heterologous uptake assay, and in the mammalian ortholog the process is demonstrated in vivo: endothelial-specific deletion significantly reduces brain uptake of intravenously injected radiolabelled choline. Reason: The process term follows correctly from an activity that is experimentally demonstrated for this gene product, and in vivo evidence in the mammalian ortholog shows the activity is used for choline movement rather than being an artefact of overexpression. Supporting Evidence: PMID:38302740 We tested the choline import function for several MFSD7c orthologs including the ones from zebrafish (DaMfsd7c_a and DaMfsd7c_b isoforms), medaka fish (MeMfsd7c), and frog (XeMfsd7c). Consistently, these MFSD7c orthologs also exhibited choline transport activity PMID:38302740 There was a significant reduction of radioactive signals in the brain of EcMfsd7c-KO mice compared to controls. file:DANRE/flvcr2a/flvcr2a-uniprot.txt Reaction=choline(out) = choline(in) file:DANRE/flvcr2a/flvcr2a-uniprot.txt Choline uniporter that specifically mediates choline uptake |
| GO:0022857 transmembrane transporter activity | IEA GO_REF:0000002 | MODIFY | Summary: GO:0022857 is the InterPro2GO mapping from the generic MFS signatures IPR011701 and IPR020846. It asserts only that flvcr2a is a transmembrane transporter. True, but the substrate is known: the zebrafish protein transports choline in uptake assays, and the human ortholog's structure shows a choline-selective binding site. Reason: A specific child within the same aspect is experimentally supported for this gene product, so the domain-level parent under-specifies what is known. The mapping itself is not wrong; it is simply superseded by the IDA row for choline transmembrane transporter activity. Proposed replacements: choline transmembrane transporter activity Supporting Evidence: PMID:38302740 We tested the choline import function for several MFSD7c orthologs including the ones from zebrafish (DaMfsd7c_a and DaMfsd7c_b isoforms), medaka fish (MeMfsd7c), and frog (XeMfsd7c). Consistently, these MFSD7c orthologs also exhibited choline transport activity file:DANRE/flvcr2a/flvcr2a-uniprot.txt Reaction=choline(out) = choline(in) PMID:38778100 key conserved tryptophan residue (W125FLVCR1/W102FLVCR2) forms a cationβΟ interaction with the ammonium and amine groups of the respective ligands. PMID:38778100 Our findings suggest that both FLVCR1 and FLVCR2 operate as uniporters, facilitating downhill ligand transport independent of sodium or pH gradients. |
| GO:0031966 mitochondrial membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: GO:0031966 places flvcr2a in the mitochondrial membrane. For the mammalian ortholog this is a reported location, referenced in the deorphanization study alongside its plasma-membrane pool, and it reaches the zebrafish entry as a similarity transfer from mouse Flvcr2 that in turn drives the SubCell keyword SL-0171. No zebrafish evidence exists, and no transport activity has been assigned to the mitochondrial pool. Reason: Kept as a genuine but secondary location. The literature supports a mitochondrial pool of the mammalian protein, so the annotation is not wrong, but every measured transport event for this protein family, including the assay on the zebrafish protein, is across the plasma membrane. Non-core. Supporting Evidence: file:DANRE/flvcr2a/flvcr2a-uniprot.txt Mitochondrion membrane file:DANRE/flvcr2a/flvcr2a-uniprot.txt Multi-pass membrane protein PMID:38302740 Besides its localization in the mitochondria, this study and our results also showed that MFSD7c was expressed in the plasma membrane PMID:38302740 our results show that MFSD7c facilitates the transport of choline and it behaves like a channel for the movement of choline via the plasma membrane |
| GO:0034229 ethanolamine transport | IEA GO_REF:0000108 | KEEP AS NON CORE | Summary: GO:0034229 is the process counterpart of ethanolamine transmembrane transporter activity, derived by inter-ontology inference from GO:0034228. Ethanolamine handling by this transporter family is real but weaker and more conditional than choline transport. Structural and transport work on the human orthologs concludes that choline and ethanolamine are the primary substrates of both FLVCR1 and FLVCR2, and mutating the shared binding-site glutamine of FLVCR2 abolishes transport of both solutes. In the MFSD7c-focused assays, however, ethanolamine uptake was not increased by the transporter alone and only became significant when ethanolamine kinase ETNK1 was co-expressed to trap the substrate intracellularly. No zebrafish ethanolamine data exist; the annotation reaches flvcr2a by similarity to human FLVCR2. Reason: Follows the action on the molecular-function row it is inferred from. The transported-solute claim is supported for the human ortholog but has no zebrafish evidence and no demonstrated physiological role, so it is kept as secondary substrate context. Supporting Evidence: PMID:38778100 we conclude that choline and ethanolamine represent primary transport substrates of both FLVCR1 and FLVCR2 PMID:38778100 Ethanolamine transport activities of human FLVCR1 and FLVCR2 PMID:38302740 transport of ethanolamine by MFSD7c was increased to a significant level when ETNK1 was co-expressed file:DANRE/flvcr2a/flvcr2a-uniprot.txt Reaction=ethanolamine(in) = ethanolamine(out) |
