FIG4

Summary: FIG4 is assigned to the Golgi membrane as the phosphatase component of the PAS complex.

Reason: Reactome:R-HSA-1676005 explicitly places human FIG4-catalysed PI(3,5)P2-to-PI3P conversion at the Golgi membrane. Retain this curated location, with the Golgi pool secondary to the better-established endosomal function. The pathway assignment does not establish constitutive Golgi residence in every human cell type.

Summary: The chimeric PAS complex contains human FIG4 at the Golgi membrane.

Reason: The full Reactome entity graph for R-NUL-1675982 identifies a chimeric complex containing human FIG4 Q92562 and VAC14 with mouse Pikfyve. Its human FIG4 entity has the Golgi-membrane compartment. The top-level mouse species label therefore does not invalidate this human location assertion. Retain as a contextual, non-core pool; the kinase reaction belongs to Pikfyve.

Summary: PI3P phosphatase activity is supported by the rat Fig4 ortholog assay.

Reason: The Ensembl donors A6KTC8 and ENSRNOP00000000343 identify rat Fig4/Sac3. PMID:17909536 reports PI3P hydrolysis in vitro and loss of activity after a catalytic-core mutation. This is positive ortholog evidence for the existing transfer, although physiological PI3P consumption by human FIG4 is not established. Keep outside the core PI(3,5)P2 5-phosphatase function. The current donor GOA lacking its historical experimental rows is not evidence that the source experiment never existed.

Summary: FIG4 hydrolyses PI(4,5)P2 in vitro, with PI(3,5)P2 as the preferred substrate.

Reason: The experimental source is consistent with PMID:17556371, whose accessible abstract reports hydrolysis of the other D5-phosphorylated polyphosphoinositides as well as the preferred PI(3,5)P2 substrate. The reviewed human UniProt record explicitly identifies PI(4,5)P2. Retain this biochemical substrate activity as non-core; the full substrate panel was not independently inspected, and normal-cell PI(4,5)P2 flux is not inferred.

Summary: FIG4 hydrolyses PI(4,5)P2 in vitro, with PI(3,5)P2 as the preferred substrate.

Reason: The RHEA:22764/EC:3.1.3.36 mapping is consistent with PMID:17556371, whose accessible abstract reports hydrolysis of the other D5-phosphorylated polyphosphoinositides as well as the preferred PI(3,5)P2 substrate. The reviewed human UniProt record explicitly identifies PI(4,5)P2. Retain this biochemical substrate activity as non-core; the full substrate panel was not independently inspected, and normal-cell PI(4,5)P2 flux is not inferred.

Summary: FIG4 dephosphorylates PIKFYVE serines in the human PAS complex.

Reason: PMID:33098764 compares reconstituted human complexes containing wild-type FIG4 or catalytic mutant C486S. Mass spectrometry identifies FIG4-dependent loss of phosphorylation at PIKFYVE S48, S1669 and S2053, with S48/S2053 corroborated in complexes isolated from Expi293F cells. PIKFYVE S2053E reduces lipid-kinase activity. GO:0004722 explicitly includes protein serine phosphatase activity as a narrow synonym, so the demonstrated serine activity supports the existing term; they do not demonstrate a broad threonine-substrate repertoire or isolated-FIG4 peptide kinetics. The modified residues are on PIKFYVE, not FIG4.

Summary: FIG4 dephosphorylates PIKFYVE serines in the human PAS complex.

Reason: PMID:33098764 compares reconstituted human complexes containing wild-type FIG4 or catalytic mutant C486S. Mass spectrometry identifies FIG4-dependent loss of phosphorylation at PIKFYVE S48, S1669 and S2053, with S48/S2053 corroborated in complexes isolated from Expi293F cells. PIKFYVE S2053E reduces lipid-kinase activity. GO:0004722 explicitly includes protein serine phosphatase activity as a narrow synonym, so the demonstrated serine activity supports the existing term; they do not demonstrate a broad threonine-substrate repertoire or isolated-FIG4 peptide kinetics. The modified residues are on PIKFYVE, not FIG4.

Summary: FIG4 associates with human PIKFYVE in the PAS complex.

Reason: PMID:17556371 reports ternary association of Sac3/FIG4 with ArPIKfyve/VAC14 and PIKfyve, independently corroborated for human proteins by PMID:33098764. The source-specific human partner Q9Y2I7 is preserved. This association can be mediated by VAC14; it does not justify direct kinase binding or transfer of PIKFYVE kinase activity to FIG4. Retain supported generic binding as non-core under the campaign policy.

