TUSC3 (Tumor suppressor candidate 3; N33) is an endoplasmic-reticulum membrane protein that functions as an accessory oxidoreductase subunit of the STT3B-containing oligosaccharyltransferase (OST-B) complex. It is a multi-pass membrane protein whose lumenal, N-terminal thioredoxin-like domain carries a redox-active CXXC motif (Cys99-Cys102). Within OST-B, TUSC3 promotes N-linked glycosylation of a subset of asparagine acceptor sites that are near cysteine residues or that are otherwise skipped during co-translational glycosylation; in its oxidized form it is thought to form transient mixed disulfides with a free thiol in a glycoprotein substrate, delaying disulfide-bond formation and giving the catalytic STT3B subunit access to the unmodified sequon. TUSC3 is the close paralog of MAGT1, the two being mutually exclusive and functionally largely redundant subunits of OST-B, and is the metazoan orthologue of yeast Ost3/Ost6. Biallelic loss-of-function of TUSC3 causes autosomal-recessive non-syndromic intellectual disability. A role in cellular magnesium uptake has been proposed by analogy to MAGT1, but the established, mechanistically supported function is the ER OST-B oxidoreductase activity.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0006487 protein N-linked glycosylation | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (PANTHER) inference that TUSC3 is involved in protein N-linked glycosylation. This is the core biological process of TUSC3 as an OST-B subunit and is strongly supported by direct experimental evidence. Reason: Correct core BP for this gene. TUSC3 is an accessory subunit of the STT3B/OST-B complex that catalyzes N-linked glycosylation; UniProt states it is "Involved in N-glycosylation of STT3B-dependent substrates." A redundant IBA of the gene's own correct core function is retained, not marked over-annotated. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt Involved in N-glycosylation of STT3B-dependent |
| GO:0008250 oligosaccharyltransferase complex | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference that TUSC3 is part of the oligosaccharyltransferase complex. Correct; TUSC3 is an accessory subunit of the STT3B-containing OST complex. Reason: Well-supported core CC. A more specific term (GO:0160227 oligosaccharyltransferase complex B) exists and is used in core_functions, but the parent term is not wrong. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt Accessory component of the STT3B-containing form of the |
| GO:1903830 magnesium ion transmembrane transport | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phylogenetic inference of magnesium ion transmembrane transport. The WITH/FROM for this IBA is a Drosophila gene (FB:FBgn0032015) in a distinct PANTHER family (PTN000976171), not the OST3/OST6 subfamily that contains TUSC3. The established function of TUSC3 is the ER OST-B oxidoreductase, not Mg2+ transport. Reason: This IBA propagates a magnesium-transport role that is at best an indirect consequence of TUSC3's glycosylation function. The mechanistic literature shows TUSC3/MAGT1 are ER-resident OST oxidoreductase subunits, not plasma-membrane channels, and that a direct dual role is "incompatible" without invoking dual localization and dual activity. Not a core function. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: FB:FBgn0032015 Β· Drosophila source gene (PANTHER PTN000976171) SUPPORTS SOURCE BUT NOT TARGET The WITH/FROM seed for this IBA is a Drosophila gene in PANTHER family PTN000976171, distinct from the OST3/OST6 (PTN000294174) subfamily that contains TUSC3; a magnesium-transport role does not transfer to the human OST-B oxidoreductase subunit. Supporting Evidence: PMID:25135935 Direct roles for MagT1 in both N-linked glycosylation and magnesium uptake are incompatible unless one invokes both a dual localization (RER and plasma membrane) and a dual activity (oxidoreductase and Mg+2 channel) |
| GO:0005789 endoplasmic reticulum membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic mapping from the UniProtKB Subcellular Location vocabulary to the ER membrane. This is the correct localization of TUSC3. Reason: Correct core CC. UniProt SUBCELLULAR LOCATION is "Endoplasmic reticulum membrane; Multi-pass membrane protein." A redundant IEA of the gene's own correct location is retained. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt Endoplasmic reticulum membrane |
| GO:0006487 protein N-linked glycosylation | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning electronic annotation to protein N-linked glycosylation, the correct core BP for TUSC3. Reason: Redundant IEA of the gene's own correct core process; retained per policy. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt Involved in N-glycosylation of STT3B-dependent |