| GO:0055085 transmembrane transport | IEA GO_REF:0000002 | MODIFY | Summary: GO:0055085 is the InterPro2GO process mapping from the MFS signature IPR011701, asserting only that flvcr2a participates in transmembrane transport. The specific process is choline transport, demonstrated for the zebrafish protein in vitro and for the mammalian ortholog in vivo. Reason: Same granularity argument as the corresponding molecular-function row: the generic domain-derived parent is correct but uninformative when the transported solute is established. Proposed replacements: choline transport Supporting Evidence: PMID:38302740 We tested the choline import function for several MFSD7c orthologs including the ones from zebrafish (DaMfsd7c_a and DaMfsd7c_b isoforms), medaka fish (MeMfsd7c), and frog (XeMfsd7c). Consistently, these MFSD7c orthologs also exhibited choline transport activity PMID:38302740 There was a significant reduction of radioactive signals in the brain of EcMfsd7c-KO mice compared to controls. file:DANRE/flvcr2a/flvcr2a-uniprot.txt Choline uniporter that specifically mediates choline uptake |
| GO:0005789 endoplasmic reticulum membrane | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: GO:0005789 places flvcr2a in the endoplasmic reticulum membrane. UniProt carries this location for the zebrafish protein only as a similarity transfer from human FLVCR2 (ECO:0000250), and it is the SubCell keyword SL-0097 that generates the IEA row. No zebrafish imaging or fractionation supports it, and the compartment where the protein's transport activity is measured is the plasma membrane. Reason: Kept, because a multi-pass MFS protein can legitimately be detected in the ER and the donor record asserts it; but this is not where the reviewed activity happens. The functional site for choline transport is the plasma membrane, where the human protein is imaged and where uptake and efflux are measured, so the ER location is retained as non-core context rather than as part of the core function. The ISS row makes the donor explicit (human FLVCR2, UniProtKB:Q9UPI3); it is the same claim as the SubCell-derived IEA row and is treated identically. Supporting Evidence: file:DANRE/flvcr2a/flvcr2a-uniprot.txt Endoplasmic reticulum membrane file:DANRE/flvcr2a/flvcr2a-uniprot.txt Multi-pass membrane protein PMID:38778100 Confocal imaging shows that FLVCR1 and FLVCR2 are localized at the plasma membrane PMID:38302740 our results show that MFSD7c facilitates the transport of choline and it behaves like a channel for the movement of choline via the plasma membrane |
| GO:0005886 plasma membrane | ISS GO_REF:0000024 | ACCEPT | Summary: GO:0005886 places flvcr2a in the plasma membrane. This is the best-supported location for the protein family: confocal imaging localizes human FLVCR1 and FLVCR2 to the plasma membrane, the mammalian ortholog is documented in the plasma membrane of CNS endothelium, choline flux is measured across the plasma membrane of transfected cells, and a transport-dead disease mutant still reaches the plasma membrane normally, showing that the assay depends on plasma-membrane delivery. Reason: Accepted as the functional site. The zebrafish protein's own demonstrated activity, choline uptake in transfected cells, requires it to be in the plasma membrane, and the orthologs are directly imaged there (PMID:38778100, PMID:38302740). The located_in ISS row transfers the location from mouse Flvcr2 (UniProtKB:Q91X85). Supporting Evidence: PMID:38778100 Confocal imaging shows that FLVCR1 and FLVCR2 are localized at the plasma membrane PMID:38302740 The reduced import of choline of the mutant was not due to reduced protein expression levels nor defective localization in the plasma membrane PMID:38302740 Besides its localization in the mitochondria, this study and our results also showed that MFSD7c was expressed in the plasma membrane PMID:38302740 our results show that MFSD7c facilitates the transport of choline and it behaves like a channel for the movement of choline via the plasma membrane file:DANRE/flvcr2a/flvcr2a-uniprot.txt Cell membrane |
