VTC-4 (NCU08110) is the Neurospora crassa ortholog of the catalytic subunit Vtc4 of the vacuolar transporter chaperone (VTC) complex, the membrane-integral polyphosphate polymerase of fungal vacuoles. The 771-residue protein has an N-terminal SPX domain, a central VTC catalytic tunnel domain and three C-terminal transmembrane helices. In characterized yeast Vtc4 the central domain transfers the ATP gamma-phosphate onto a growing inorganic polyphosphate (polyP) chain, and synthesis is coupled to translocation of the polymer through the complex's transmembrane channel into the vacuole lumen; the SPX domain binds inositol polyphosphates, which stimulate synthesis and link polyP storage to cellular phosphate status. VTC-4 retains every mapped catalytic and ATP-binding residue of yeast Vtc4, including the active-site lysine that the non-catalytic Vtc2/Vtc3 paralogs have lost. Fluorescently tagged VTC-4 localizes to vacuolar membranes in N. crassa hyphae, including prevacuolar compartments near the hyphal tip and the tubular and spherical vacuolar network. Yeast VTC also occurs in the endoplasmic reticulum and contributes to microautophagy, vacuole fusion and calmodulin binding; these are inferred for VTC-4 by orthology only.
Summary: Fungal-type vacuole membrane is supported both by the PAINT inference and by direct localization of tagged VTC-4 in N. crassa.
Reason: The IBA places VTC subunits on the fungal vacuole membrane from the yeast VTC experimental record. Target-specific microscopy (PMID:26453652) observed tagged VTC-4 with the V-ATPase subunit VMA-1 in prevacuolar compartments and the tubular vacuolar network. VTC-4 is a multi-pass protein (three predicted TM helices), so membrane residence is the expected state. This is the location of the core polymerase/translocase activity.
Summary: InsP6 binding by the Vtc4 SPX domain is well established structurally in the close Sordariomycete ortholog (Chaetomium thermophilum Vtc4, PDB 5IJP) and functionally in yeast; the target conserves the SPX basic binding motif. Kept as a regulatory, non-core activity.
Reason: The Ensembl Compara transfer is from yeast Vtc4, which is in the same Vtc4 clade (PANTHER PTHR46140:SF1; NJ tree, RESULTS-rereview.md) as the target. The closest structurally characterized ortholog, C. thermophilum Vtc4 (80.5% identity to VTC-4), was crystallized with InsP6 bound to its SPX domain, and the yeast Vtc4 InsP-binding motif (residues 126-133) is conserved in VTC-4 (G129-K136). SPX-InsP binding regulates polyP synthesis (PMID:27080106) rather than being the catalytic function itself; InsP6 binding is real in vitro but inositol pyrophosphates are the more potent physiological activators, so this is kept as a non-core regulatory activity.
Supporting Evidence:
file:NEUCR/vtc-4/vtc-4-PDB-5IJP.json
Crystal structure of the SPX domain of Chaetomium thermophilum Vtc4 in complex with inositol hexakisphosphate (InsP6).
SPX domains--which are found in eukaryotic phosphate transporters, signaling proteins, and inorganic polyphosphate polymerases--provide a basic binding surface for inositol polyphosphate signaling molecules (InsPs)
Summary: The yeast Vtc4 central domain binds calmodulin in vitro; transfer to the N. crassa ortholog is reasonable but the binding is peripheral to polyP synthesis.
Reason: Direct binding of purified yeast Vtc2, Vtc3 and Vtc4 central domains to immobilized calmodulin (Cmd1) was shown in PMID:17079729. Because Vtc2 and Vtc3 central domains (PANTHER SF2) bind as well, the property is shared across the VTC core-domain paralogs and transfer to the Vtc4 ortholog VTC-4 is supported, but its physiological role is unclear and it is not part of the catalytic function. Not tested in N. crassa.
Supporting Evidence:
file:NEUCR/vtc-4/vtc-4-primary-excerpts.md
bound to Cmd1p-Sepharose in the presence and in absence of free Ca2+.