| GO:0008250 oligosaccharyltransferase complex | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA electronic annotation placing TUSC3 in the oligosaccharyltransferase complex. Correct core CC. Reason: Redundant IEA of the gene's own correct complex membership; retained. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt Accessory component of the STT3B-containing form of the |
| GO:0015095 magnesium ion transmembrane transporter activity | IEA GO_REF:0000117 | REMOVE | Summary: ARBA electronic annotation assigning magnesium ion transmembrane transporter activity as a molecular function. TUSC3 has no demonstrated channel/transporter activity; its established MF is a thioredoxin-like oxidoreductase activity, and any Mg2+ effect is indirect. Reason: This is a clearly-wrong electronic (IEA/ARBA) molecular-function assignment. TUSC3 is an ER OST-B oxidoreductase subunit; the Mg2+ transporter-activity MF conflicts with the mechanistic evidence and UniProt only states it "Could indirectly play a role in Mg(2+) transport." REMOVE is appropriate for an unsupported IEA MF (not applied to the experimental Mg2+ annotation, which is kept below). Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt a role in Mg(2+) transport |
| GO:0055085 transmembrane transport | TAS Reactome:R-HSA-5223345 | MARK AS OVER ANNOTATED | Summary: Reactome TAS to the generic "transmembrane transport" process, derived from the proposed magnesium-transport role. Uninformative and reflecting a role that is peripheral (at most indirect) to TUSC3's core OST function. Reason: Very general BP tied to the magnesium-transport hypothesis rather than the established OST-B oxidoreductase/glycosylation function. Not core. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt a role in Mg(2+) transport |
| GO:0005789 endoplasmic reticulum membrane | NAS PMID:31831667 Cryo-electron microscopy structures of human oligosaccharylt... | ACCEPT | Summary: ComplexPortal NAS localizing TUSC3 to the ER membrane, consistent with the cryo-EM analysis of human OST-A/OST-B and with UniProt. Correct core CC. Reason: Correct location supported by structural work on the human OST complexes and by UniProt SUBCELLULAR LOCATION. Supporting Evidence: PMID:31831667 Oligosaccharyltransferase (OST) catalyzes the transfer of a high-mannose glycan onto secretory proteins in the endoplasmic reticulum |
| GO:0006487 protein N-linked glycosylation | IDA PMID:31831667 Cryo-electron microscopy structures of human oligosaccharylt... | ACCEPT | Summary: ComplexPortal IDA (from the human OST-A/OST-B cryo-EM structures) that TUSC3, as an OST-B subunit, is involved in protein N-linked glycosylation. This is the core BP. Reason: Directly supported experimental annotation to the gene's core process. Supporting Evidence: PMID:31831667 Mammals express two distinct OST complexes that act in a cotranslational (OST-A) or posttranslocational (OST-B) manner |
| GO:0008250 oligosaccharyltransferase complex | IPI PMID:31831667 Cryo-electron microscopy structures of human oligosaccharylt... | ACCEPT | Summary: ComplexPortal IPI establishing TUSC3 as part of the oligosaccharyltransferase complex, based on the cryo-EM structure of human OST-B in which the MAGT1/TUSC3 subunit is resolved. Core CC. Reason: Physical-interaction evidence for OST complex membership; a more specific OST-B term is captured in core_functions. Supporting Evidence: PMID:31831667 structural differences in the catalytic subunits STT3A and STT3B facilitate contacts to distinct OST subunits, DC2 in OST-A and MAGT1 in OST-B |
| GO:0015693 magnesium ion transport | IMP PMID:19717468 Mammalian MagT1 and TUSC3 are required for cellular magnesiu... | KEEP AS NON CORE | Summary: Experimental (IMP) annotation from Zhou & Clapham 2009: knockdown of TUSC3 lowers intracellular Mg2+ and morpholino knockdown in zebrafish arrests development, rescuable with excess Mg2+. This supports a role of TUSC3 upstream of or within magnesium ion transport, though later work reinterprets this as an indirect consequence of the OST oxidoreductase function. Reason: Experimental annotation - not removed. However the acts_upstream_of_or_within qualifier is appropriately weak, and the established core function is the ER OST-B oxidoreductase; any magnesium-transport contribution is at most indirect (UniProt: "a role in Mg(2+) transport"). Retained as a non-core role. Supporting Evidence: PMID:19717468 Knockdown of either MagT1 or TUSC3 protein significantly lowers the total and free intracellular Mg(2+) concentrations in mammalian cell lines |