Summary: Human FIG4 associates with mouse Pikfyve in a mixed-species PAS complex.

Reason: The source partner Q9Z1T6 is mouse Pikfyve and remains explicitly cross-species. PMID:17556371 reports the mammalian ternary association; the follow-up Methods in PMID:18950639 specify human Sac3 and VAC14 with mouse PIKfyve constructs. Retain the interaction context without rewriting its partner as human or claiming an isolated direct binary interaction. Generic association is non-core and does not supply kinase activity.

Summary: FIG4 binds the VAC14 scaffold of the PAS complex.

Reason: PMID:19037259 Results and Figure 2B explicitly report a yeast two-hybrid interaction between human VAC14 and FIG4; VAC14 L156R retains FIG4 interaction while disrupting the PIKFYVE interaction. Independent human complex reconstitution and structural analysis in PMID:33098764 establish VAC14 as the scaffold and place FIG4 against two VAC14 subunits. Together these support GO:0097110 scaffold protein binding, a more informative description of the tested interaction. This assigns binding to a scaffold, not VAC14 scaffold activity to FIG4. The absence of a current IntAct pair does not negate the primary human experiment.

Proposed replacements: scaffold protein binding

Summary: FIG4 binds the VAC14 scaffold of the PAS complex.

Reason: The exact PMID:28514442 IntAct record EBI-21634069/IM-25778-3735 reports positive anti-tag coimmunoprecipitation of human FIG4 and VAC14. That screen establishes association, not an isolated binary interface. Independent human binary evidence in PMID:19037259 and human scaffold-complex architecture in PMID:33098764 establish the informative scaffold protein binding subtype. MODIFY integrates that independent mechanistic evidence without relabeling the original screen as a direct-binding assay or assigning VAC14 scaffold activity to FIG4.

Proposed replacements: scaffold protein binding

Summary: FIG4 binds the VAC14 scaffold of the PAS complex.

Reason: The exact human FIG4–VAC14 pair in PMID:32296183 is supported by IntAct records EBI-22337401, EBI-22795651 and EBI-24289640, covering prey-pooling, array and validated two-hybrid assays. These are three assay records within one study, not independent biological replications. Human VAC14 scaffold architecture and FIG4 contacts are independently established by PMID:33098764. The combination supports GO:0097110 scaffold protein binding rather than generic protein binding, without assigning the partner scaffold function to FIG4.

Proposed replacements: scaffold protein binding

Summary: Human FIG4 physically associates with GORASP2 in binary interaction assays.

Reason: PMID:32296183 is joined to the exact human FIG4–GORASP2 pair through IntAct records EBI-25196395, EBI-24163506 and EBI-24777480. The records cover prey-pooling, array and validated two-hybrid methods and are positive physical associations. These are assay records from the same study, not three independent biological replications. Retain the supported generic interaction as non-core; neither partner identity nor the interaction alone establishes an additional catalytic, scaffold or trafficking activity for FIG4.

Summary: FIG4 binds the VAC14 scaffold of the PAS complex.

Reason: PMID:33961781 has exact positive human FIG4–VAC14 records EBI-54558089, EBI-54901807 and EBI-55017599 detected by anti-tag coimmunoprecipitation. They establish cellular complex association, not necessarily direct binary contact. The independent human two-hybrid result in PMID:19037259 and scaffold-complex architecture in PMID:33098764 support the more informative GO:0097110 scaffold protein binding interpretation. Preserve the source-specific assay boundary and do not count the records as independent replications or transfer VAC14 scaffold activity to FIG4.

Proposed replacements: scaffold protein binding

Summary: Human FIG4 associates with PIKFYVE in the proteome-scale interaction data.

Reason: PMID:33961781 has exact positive human-pair IntAct records EBI-54729614 and EBI-55017599, detected by anti-tag coimmunoprecipitation. These support association within cellular protein complexes, not necessarily direct binary contact. The PAS association is independently supported by PMID:33098764. Keep generic binding as non-core; do not infer the partner's enzymatic or scaffold function, and do not count database records as independent replications.

Summary: The FIG4–PIKFYVE association is supported independently of the unresolved OpenCell table entry.

