SET6 (systematic name YPL165C) is a 373-residue Saccharomyces cerevisiae protein belonging to the SET-domain (Su(var)3-9, Enhancer-of-zeste, Trithorax) class of the class V-like SAM-binding methyltransferase superfamily. It is one of the two budding-yeast members of the SMYD subfamily (its paralog being Set5) and is annotated as a putative protein-lysine methyltransferase. Its SET domain retains the invariant catalytic and S-adenosyl-L-methionine-binding residues that the catalytically dead yeast SET proteins Set3 and Set4 have lost, so SET6 is considered a likely catalytically competent enzyme, and it carries a cysteine-rich SMYD-type zinc-binding architecture. However, no methyltransferase activity, protein substrate, subcellular localization, or defined biological role has been experimentally established for SET6; the only reported phenotype is a drug-conditional fitness effect (resistance to compounds targeting ergosterol biosynthesis) from a genome-wide chemogenomic screen, whose molecular basis is unclear.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0016279 protein-lysine N-methyltransferase activity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Best-supported molecular function for SET6: a putative, likely catalytically competent protein-lysine methyltransferase. The SET/SMYD active site is intact (unlike Set3/Set4) and the literature singles out Set6 as the yeast SET protein most likely to be a genuine protein methyltransferase. Retained as the enzyme's likely function but kept non-core because no methyltransferase activity or substrate has been demonstrated. Reason: Phylogenetically inferred (IBA) but strongly corroborated. On sequence, SET6 retains the invariant catalytic/SAM-binding residues (NHSC pocket, catalytic Tyr337) that the catalytically dead Set3/Set4 have lost, and the literature explicitly calls Set6 the most likely genuine yeast protein methyltransferase. This lysine-KMT level (rather than the more specific histone-MTase term) is the defensible molecular function. It remains putative because no in-vitro/in-vivo activity or substrate has been shown, so it is kept as a non-core, likely-true function. Supporting Evidence: PMID:37252976 Of these three putative methyltransferases, the most likely protein methyltransferase is Set6, as the SET domain invariably methylates proteins. file:yeast/SET6/SET6-bioinformatics/RESULTS.md The invariant Asn-His of the "NHSC" AdoMet/catalytic motif is **conserved in SET6** (Asn303-His304-Ser305-Cys306), exactly as in the active enzymes Set1, Set2, Set5, SMYD2 and SMYD3. |
| GO:0042054 histone methyltransferase activity | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Over-specific substrate assignment. The defensible function for SET6 is protein-lysine methyltransferase activity (GO:0016279); no histone substrate has been demonstrated, and the SMYD subfamily to which Set6 belongs (and human SETD6) act largely on non-histone targets. The histone-specific term is an over-annotation. Reason: This IBA term over-specifies the substrate to histones. Set6 has no identified substrate of any kind, and the SMYD family it belongs to has well-documented non-histone targets, so restricting the predicted activity to histones is not supported. The parent-level protein-lysine methyltransferase activity (GO:0016279) already captures the family-inferred function without the unwarranted histone specificity. Prefer the generic KMT term over this one. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN008534456 · SMYD-family PANTHER node (PTHR12197) from which the IBA histone-MTase term was propagated SUPPORTS SOURCE BUT NOT TARGET Family node is a bona fide protein-lysine methyltransferase group, but the histone-specific substrate does not transfer to Set6, whose substrate is unknown and whose SMYD subfamily acts largely on non-histone targets. Proposed replacements: protein-lysine N-methyltransferase activity Supporting Evidence: PMID:31642774 Set5, and another member of this sub-group, Set6, are homologous to the mammalian SMYD proteins [6], which have been reported to have both histone and non-histone targets. PMID:37252976 If they are in fact protein methyltransferases, they are most likely to target as-yet undiscovered methylation sites |