| GO:0006487 protein N-linked glycosylation | ISS GO_REF:0000024 | ACCEPT | Summary: Sequence-similarity (ISS) transfer of the protein N-linked glycosylation process to TUSC3. Correct core BP. Reason: Redundant ISS of the gene's own correct core process; retained. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt Involved in N-glycosylation of STT3B-dependent |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-446209 | ACCEPT | Summary: Reactome TAS localizing TUSC3 to the ER membrane in the asparagine N-linked glycosylation pathway. Correct core CC. Reason: Correct ER-membrane location, consistent with UniProt and structural data. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt Endoplasmic reticulum membrane |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-9694793 | ACCEPT | Summary: Reactome TAS (SARS-CoV-2 spike protein N-glycosylation pathway) placing TUSC3 at the ER membrane. Location is correct; the specific viral pathway context reflects OST acting on a viral glycoprotein substrate. Reason: Correct ER-membrane location. The viral-pathway framing is a substrate context, not a change to TUSC3's localization or function. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt Endoplasmic reticulum membrane |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-9918962 | ACCEPT | Summary: Reactome TAS (viral envelope E protein N-glycosylation) placing TUSC3 at the ER membrane. Correct location. Reason: Correct ER-membrane location; viral-substrate pathway context only. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt Endoplasmic reticulum membrane |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-9918988 | ACCEPT | Summary: Reactome TAS (viral pre-M protein N-glycosylation) placing TUSC3 at the ER membrane. Correct location. Reason: Correct ER-membrane location; viral-substrate pathway context only. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt Endoplasmic reticulum membrane |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-9919011 | ACCEPT | Summary: Reactome TAS (viral pre-NS1 folding and N-glycosylation) placing TUSC3 at the ER membrane. Correct location. Reason: Correct ER-membrane location; viral-substrate pathway context only. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt Endoplasmic reticulum membrane |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-9931286 | ACCEPT | Summary: Reactome TAS (CD274/PD-L1 N-linked glycosylation in ER) placing TUSC3 at the ER membrane. Correct location. Reason: Correct ER-membrane location; substrate-pathway context only. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt Endoplasmic reticulum membrane |
| GO:0015095 magnesium ion transmembrane transporter activity | TAS Reactome:R-HSA-5339528 | MARK AS OVER ANNOTATED | Summary: Reactome TAS asserting TUSC3 enables magnesium ion transmembrane transporter activity (in a reaction "TUSC3 transports Mg2+ from extracellular region to cytosol"). TUSC3 has no demonstrated intrinsic transporter/channel activity; its established MF is a thioredoxin-like oxidoreductase. Reason: Over-annotation of a molecular function that has not been demonstrated for TUSC3. As this is a TAS (not IEA) rooted in the Mg2+ hypothesis, it is marked over-annotated rather than removed. The established MF and the indirect nature of the Mg2+ role argue against a direct transporter activity. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt a role in Mg(2+) transport |
| GO:0006487 protein N-linked glycosylation | IMP PMID:26864433 Mammalian cells lacking either the cotranslational or posttr... | ACCEPT | Summary: Experimental IMP from CRISPR MagT1/TUSC3 double-knockout HEK293 cells: loss of both oxidoreductase subunits dramatically reduces glycosylation of STT3B-dependent substrates (e.g. SHBG, pCatC), demonstrating TUSC3 involvement in protein N-linked glycosylation. This is the strongest experimental evidence for the core BP. Reason: Direct genetic evidence (double-KO) for the gene's core process. Supporting Evidence: PMID:26864433 The absence of both oxidoreductase subunits (MagT1(β/β) TUSC3(β/β)), or elimination of STT3B, causes a dramatic reduction in SHBG glycosylation |
| GO:0005886 plasma membrane | TAS Reactome:R-HSA-5339528 | MARK AS OVER ANNOTATED | Summary: Reactome TAS placing TUSC3 in the plasma membrane, tied to the proposed Mg2+-transport reaction. This contradicts the established ER-membrane localization; TUSC3 is an ER OST subunit and shows minimal cell-surface exposure. Reason: Plasma-membrane localization is an over-annotation from the magnesium-transport hypothesis. Direct cell-surface biotinylation experiments and colocalization with calreticulin show the paralog MagT1 (and by homology TUSC3) is ER-resident, not plasma-membrane. Supporting Evidence: PMID:25135935 Colocalization of MagT1-V5 and calreticulin confirmed that MagT1 is an ER resident protein |