Reason: The fetched PMID:35271311 text describes the endogenous-tagging interaction resource but does not expose this exact pair, and the current IntAct query yielded no matching record. This limitation is retained rather than treated as negative evidence. Independent human PAS-complex purification and structural-biochemical work in PMID:33098764 establishes the same FIG4–PIKFYVE complex association. Retain the biological interaction as non-core on that independent evidence, without claiming the exact OpenCell assay was verified or that the proteins bind directly in isolation.

Summary: The broad phosphatidylinositol-biosynthesis assertion exceeds the established human pathway evidence.

Reason: GO:0006661 retains exact phosphoinositide-biosynthesis synonyms, but its definition names phosphatidylinositol and the ontology distinguishes phosphatidylinositol phosphate biosynthesis in GO:0046854. The latter and GO:0036092 have no is_a/part_of path to GO:0006661; missing ancestry alone is not proof of disjointness. More decisively, the established human core reaction removes the 5-phosphate from PI(3,5)P2 to form PI3P, not unphosphorylated PI. FIG4 also supports PIKFYVE activity, but neither observation establishes this broader PI-biosynthesis assertion. The rat donor substrate breadth retained elsewhere does not establish the human pathway-specific claim. Mark the incoming term as over-annotated while retaining the existing phosphatidylinositol-dephosphorylation annotations. ARBA:ARBA00028655 is an unvetted machine-learning association, assessed against the primary chemistry rather than treated as independent experimental support.

Summary: The broad phosphatidylinositol-biosynthesis assertion exceeds the established human pathway evidence.

Reason: GO:0006661 retains exact phosphoinositide-biosynthesis synonyms, but its definition names phosphatidylinositol and the ontology distinguishes phosphatidylinositol phosphate biosynthesis in GO:0046854. The latter and GO:0036092 have no is_a/part_of path to GO:0006661; missing ancestry alone is not proof of disjointness. More decisively, the established human core reaction removes the 5-phosphate from PI(3,5)P2 to form PI3P, not unphosphorylated PI. FIG4 also supports PIKFYVE activity, but neither observation establishes this broader PI-biosynthesis assertion. The rat donor substrate breadth retained elsewhere does not establish the human pathway-specific claim. Mark the incoming term as over-annotated while retaining the existing phosphatidylinositol-dephosphorylation annotations. The Reactome pathway includes phosphatases and kinases; its broad title does not identify the chemical step performed by FIG4.

Summary: The broad phosphatidylinositol-biosynthesis assertion exceeds the established human pathway evidence.

Reason: GO:0006661 retains exact phosphoinositide-biosynthesis synonyms, but its definition names phosphatidylinositol and the ontology distinguishes phosphatidylinositol phosphate biosynthesis in GO:0046854. The latter and GO:0036092 have no is_a/part_of path to GO:0006661; missing ancestry alone is not proof of disjointness. More decisively, the established human core reaction removes the 5-phosphate from PI(3,5)P2 to form PI3P, not unphosphorylated PI. FIG4 also supports PIKFYVE activity, but neither observation establishes this broader PI-biosynthesis assertion. The rat donor substrate breadth retained elsewhere does not establish the human pathway-specific claim. Mark the incoming term as over-annotated while retaining the existing phosphatidylinositol-dephosphorylation annotations. The Reactome pathway includes phosphatases and kinases; its broad title does not identify the chemical step performed by FIG4.

Summary: FIG4 functions at endosomal membranes as a PAS-complex component.

Reason: PMID:17556371 reports ectopically expressed Sac3 colocalizing with EEA1-positive endosomes and biochemical functions in early-endosome carrier formation; the human UniProt subcellular-location record states that localization requires VAC14 and PIKFYVE. Endosomal recruitment of the complex and FIG4 lipid catalysis are consistent with PMID:33098764. Retain this meaningful broader compartment, without inferring a transmembrane FIG4 topology or assigning membrane recruitment to an independent FIG4 targeting domain.

Summary: FIG4 functions at endosomal membranes as a PAS-complex component.

Reason: PMID:17556371 reports ectopically expressed Sac3 colocalizing with EEA1-positive endosomes and biochemical functions in early-endosome carrier formation; the human UniProt subcellular-location record states that localization requires VAC14 and PIKFYVE. Endosomal recruitment of the complex and FIG4 lipid catalysis are consistent with PMID:33098764. Retain this meaningful broader compartment, without inferring a transmembrane FIG4 topology or assigning membrane recruitment to an independent FIG4 targeting domain.