Summary: Correct but general; the fungal-type vacuole membrane child term is supported directly and already carried by the gene (IBA).
Reason: VTC-4 is a fungal protein observed on vacuolar membranes in N. crassa (PMID:26453652); the more specific is_a descendant fungal-type vacuole membrane (GO:0000329), already annotated by IBA, is preferred.
Summary: Yeast VTC complexes occur on the ER and cell periphery as well as vacuoles; the PAINT inference is accepted as a secondary location for the N. crassa ortholog.
Reason: The ER location in yeast is documented for the VTC complex (PMID:17079729), with the Vtc2-containing subcomplex enriched at the cell periphery/ER. The N. crassa experiment (PMID:26453652) documents vacuolar and prevacuolar membranes, and does not report or exclude an ER pool. The PAINT node placement is not contradicted by target-specific evidence, so the annotation is retained but is not where the core activity is documented in N. crassa.
The VTC complex is present on the ER and vacuoles and at the cell periphery. On induction of autophagy by nutrient limitation the VTC complex is recruited to and concentrated on vacuoles.
Summary: Correct but general; Vtc4 synthesizes polyP, so the is_a child polyphosphate biosynthetic process (already annotated by IBA and IEA) is the precise term.
Reason: The catalytic Vtc4 subunit polymerizes polyP from ATP (PMID:19390046); VTC-4 sits in the Vtc4 clade and retains the catalytic lysine and all mapped ATP-binding residues (RESULTS-rereview.md).
Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains.
Summary: Core process. The target is the N. crassa Vtc4 ortholog and conserves the catalytic machinery of the characterized yeast enzyme.
Reason: PAINT placed this term at node PTN001646574, which includes VTC subunits from yeasts and trypanosomes. For VTC-4 the inference is especially well grounded because the protein is in the Vtc4 clade (PANTHER PTHR46140:SF1, grouping with S. cerevisiae, S. pombe and C. thermophilum Vtc4, separate from the SF2 Vtc2/Vtc3 clade that contains the N. crassa paralog NCU01745) and retains the active-site lysine (K462, = yeast K458) and all seven mapped ATP-binding residues (RESULTS-rereview.md). The yeast enzyme makes polyP chains from ATP (PMID:19390046). Note that PANTHER SF1 also contains short Vtc1 and Vtc5 entries, so the subfamily label alone is not the boundary for the activity; the tree and residue conservation are.
Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains.
Summary: Same core process as the IBA row; consistent with ortholog transfer from yeast and fission yeast Vtc4.
Reason: The donors (S. cerevisiae Vtc4, S. pombe vtc4) are in the same Vtc4 clade as VTC-4 (RESULTS-rereview.md); the catalytic residues are conserved.
Summary: The VTC complex couples polyP synthesis to translocation of the polymer into the vacuole lumen, which is directed movement of a substance into the vacuole. No more specific GO term for polyphosphate import exists, so the term is kept.
Reason: GO:0007034 is defined as directed movement of substances into, out of or within a vacuole. In yeast, polyP made by VTC is imported into vacuoles and synthesis is obligatorily coupled to import through the VTC transmembrane domains (PMID:25315834; PMID:19390046). VTC-4 contributes one of the transmembrane modules of that channel and is the synthesizing subunit, so it performs part of the transport. No GO term for polyphosphate transmembrane transport was found (OLS search, 2026-10-21), so this broad term is as precise as available. Inference by orthology (same Vtc4 clade); not assayed in N. crassa.
Mutations in the VTC transmembrane regions, which are likely to constitute the translocation channel, block not only polyP translocation but also synthesis.
Summary: Core molecular function. GO:0008976 is the ATP + polyP(n) = ADP + polyP(n+1) activity performed by the Vtc4 catalytic domain; VTC-4 conserves the full catalytic site.