| GO:0031966 mitochondrial membrane | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: GO:0031966 places flvcr2a in the mitochondrial membrane. For the mammalian ortholog this is a reported location, referenced in the deorphanization study alongside its plasma-membrane pool, and it reaches the zebrafish entry as a similarity transfer from mouse Flvcr2 that in turn drives the SubCell keyword SL-0171. No zebrafish evidence exists, and no transport activity has been assigned to the mitochondrial pool. Reason: Kept as a genuine but secondary location. The literature supports a mitochondrial pool of the mammalian protein, so the annotation is not wrong, but every measured transport event for this protein family, including the assay on the zebrafish protein, is across the plasma membrane. Non-core. The ISS row names mouse Flvcr2 (UniProtKB:Q91X85) as donor and is the same claim as the SubCell-derived IEA row. Supporting Evidence: file:DANRE/flvcr2a/flvcr2a-uniprot.txt Mitochondrion membrane file:DANRE/flvcr2a/flvcr2a-uniprot.txt Multi-pass membrane protein PMID:38302740 Besides its localization in the mitochondria, this study and our results also showed that MFSD7c was expressed in the plasma membrane PMID:38302740 our results show that MFSD7c facilitates the transport of choline and it behaves like a channel for the movement of choline via the plasma membrane |
| GO:0034228 ethanolamine transmembrane transporter activity | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: GO:0034228 claims flvcr2a can translocate ethanolamine. Ethanolamine handling by this transporter family is real but weaker and more conditional than choline transport. Structural and transport work on the human orthologs concludes that choline and ethanolamine are the primary substrates of both FLVCR1 and FLVCR2, and mutating the shared binding-site glutamine of FLVCR2 abolishes transport of both solutes. In the MFSD7c-focused assays, however, ethanolamine uptake was not increased by the transporter alone and only became significant when ethanolamine kinase ETNK1 was co-expressed to trap the substrate intracellularly. No zebrafish ethanolamine data exist; the annotation reaches flvcr2a by similarity to human FLVCR2. Reason: The capability is supported for the human donor and shares a binding site with choline transport, so it is retained; but for this gene product choline is the solute with direct species-level evidence and with an in vivo physiological role, and ethanolamine uptake by the FLVCR2 ortholog required a trapping kinase to be detectable. Non-core rather than accepted as a core activity. Supporting Evidence: PMID:38778100 we conclude that choline and ethanolamine represent primary transport substrates of both FLVCR1 and FLVCR2 PMID:38778100 Ethanolamine transport activities of human FLVCR1 and FLVCR2 PMID:38778100 alanine substitution of the analogous Q191FLVCR2 residue abolishes the transport of both ethanolamine and choline in FLVCR2 PMID:38302740 MFSD7c did not increase uptake of ethanolamine PMID:38302740 transport of ethanolamine by MFSD7c was increased to a significant level when ETNK1 was co-expressed file:DANRE/flvcr2a/flvcr2a-uniprot.txt Reaction=ethanolamine(in) = ethanolamine(out) |
| GO:0015220 choline transmembrane transporter activity | IDA PMID:38302740 MFSD7c functions as a transporter of choline at the blood-br... | ACCEPT | Summary: GO:0015220 claims flvcr2a can translocate choline across a membrane. This is the activity the zebrafish protein itself was tested for: both zebrafish Mfsd7c isoforms (DaMfsd7c_a, DaMfsd7c_b), along with medaka and frog orthologs, exhibited choline transport activity in the same heterologous uptake assay used to deorphanize the human protein. Mechanistically the transport is facilitative and uncoupled: substituting lithium for sodium does not affect it, it proceeds across a broad pH range, it is electrogenic, and it reverses when the choline gradient is inverted. Reason: Core molecular function, with species-specific experimental support rather than inference alone. UniProt records the reaction choline(out) = choline(in) with experimental evidence from the same study, and structural work on the human ortholog identifies the binding site that makes choline the substrate (a conserved tryptophan forming a cation-pi interaction with the quaternary ammonium group). Supporting Evidence: PMID:38302740 We tested the choline import function for several MFSD7c orthologs including the ones from zebrafish (DaMfsd7c_a and DaMfsd7c_b isoforms), medaka fish (MeMfsd7c), and frog (XeMfsd7c). Consistently, these MFSD7c orthologs also exhibited choline transport activity file:DANRE/flvcr2a/flvcr2a-uniprot.txt Reaction=choline(out) = choline(in) file:DANRE/flvcr2a/flvcr2a-uniprot.txt Choline uniporter that specifically mediates choline uptake PMID:38302740 Replacement of sodium with lithium did not affect choline import by MFSD7c PMID:38302740 These results show that the import of choline by MFSD7c is electrogenic. PMID:38778100 key conserved tryptophan residue (W125FLVCR1/W102FLVCR2) forms a cationβΟ interaction with the ammonium and amine groups of the respective ligands. PMID:38778100 Our findings suggest that both FLVCR1 and FLVCR2 operate as uniporters, facilitating downhill ligand transport independent of sodium or pH gradients. |