Summary: Endosome-membrane localization is consistent with FIG4 membership in the PIKFYVE complex.

Reason: This ComplexPortal-derived NAS row cites PMID:16954148. The inspected localization experiments concern GFP-PIKfyve in human HeLa cells and do not independently image FIG4. The broader FIG4 location is nevertheless directly supported by the Sac3 endosome experiments in PMID:17556371 and by its established PAS-complex membership. Retain the correct location with the source limitation explicit; do not relabel the NAS source as a FIG4-specific IDA experiment.

Summary: Rat Fig4 phosphatase activity promotes neurite outgrowth in differentiating PC12 cells.

Reason: The Ensembl donor is rat Fig4/Sac3. PMID:17909536 reports increased neurite outgrowth after rSac3 expression, reduced outgrowth after antisense inhibition, and loss of the promoting effect with a catalytic-core mutant during NGF-dependent PC12 differentiation. This provides positive catalytic-function-dependent evidence for the existing ortholog transfer, beyond a generic disease phenotype. Keep the contextual neuronal outcome non-core; the proposed detailed trafficking mechanism and an equivalent human-neuron effect were not demonstrated by the accessible abstract.

Summary: The generic phosphatase activity is entailed by FIG4’s established lipid and protein phosphatase activities.

Reason: InterPro:IPR002013 identifies the Sac phosphatase domain. The claim is correct but substantially less informative than the accepted PI(3,5)P2 5-phosphatase and protein-serine/threonine-phosphatase activities supported by human biochemical experiments. TOO_GENERAL concerns this term’s specificity, not a false domain assignment.

Summary: FIG4 catalyses PI(3,5)P2 dephosphorylation at the early endosome membrane.

Reason: Reactome:R-HSA-1676174 explicitly places the human FIG4 phosphatase component at the early endosome membrane in the PAS complex. This is consistent with the endosomal phosphoinositide interconversion mechanism and the broader Sac3 endosome experiments. Retain the curated compartment; the biochemical complex study does not by itself resolve all endogenous early-versus-late localization dynamics.

Summary: Human FIG4 is present in a chimeric PAS complex assigned to the early endosome membrane.

Reason: The actual Reactome:R-NUL-1675886 catalyst-to-complex-to-protein graph is explicitly chimeric and includes human FIG4 Q92562, human VAC14 and mouse Pikfyve. The FIG4 entity carries the early-endosome-membrane location matching this row. Thus the top-level mouse label does not constitute wrong-species annotation. Retain the location while keeping the reaction’s kinase activity on Pikfyve and avoiding an unsupported claim of direct endogenous human imaging.

Summary: FIG4 catalyses PI(3,5)P2 dephosphorylation at the late endosome membrane.

Reason: Reactome:R-HSA-1676020 explicitly places the human FIG4 phosphatase component at the late endosome membrane in the PAS complex. This is consistent with the endosomal phosphoinositide interconversion mechanism and the broader Sac3 endosome experiments. Retain the curated compartment; the biochemical complex study does not by itself resolve all endogenous early-versus-late localization dynamics.

Summary: Human FIG4 is present in a chimeric PAS complex assigned to the late endosome membrane.

Reason: The actual Reactome:R-NUL-1675925 catalyst-to-complex-to-protein graph is explicitly chimeric and includes human FIG4 Q92562, human VAC14 and mouse Pikfyve. The FIG4 entity carries the late-endosome-membrane location matching this row. Thus the top-level mouse label does not constitute wrong-species annotation. Retain the location while keeping the reaction’s kinase activity on Pikfyve and avoiding an unsupported claim of direct endogenous human imaging.

Summary: Human FIG4 is present in a chimeric PAS complex assigned to the late endosome membrane.

Reason: The actual Reactome:R-NUL-1676051 catalyst-to-complex-to-protein graph is explicitly chimeric and includes human FIG4 Q92562, human VAC14 and mouse Pikfyve. The FIG4 entity carries the late-endosome-membrane location matching this row. Thus the top-level mouse label does not constitute wrong-species annotation. Retain the location while keeping the reaction’s kinase activity on Pikfyve and avoiding an unsupported claim of direct endogenous human imaging.

Summary: FIG4 can dephosphorylate PI(3,4,5)P3 at position 5 in vitro.