Reason: The PAINT donors are yeast Vtc4 (SGD:S000003549) and T. brucei vtc4, i.e. the catalytic Vtc4 members only; curators did not use the non-catalytic Vtc2/Vtc3 for this term, consistent with the loss of the catalytic lysine in those paralogs. VTC-4 groups with the Vtc4 orthologs and retains K462 (yeast active-site K458) plus the ATP-binding residues (RESULTS-rereview.md). The yeast enzyme transfers the ATP gamma-phosphate to a growing polyP chain (PMID:19390046).
Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains.
Summary: Same core activity as the IBA row; EC 2.7.4.1 mapping is correct for the Vtc4 polymerase.
Reason: EC 2.7.4.1 ([phosphate](n) + ATP = [phosphate](n+1) + ADP) is the reaction catalysed by yeast Vtc4 (PMID:19390046) and maps to GO:0008976; VTC-4 retains the catalytic residues.
Summary: The yeast VTC complex is a constituent of autophagic tubes and is required for scission of microautophagic vesicles; transfer to the N. crassa ortholog is kept as a secondary, non-core process.
Reason: PMID:17079729 shows that deleting VTC blocks microautophagic uptake at the vesicle scission step, and the complex is present on the autophagic tube membranes, so VTC acts at the membrane where the step occurs rather than being a distant requirement. The evidence is from yeast, by orthology; microautophagy has not been studied for VTC-4 in N. crassa, and whether the defect reflects loss of polyP synthesis or a separate membrane role is not resolved. Kept as non-core.
Deletion of the VTC complex blocks microautophagic uptake into vacuoles. The mutants still form autophagic tubes but the production of microautophagic vesicles from their tips is impaired.
Summary: VTC-4 is the Vtc4 ortholog and the complex's catalytic subunit; membership in the VTC complex is core.
Reason: In yeast the VTC polyP polymerase is a membrane-integral complex whose catalytic subunit is Vtc4 (PMID:19390046). N. crassa encodes the corresponding Vtc1-type (NCU03883) and Vtc2/3-type (NCU01745) partners, and VTC-4 has the same SPX/catalytic/three-TM architecture as yeast Vtc4, grouping with it phylogenetically (RESULTS-rereview.md). Complex composition has not been determined biochemically in N. crassa.
we identified a eukaryotic polyphosphate polymerase within the membrane-integral vacuolar transporter chaperone (VTC) complex
Core Functions
Catalytic subunit of the vacuolar VTC polyphosphate polymerase; transfers the ATP gamma-phosphate to a growing polyphosphate chain at the vacuolar membrane, coupled to polymer translocation into the vacuole lumen.
Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains.
Q: Does N. crassa VTC-4 form distinct subcomplexes with VTC-1 (NCU03883) and the single Vtc2/3-type paralog NCU01745, given that yeast has separate Vtc2 and Vtc3 subunits?
Suggested Experiments
Experiment: Measure polyP accumulation in a vtc-4 deletion and in a K462A active-site mutant of N. crassa to confirm that VTC-4 is the catalytic polyP polymerase in this fungus.
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
The single ProtNLM2 GO prediction (vacuolar membrane) is correct for the catalytic VTC polyphosphate polymerase subunit, but it is not novel and is less specific than the fungal-type vacuole membrane annotation the gene already carries.
Review rationale: Tagged VTC-4 co-localizes with the vacuolar ATPase subunit VMA-1 in prevacuolar compartments and the tubular vacuolar network of N. crassa hyphae (PMID:26453652 abstract; primary excerpts), which directly supports residence of this multi-pass VTC subunit on vacuolar membranes. The exact term is already in GOA for the gene (IEA), and the gene also carries its is_a descendant fungal-type vacuole membrane (GO:0000329, IBA), which the gene review accepts as the core location. The prediction is therefore correct but less specific than an existing supported annotation.
Supporting Evidence:
PMID:26453652: "Vacuolar membrane proteins, such as the vacuolar ATPase (VMA-1) and the polyphosphate polymerase (VTC-4), were observed in the PVCs."