| GO:0150104 transport across blood-brain barrier | ISS GO_REF:0000024 | ACCEPT | Summary: GO:0150104 claims flvcr2a participates in moving a solute across the blood-brain barrier. The donor is mouse Flvcr2/Mfsd7c, which is expressed in blood-brain barrier endothelium and in CNS vascular endothelial cells, and whose tamoxifen-induced endothelial-specific deletion (Cdh5-CreERT2) significantly reduces brain uptake of intravenously injected radiolabelled choline while peripheral organ signals are unaffected. That is direct in vivo evidence that the transporter performs the barrier-crossing step itself, not merely that the barrier needs it. For zebrafish this remains a conservation-based inference: the zebrafish protein has the transport activity, but no in vivo zebrafish barrier flux has been measured. Reason: The gene product executes the transport step at the barrier in the donor species, satisfying the participation requirement, and the zebrafish protein has the same demonstrated choline-transport activity, making the similarity transfer well founded. Accepted as part of the core function, with the caveat that the zebrafish in vivo test has not been done. Supporting Evidence: PMID:38302740 There was a significant reduction of radioactive signals in the brain of EcMfsd7c-KO mice compared to controls. PMID:38302740 we generated conditional KO mice for Mfsd7c in the endothelial cells using Cdh5-CreERT2 PMID:38302740 MFSD7c was expressed in endothelial cells of the CNS vasculature PMID:32369449 We identified the mouse ortholog Mfsd7c as a gene expressed in the blood-brain barrier. PMID:38302740 We tested the choline import function for several MFSD7c orthologs including the ones from zebrafish (DaMfsd7c_a and DaMfsd7c_b isoforms), medaka fish (MeMfsd7c), and frog (XeMfsd7c). Consistently, these MFSD7c orthologs also exhibited choline transport activity file:DANRE/flvcr2a/flvcr2a-deep-research-falcon.md there is **no direct in vivo zebrafish mutant phenotype, tissue expression map, or subcellular localization imaging** for flvcr2a |
| GO:0005886 plasma membrane | ISS GO_REF:0000024 | ACCEPT | Summary: GO:0005886 places flvcr2a in the plasma membrane. This is the best-supported location for the protein family: confocal imaging localizes human FLVCR1 and FLVCR2 to the plasma membrane, the mammalian ortholog is documented in the plasma membrane of CNS endothelium, choline flux is measured across the plasma membrane of transfected cells, and a transport-dead disease mutant still reaches the plasma membrane normally, showing that the assay depends on plasma-membrane delivery. This row makes the stronger is_active_in claim: the plasma membrane is where the transport activity itself occurs. Reason: Accepted, and the is_active_in form is the more accurate of the two plasma-membrane rows: choline translocation by this protein is measured as movement across the plasma membrane, in both the import direction and, on inverting the gradient, the export direction. Supporting Evidence: PMID:38778100 Confocal imaging shows that FLVCR1 and FLVCR2 are localized at the plasma membrane PMID:38302740 our results show that MFSD7c facilitates the transport of choline and it behaves like a channel for the movement of choline via the plasma membrane PMID:38778100 indicating a bidirectional choline transport activity mediated by FLVCR2 PMID:38302740 The reduced import of choline of the mutant was not due to reduced protein expression levels nor defective localization in the plasma membrane |
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Download this section (compressed HTML)Q: Do the two zebrafish co-orthologs flvcr2a and flvcr2b divide the choline-transport role between tissues, and does either carry choline across the zebrafish blood-brain barrier in vivo as endothelial deletion of the mammalian ortholog shows for mouse?
Q: Is the mitochondrial and endoplasmic-reticulum pool of the protein transport-competent, and if so which solute does it move in those membranes?
Q: Does zebrafish flvcr2a transport ethanolamine, and does it require a trapping kinase to be detectable as the mammalian ortholog does?
Experiment: Express flvcr2a in HEK293 cells and measure radiolabelled choline uptake with sodium replaced by lithium and across a pH range, plus whole-cell patch clamp for choline-evoked current, and a washout assay to test bidirectionality.
Hypothesis: Zebrafish flvcr2a mediates facilitative, sodium-independent, electrogenic choline uptake.
Experiment: Generate flvcr2a mutants (and flvcr2a;flvcr2b double mutants to control for co-ortholog redundancy), inject deuterated choline into the circulation, and quantify labelled choline and choline-containing lipids in brain versus peripheral tissue by LC-MS/MS; score cerebral vascular patterning in parallel.
Hypothesis: flvcr2a supplies choline to the zebrafish brain across the vascular endothelium.
Experiment: Compare heme and bilirubin levels and heme-analog (ZnMP) uptake in flvcr2a mutant versus wild-type larvae, and test flvcr2a alongside a bona fide heme transporter in a side-by-side heme flux assay.
Hypothesis: flvcr2a does not perform heme transport in vivo despite the family-level annotations.
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