Reason: PMID:17556371 reports activity against D5-phosphorylated polyphosphoinositides, with preference for PI(3,5)P2; the human UniProt catalytic-activity record explicitly identifies PI(3,4,5)P3 and RHEA:25528. Retain the experimental annotation and its electronic mapping as supported non-core biochemical substrate breadth. The abstract does not establish that FIG4 is a principal physiological PIP3-turnover enzyme or that it substitutes for other phosphatases in growth-factor signaling.

Summary: FIG4 can dephosphorylate PI(3,4,5)P3 at position 5 in vitro.

Reason: PMID:17556371 reports activity against D5-phosphorylated polyphosphoinositides, with preference for PI(3,5)P2; the human UniProt catalytic-activity record explicitly identifies PI(3,4,5)P3 and RHEA:25528. Retain the experimental annotation and its electronic mapping as supported non-core biochemical substrate breadth. The abstract does not establish that FIG4 is a principal physiological PIP3-turnover enzyme or that it substitutes for other phosphatases in growth-factor signaling.

Summary: Bisphosphoinositide phosphatase activity is correct but less specific than the accepted PI(3,5)P2 5-phosphatase activity.

Reason: The rat donor PMID:17909536 directly reports PI(3,5)P2 hydrolysis, and human FIG4 studies independently establish the same substrate class and the 5-phosphate reaction. Retain the biological validity of the Ensembl transfer while marking the broad activity term TOO_GENERAL because GO:0043813 already specifies FIG4’s core substrate and reaction. This is not a rejection based on donor species or missing current donor annotations.

Summary: PI3P is a direct product of FIG4 catalysis, retained as a non-core process annotation.

Reason: GO:0043813 removes the 5-phosphate from PI(3,5)P2 and produces PI3P. The current ontology explicitly places this activity part_of GO:0036092, so the GO_REF:0000108 inference is valid chemical-product formation, not an inference from knockout necessity. The merged yeast FIG4 review questions the annotation because VPS34 supplies bulk PI3P; that flux argument does not invalidate the reaction or the explicit GO relationship. Retain this human annotation as non-core to distinguish the product-based process from the defining physiological role in PI(3,5)P2 turnover. No claim is made that FIG4 supplies most cellular PI3P or determines its net steady-state concentration.

Summary: PI4P phosphatase activity is reported for rat Fig4/Sac3 in vitro.

Reason: The source identifiers resolve to the rat Fig4 ortholog, and PMID:17909536 explicitly reports PI4P hydrolysis with loss of activity after a catalytic-core mutation. This supplies positive biochemical evidence for the existing Ensembl transfer, despite the lack of those historical experimental rows in the current donor GOA response. Retain as non-core ortholog-derived substrate breadth; physiological PI4P consumption by human FIG4 is not established by that rat assay.

Summary: FIG4 removes the 5-phosphate from PI(3,5)P2 to form PI3P.

Reason: This is FIG4’s established lipid-phosphatase reaction. PMID:17556371 reports preferential PI(3,5)P2 hydrolysis, and PMID:33098764 directly assays soluble diC8-PI(3,5)P2 turnover by the human complex. Robust activity in the latter assay requires the ternary complex with active PIKFYVE; it is not evidence that isolated FIG4 has identical activity. The reaction and its curated endosomal or RHEA/InterPro mapping are supported without transferring PIKFYVE kinase activity to FIG4.

Summary: FIG4 removes the 5-phosphate from PI(3,5)P2 to form PI3P.

Reason: This is FIG4’s established lipid-phosphatase reaction. PMID:17556371 reports preferential PI(3,5)P2 hydrolysis, and PMID:33098764 directly assays soluble diC8-PI(3,5)P2 turnover by the human complex. Robust activity in the latter assay requires the ternary complex with active PIKFYVE; it is not evidence that isolated FIG4 has identical activity. The reaction and its curated endosomal or RHEA/InterPro mapping are supported without transferring PIKFYVE kinase activity to FIG4.

Summary: The ancestral PI(3,5)P2 5-phosphatase assertion is inherited by human FIG4.

Reason: The complete official PTHR45738 topology places PTN000127900 on the verified human Q92562 path. The original IBD.gaf contains the matching positive GO:0043813 assertion at that node, recovered with the repository parser and serializer. Human Q92562 occurs among its experimental seeds; this is expected target grounding, not circular transfer. Direct human FIG4 biochemistry independently agrees. No claim depends on the number of donor entries or on an inferred family name.