| GO:0005634 nucleus | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Plausible location by SMYD-family phylogenetic transfer, but the subcellular localization of Set6 has never actually been measured, so this is a low-confidence, non-core annotation. Its paralog Set5 is found in both nucleus and cytoplasm. Reason: IBA family transfer to nucleus is reasonable for a SET/SMYD protein, but the localization of Set6 specifically is explicitly unknown, and its SMYD paralog Set5 is in fact dual nuclear/cytoplasmic, which would be consistent with non-histone substrates. Retain as a non-core, unverified location rather than a defining feature. Supporting Evidence: PMID:31642774 While the subcellular localization of Set6 is not yet known, Set5 is found in both the nucleus and cytoplasm [19], suggesting it is also likely to have non-histone substrates. |
| GO:0006338 chromatin remodeling | IEA GO_REF:0000108 | REMOVE | Summary: Automatic inter-ontology inference chained off the over-annotated histone methyltransferase term. There is no evidence that Set6 participates in chromatin remodeling; the biological process is unknown. Remove along with its over-specific parent MF term. Reason: This IEA annotation is derived by logical inference (GO_REF:0000108) from GO:0042054 histone methyltransferase activity (see the with/from field GO:0042054 in GOA), which is itself an over-annotation for SET6. Set6 has no demonstrated histone activity, no identified substrate, and no measured chromatin role; deletion has no standalone growth phenotype. The inference collapses once the histone-MTase premise is removed, so the chromatin-remodeling process assignment is unsupported and should be removed. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: GO:0042054 · histone methyltransferase activity (the over-annotated MF term this IEA process was logically inferred from) SOURCE WEAK OR INFERRED The inter-ontology IEA process is chained off an over-specific, itself-inferred MF term; it has no independent support for a chromatin role in Set6. Supporting Evidence: PMID:37252976 If they are in fact protein methyltransferases, they are most likely to target as-yet undiscovered methylation sites |
| GO:0008270 zinc ion binding | RCA PMID:30358795 The cellular economy of the Saccharomyces cerevisiae zinc pr... | KEEP AS NON CORE | Summary: Computational (zinc-proteome) prediction that SET6 binds zinc, independently corroborated by its cysteine-rich SMYD-type zinc-knot/post-SET architecture. A plausible structural molecular function; kept as non-core because it is a structural, not the defining, activity. Reason: The RCA annotation comes from a genome-scale zinc-proteome analysis. It is supported by the local bioinformatics finding that SET6 is markedly Cys-rich (17 Cys, 4.6%) in three clusters (N-terminal CxxC, an internal-insert cluster, and a C-terminal post-SET C-x-C...C) matching the expected SMYD-family zinc-binding architecture. This is a structural cofactor-binding function typical of SMYD SET domains rather than SET6's defining catalytic activity, so it is retained as non-core. Supporting Evidence: file:yeast/SET6/SET6-bioinformatics/RESULTS.md This clustered pattern is the expected SMYD-family zinc-knot / post-SET metal-binding architecture and is consistent with the computational zinc-ion-binding prediction (RCA, PMID:30358795). |
| GO:0005575 cellular_component | ND GO_REF:0000015 | ACCEPT | Summary: Root cellular_component term with the ND (No biological Data) evidence code, used as a placeholder indicating no localization data. Accept as-is per GO convention. Reason: This is a standard ND root-term placeholder recording that no cellular-component data is available; it is not an informative annotation to modify or remove. |
| GO:0008150 biological_process | ND GO_REF:0000015 | ACCEPT | Summary: Root biological_process term with the ND evidence code, a placeholder indicating no process data. Accept as-is per GO convention. Reason: Standard ND root-term placeholder recording that no biological-process data is available for SET6; not an informative annotation to modify or remove. |
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Download this section (compressed HTML)Q: Does SET6 catalyze protein-lysine methylation, and if so, what is its physiological protein substrate - a histone, or (as its SMYD family suggests) a non-histone target?
Suggested experts: Hamey JJ, Wilkins MR
Q: What is the subcellular localization of SET6, and does it (like its paralog Set5) distribute between the nucleus and cytoplasm, consistent with a non-histone substrate?