| GO:0005739 mitochondrion | ISS GO_REF:0000024 | REMOVE | Summary: ISS annotation to mitochondrion, transferred from a mouse ortholog (UniProtKB:Q8BTV1). There is no experimental support for mitochondrial localization of human TUSC3; its established location is the ER membrane. Reason: Clearly-wrong electronic/ISS localization. UniProt SUBCELLULAR LOCATION is ER membrane (multi-pass), and TUSC3 is an OST subunit; a mitochondrial location is inconsistent with all functional and structural evidence. Supporting Evidence: file:human/TUSC3/TUSC3-uniprot.txt Endoplasmic reticulum membrane |
| GO:0006487 protein N-linked glycosylation | NAS PMID:18455129 Oligosaccharyltransferase-subunit mutations in nonsyndromic ... | ACCEPT | Summary: NAS (Molinari et al. 2008) that TUSC3 encodes an OST subunit involved in N-glycosylation. Correct core BP; the paper established TUSC3 as an OTase subunit while showing bulk N-glycosylation was normal in patient fibroblasts (consistent with MAGT1 redundancy). Reason: Supports the core process. Non-experimental (NAS), but consistent with the strong experimental evidence for the same term. Supporting Evidence: PMID:18455129 the gene N33/TUSC3 encoding one subunit of the oligosaccharyltransferase (OTase) complex, which catalyzes the transfer of an oligosaccharide chain on nascent proteins, the key step of N-glycosylation |
| GO:0050890 cognition | IMP PMID:18455129 Oligosaccharyltransferase-subunit mutations in nonsyndromic ... | KEEP AS NON CORE | Summary: Experimental IMP (from human genetics) to "cognition": biallelic loss-of-function TUSC3 variants cause non-syndromic autosomal-recessive intellectual disability (MRT7), indicating TUSC3 is required for normal cognitive development. This is a downstream, tissue-level phenotype rather than a core molecular/cellular function. Reason: Experimental annotation grounded in disease genetics - not removed. The cognition phenotype is a consequence of impaired OST-B glycosylation in the developing nervous system, not itself a core function of the gene product. Kept as a non-core, pleiotropic/organismal role. Supporting Evidence: PMID:18455129 fine regulation of OTase activity is essential for normal cognitive-function development |
| GO:0005886 plasma membrane | NAS PMID:19717468 Mammalian MagT1 and TUSC3 are required for cellular magnesiu... | MARK AS OVER ANNOTATED | Summary: NAS plasma-membrane localization from Zhou & Clapham 2009, based on the proposed role of TUSC3 as a plasma-membrane Mg2+ transporter. Contradicted by the established ER-resident OST localization. Reason: Over-annotation stemming from the magnesium-transport hypothesis. Direct localization studies place the protein (and its paralog MagT1) in the ER, not the plasma membrane. Supporting Evidence: PMID:25135935 Colocalization of MagT1-V5 and calreticulin confirmed that MagT1 is an ER resident protein |
| GO:0008250 oligosaccharyltransferase complex | IDA PMID:15835887 Proteomic analysis of mammalian oligosaccharyltransferase re... | ACCEPT | Summary: HGNC-UCL IDA from the proteomic analysis of mammalian OST subcomplexes (Shibatani et al. 2005) placing TUSC3 in the oligosaccharyltransferase complex. Core CC supported by direct proteomic identification of OST subunits. Reason: Direct experimental (proteomics) evidence for OST complex membership. Core complex localization for TUSC3. Supporting Evidence: PMID:15835887 Our results identify two potential new subunits of mammalian OST and demonstrate a remarkable heterogeneity in OST composition |
| GO:0015035 protein-disulfide reductase activity | IDA PMID:25135935 Oxidoreductase activity is necessary for N-glycosylation of ... | NEW | Summary: Proposed new molecular-function annotation. TUSC3's lumenal thioredoxin domain (CXXC motif, Cys99-Cys102) is a redox-active protein-disulfide oxidoreductase: in OST-B it forms transient mixed disulfides with glycoprotein substrates and its CXXC motif is required for activity, as shown by TUSC3 fully complementing MagT1 in a CXXC-dependent manner. This core MF is not currently in GOA and should be added. Reason: Captures the established core molecular function of TUSC3 (thioredoxin-like protein-disulfide oxidoreductase), grounded in the CXXC mutagenesis/ complementation experiments and the crystal structure of the thioredoxin domain. Missing from the current GOA annotation set. Supporting Evidence: PMID:25135935 Mutants that lacked both active site cysteine residues (e.g., MagT1 m1) were not able to fully restore glycosylation of pCatCΞ234-HA, which indicates that the CXXC motif is necessary for MagT1 and TUSC3 activity file:human/TUSC3/TUSC3-uniprot.txt In its oxidized form proposed to form |