Summary: FIG4 removes the 5-phosphate from PI(3,5)P2 to form PI3P.

Reason: This is FIG4’s established lipid-phosphatase reaction. PMID:17556371 reports preferential PI(3,5)P2 hydrolysis, and PMID:33098764 directly assays soluble diC8-PI(3,5)P2 turnover by the human complex. Robust activity in the latter assay requires the ternary complex with active PIKFYVE; it is not evidence that isolated FIG4 has identical activity. The reaction and its curated endosomal or RHEA/InterPro mapping are supported without transferring PIKFYVE kinase activity to FIG4.

Summary: FIG4 removes the 5-phosphate from PI(3,5)P2 to form PI3P.

Reason: This is FIG4’s established lipid-phosphatase reaction. PMID:17556371 reports preferential PI(3,5)P2 hydrolysis, and PMID:33098764 directly assays soluble diC8-PI(3,5)P2 turnover by the human complex. Robust activity in the latter assay requires the ternary complex with active PIKFYVE; it is not evidence that isolated FIG4 has identical activity. The reaction and its curated endosomal or RHEA/InterPro mapping are supported without transferring PIKFYVE kinase activity to FIG4.

Summary: FIG4 removes the 5-phosphate from PI(3,5)P2 to form PI3P.

Reason: This is FIG4’s established lipid-phosphatase reaction. PMID:17556371 reports preferential PI(3,5)P2 hydrolysis, and PMID:33098764 directly assays soluble diC8-PI(3,5)P2 turnover by the human complex. Robust activity in the latter assay requires the ternary complex with active PIKFYVE; it is not evidence that isolated FIG4 has identical activity. The reaction and its curated endosomal or RHEA/InterPro mapping are supported without transferring PIKFYVE kinase activity to FIG4.

Summary: Human FIG4 inherits a supported phosphatidylinositol-dephosphorylation function.

Reason: The original positive GO:0046856 IBD assertion is at PTN000127900, verified on the human FIG4 path in the complete official family topology. Its SGD:S000005269 experimental seed supports the curator’s ancestral placement; a single seed is not intrinsically weak evidence. Human PI(3,5)P2-to-PI3P catalysis independently confirms participation in phosphoinositide dephosphorylation. The third cellular-component IBD in the family slice is not being manufactured into a new annotation.

Summary: FIG4 directly dephosphorylates phosphoinositides.

Reason: The InterPro:IPR043573 mapping agrees with demonstrated human PI(3,5)P2 5-phosphatase activity. FIG4 itself performs the dephosphorylation chemistry, so the process attribution is grounded in catalysis rather than necessity alone. The broader process remains a useful description of its lipid function alongside the substrate-specific molecular activity.

Summary: Rat Fig4/Sac3 localization supports a recycling-endosome pool.

Reason: PMID:17909536 explicitly reports rSac3 in recycling endosomes, as well as Golgi and endoplasmic-reticulum compartments. The Ensembl source identifiers resolve to that rat ortholog. Retain this positive location transfer as non-core, with the species and accessible-abstract limits explicit; it does not establish a dominant recycling-endosome pool in all human tissues. Do not add the other donor compartments as new human annotations.

Summary: FIG4 is the phosphatase subunit of the PIKFYVE–FIG4–VAC14 PAS complex.

Reason: PMID:33098764 characterizes human complexes containing one PIKFYVE, one FIG4 and five VAC14 molecules in the reported preparations. FIG4 supplies lipid and protein phosphatase activities, while VAC14 provides the pentameric scaffold. Tagged expression, purification, structural analysis and activity comparisons directly establish complex membership. The observed stoichiometry is not asserted to be invariant in every cellular state.

Summary: FIG4 contributes both catalysis and complex stability to PI(3,5)P2 metabolism.

Reason: PMID:18950639 uses human Sac3/FIG4 and VAC14 with mouse PIKfyve in COS7 constructs, as well as endogenous human HEK293 depletion. Sac3 loss reduces PIKfyve–VAC14 coassociation, and Sac3 coexpression promotes complex assembly. These experiments support FIG4 participation in the machinery for PI(3,5)P2 metabolism; the measured kinase reaction is PIKfyve’s. Independent human-complex studies establish FIG4’s own lipid and protein phosphatase activities. VAC14, not FIG4, is the principal scaffold.