Suggested experts: Green EM
Experiment: Perform heavy-methyl SILAC quantitative mass spectrometry comparing the methylproteome of a wild-type strain with a set6-delta strain to identify SET6-dependent methylation site(s); confirm candidates with in-vitro methyltransferase assays using recombinant SET6, AdoMet, and active-site point mutants as negative controls.
Hypothesis: SET6 is a catalytically active protein-lysine methyltransferase whose substrate is a (likely non-histone) yeast protein.
Type: comparative methylproteomics + in-vitro methyltransferase assay
Experiment: Recombinantly express wild-type SET6 and NHSC-pocket / catalytic-Tyr337 point mutants and compare methyl-transfer activity against a candidate substrate panel to test whether the conserved residues are functionally required.
Hypothesis: SET6 retains intact catalytic residues, so active-site mutation should abolish any detectable methyltransferase activity.
Type: structure-function mutagenesis
Experiment: Endogenously tag SET6 with a fluorescent protein and image its subcellular distribution across growth conditions, including in the presence of ergosterol-biosynthesis inhibitors that produce its chemogenomic phenotype.
Hypothesis: SET6 localizes within the yeast cell in a pattern consistent with its (unknown) substrate compartment.
Type: fluorescence microscopy / localization
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The protein substrate of SET6 is unknown: no methylation site on any histone or non-histone protein has ever been attributed to SET6, so the specific methyl-acceptor it acts on is undetermined.
OPEN BIOLOGY MF_DARK
What is known: Established: SET6 is a SMYD-subfamily SET-domain protein whose catalytic/SAM-binding residues are intact (unlike the dead Set3/Set4), and it is the yeast SET protein most likely to be a genuine protein-lysine methyltransferase. Unknown: which protein (and which lysine) it methylates. If it is active, it most likely targets an as-yet undiscovered methylation site.
Significance: SET6 is one of only a handful of putative protein methyltransferases keeping the otherwise near-complete yeast protein-methylation enzyme-substrate network incomplete; identifying its substrate would help close that network.
What would resolve it: In-vitro methyltransferase assays with recombinant SET6 against candidate substrates and heavy-methyl SILAC / mass-spectrometry comparison of wild-type vs set6-delta methylproteomes to detect SET6-dependent methylation site(s).
Provenance (the field's own admissions):
Gap: Whether SET6 actually catalyzes methyl transfer in vitro or in vivo has never been demonstrated; its catalytic competence is inferred solely from conserved active-site residues.
OPEN BIOLOGY MF_DARK
What is known: Established: the SET-domain active-site residues required for catalysis and AdoMet binding are conserved in SET6, unlike in the catalytically dead Set3/Set4. Unknown: whether SET6 transfers a methyl group to any acceptor. Sequence conservation is necessary but not sufficient for enzymatic activity.
Significance: Confirming (or refuting) methyltransferase activity would resolve whether SET6 is a bona fide enzyme or, despite intact motifs, another inactive yeast SET protein.
What would resolve it: Direct enzymatic assay (radiometric or MS-based methyl-transfer) using recombinant SET6 and AdoMet, with active-site point mutants as controls.
Provenance (the field's own admissions):
Gap: The biological role and subcellular localization of SET6 are unknown: deletion produces no standalone growth phenotype, and where in the cell SET6 acts has not been measured.
OPEN BIOLOGY BP_DARK
What is known: Established: the only reported phenotype is drug-conditional fitness (resistance to ergosterol-biosynthesis-targeting compounds) in a chemogenomic screen, of uncertain mechanism, and SET6's SMYD paralog Set5 is dual nuclear/cytoplasmic. Unknown: the biological process SET6 participates in and its localization.
Significance: Without a biological process or localization, SET6 cannot be placed in a pathway; its drug-conditional phenotype hints at a role only indirectly.
What would resolve it: Fluorescent-protein tagging / microscopy to determine localization, and phenotypic profiling (including in the presence of ergosterol-biosynthesis inhibitors and in sensitized genetic backgrounds) to define a biological process.
Provenance (the field's own admissions):
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