| GO:0160227 oligosaccharyltransferase complex B | IPI PMID:31831667 Cryo-electron microscopy structures of human oligosaccharylt... | NEW | Summary: Proposed new, more specific complex annotation. TUSC3 is an accessory subunit specifically of the STT3B-containing OST-B complex (mutually exclusive with MAGT1), as resolved in the cryo-EM structure of human OST-B and captured by ComplexPortal CPX-8738 ("Oligosaccharyltransferase complex B, TUSC3 variant"). This is more precise than the generic GO:0008250 already annotated. Reason: Adds the STT3B-specific OST-B complex term, which more precisely reflects TUSC3 membership than the parent GO:0008250. Not currently in GOA. Supporting Evidence: PMID:31831667 structural differences in the catalytic subunits STT3A and STT3B facilitate contacts to distinct OST subunits, DC2 in OST-A and MAGT1 in OST-B file:human/TUSC3/TUSC3-uniprot.txt Accessory component of the STT3B-containing form of the |
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Download this section (compressed HTML)Q: Does TUSC3 transport Mg2+ directly, or is the reduced cellular Mg2+ on TUSC3 knockdown an indirect consequence of hypoglycosylation of genuine Mg2+ transporters? The current GO:0015095 and GO:0005886 annotations depend entirely on the 2009 interpretation, which later ER-localization work does not support.
Suggested experts: Clapham DE, Zhou H, Gilmore R, Cherepanova NA
Q: TUSC3 and MAGT1 are functionally redundant in cultured cells, yet only TUSC3 loss causes autosomal-recessive intellectual disability. Which cell types express TUSC3 without MAGT1, and which glycoprotein substrates in those cells are TUSC3-dependent?
Suggested experts: Gilmore R, Cherepanova NA, Molinari F, Colleaux L
Q: The ISS annotation of TUSC3 to mitochondrion (GO:0005739) has no experimental support in human and conflicts with the ER-membrane localization established structurally. Is there any orthologue evidence that justifies it, or should it be removed?
Suggested experts: Glockshuber R, Mohorko E
Q: Does the peptide-binding groove adjacent to the TUSC3 active site confer a substrate preference distinct from that of MAGT1, which would explain why the two paralogues are not interchangeable in every tissue despite complementing in HEK293 cells?
Suggested experts: Glockshuber R, Mohorko E, Aebi M
Experiment: Reconstitute purified TUSC3 into proteoliposomes and assay Mg2+ flux directly with a fluorescent Mg2+ indicator, using a characterized Mg2+ channel (e.g. CorA or MRS2) as a positive control. In parallel, compare cellular Mg2+ in TUSC3-null cells rescued with wild-type TUSC3 versus a CXXC-motif mutant that is oxidoreductase-dead but structurally intact: if the Mg2+ phenotype is secondary to glycosylation, only the wild-type rescue should restore Mg2+ levels.
Hypothesis: TUSC3 has no intrinsic Mg2+ transport activity; the Mg2+ phenotype of TUSC3 loss is secondary to defective N-glycosylation of bona fide magnesium transporters.
Type: proteoliposome transport assay with separation-of-function rescue
Experiment: Profile TUSC3 and MAGT1 expression across human neural cell types (single-cell RNA-seq and targeted proteomics), then in TUSC3-null iPSC-derived neurons perform site-specific N-glycoproteomics to identify sequons losing occupancy, scoring each for proximity to disulfides and for internal-cysteine (NCT/S) context. Validate the top hits by rescue with wild-type versus CXXC-mutant TUSC3.
Hypothesis: TUSC3-dependent glycoproteins in neural tissue account for the intellectual-disability phenotype, and these substrates are cysteine-proximal sequons not covered by MAGT1 in cells where MAGT1 expression is low.
Type: cell-type-resolved glycoproteomics with rescue validation
Experiment: In MagT1/TUSC3 double-null HEK293 cells, rescue separately with TUSC3, MAGT1, and reciprocal groove-swap chimeras, then compare site-occupancy across the glycoproteome by quantitative glycoproteomics. Complement with in vitro mixed-disulfide trapping on a peptide library to derive each paralogue's sequence preference and test whether the chimeras track the donor groove.
Hypothesis: TUSC3 and MAGT1 have overlapping but non-identical substrate repertoires dictated by the peptide-binding groove flanking their active sites.
Type: paralogue swap rescue with glycoproteomics and peptide-library profiling
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