LEE1 (YPL054W) is a 301-residue Saccharomyces cerevisiae protein whose only well-supported molecular property is the presence of two tandem CCCH-type (C3H1) zinc fingers, which coordinate zinc. CCCH zinc fingers are canonically nucleic-acid-binding (most often RNA-binding) modules, and LEE1 was independently identified in a proteome-wide survey as a predicted zinc-binding protein of the yeast zinc proteome. Beyond zinc coordination, its molecular activity, physiological substrate or binding target, and biological role remain uncharacterized; global GFP studies report a cytoplasmic localization. The protein is phosphorylated at several serine residues (Ser-21, Ser-30, Ser-282) and has a disordered, polar-residue-rich N-terminus. It is a non-essential protein; deletion is viable and yields only pleiotropic, high-throughput-screen phenotypes (including in a minisatellite-stability screen) with no defined pathway assignment. Its transcription is nitrogen-source responsive (a nitrogen catabolite repression target). LEE1 belongs to the makorin-related family (PANTHER PTHR11224 / InterPro MKRN-like) by sequence of its zinc fingers, but unlike the metazoan makorin E3 ubiquitin ligases it lacks the catalytic RING domain that those proteins use for ubiquitin transfer.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0061630 ubiquitin protein ligase activity | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phylogenetic (IBA) propagation of E3 ubiquitin ligase activity from the makorin family (PANTHER PTHR11224, node PTN000131854). The IBD seeds are metazoan makorins that carry a catalytic C3HC4 RING domain (human MKRN1 Q9UHC7, human MKRN3 Q13064, mouse Mkrn2 MGI:1914277). LEE1 possesses only the CCCH zinc fingers and has NO RING domain, which is the module that actually catalyzes ubiquitin transfer in makorins. Ubiquitin ligase activity is therefore an over-annotation for LEE1: the catalytic domain required for the function is absent, so this is a family/subfamily over-propagation of a catalytic activity to a member lacking the catalytic module. Reason: LEE1 lacks the RING (zf-C3HC4) domain that carries makorin E3 ligase catalysis; only CCCH zinc fingers are present (verified by direct InterPro query on Q02799, which returns no RING/IPR001841 signature). Zinc-finger membership in the makorin family does not confer the ligase activity, and there is no experimental evidence LEE1 is a ubiquitin ligase. Propagation Review Root cause: PROPAGATION BAD Failure modes: PSEUDO OR SUBACTIVITY LOSS WRONG ORTHOLOG OR PARALOG Sources checked: UniProtKB:Q9UHC7 · human MKRN1 SUPPORTS SOURCE BUT NOT TARGET RING-containing makorin E3 ligase; catalytic RING absent in LEE1 UniProtKB:Q13064 · human MKRN3 SUPPORTS SOURCE BUT NOT TARGET RING-containing makorin; LEE1 has no RING module MGI:MGI:1914277 · mouse Mkrn2 SUPPORTS SOURCE BUT NOT TARGET RING-containing makorin; ligase catalysis not transferable to LEE1 PANTHER:PTN000131854 · PTHR11224 makorin family node SUPPORTS SOURCE BUT NOT TARGET family node seeded by RING makorins; subfamily SF10 also contains RING-less members |
| GO:0016567 protein ubiquitination | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phylogenetic (IBA) propagation of the ubiquitination process that accompanies the makorin family ligase-activity assignment. It rests on the same makorin RING E3 ligase inference that does not apply to LEE1, because LEE1 lacks the catalytic RING domain. Without support for LEE1 acting as a ubiquitin ligase, participation in protein ubiquitination is an over-annotation. Reason: This process term is the biological-process shadow of the makorin ligase activity that does not transfer to LEE1 (no RING domain). No experimental evidence links LEE1 to protein ubiquitination. Propagation Review Root cause: PROPAGATION BAD Failure modes: PSEUDO OR SUBACTIVITY LOSS WRONG ORTHOLOG OR PARALOG Sources checked: PANTHER:PTN000131854 · PTHR11224 makorin family node SUPPORTS SOURCE BUT NOT TARGET process co-propagated with RING-dependent ligase activity absent in LEE1 |
| GO:0000209 protein polyubiquitination | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro-to-GO (IEA) mapping of protein polyubiquitination from the MKRN-like signature (IPR045072). The mapping reflects the makorin family's RING-dependent E3 ligase role. LEE1 matches the MKRN-like/CCCH signatures by its zinc fingers but lacks the RING domain, so the polyubiquitination process is not supportable for it and represents an over-annotation. Reason: IEA mapping derives from the makorin RING E3 ligase function; the RING catalytic domain is absent in LEE1, so polyubiquitination should not be inferred. No experimental support exists. Propagation Review Root cause: PROPAGATION BAD Failure modes: PSEUDO OR SUBACTIVITY LOSS Sources checked: InterPro:IPR045072 · MKRN-like SUPPORTS SOURCE BUT NOT TARGET family signature maps polyubiquitination via RING makorins; RING absent in LEE1 |
| GO:0046872 metal ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro-to-GO (IEA) mapping of metal ion binding from the CCCH zinc finger signatures (IPR000571, IPR036855). This is correct and domain-defensible: LEE1 has two CCCH-type zinc fingers that coordinate zinc. It is a generic parent of the more specific zinc ion binding, which is also annotated. Reason: LEE1's two CCCH zinc fingers coordinate a metal ion (zinc); the term is correct though generic. The more specific GO:0008270 (zinc ion binding) is the preferred core term. |
| GO:0061630 ubiquitin protein ligase activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro-to-GO (IEA) mapping of ubiquitin protein ligase activity from the MKRN-like signature (IPR045072). As with the IBA duplicate of this term, the activity depends on the makorin RING domain, which LEE1 does not have. This is an over-annotation for a RING-less family member. Reason: Same over-propagation as the IBA ligase-activity annotation, arriving via the InterPro MKRN-like signature. LEE1 lacks the catalytic RING domain, so the ligase activity is not supportable. Propagation Review Root cause: PROPAGATION BAD Failure modes: PSEUDO OR SUBACTIVITY LOSS WRONG ORTHOLOG OR PARALOG Sources checked: InterPro:IPR045072 · MKRN-like SUPPORTS SOURCE BUT NOT TARGET signature maps ligase activity from RING makorins; RING absent in LEE1 |
| GO:0008270 zinc ion binding | RCA PMID:30358795 The cellular economy of the Saccharomyces cerevisiae zinc pr... | ACCEPT | Summary: Reviewed computational analysis (RCA) assignment of zinc ion binding, sourced from the proteome-wide yeast zinc-proteome study that catalogued LEE1 among predicted zinc-binding proteins. This is fully consistent with LEE1's two CCCH-type (C3H1) zinc fingers (each coordinating Zn2+ via three Cys and one His). This is the best-supported molecular function of LEE1 and is retained as core. Reason: LEE1 has two CCCH-type zinc fingers (UniProt ZN_FING 87-114 and 123-145) and was independently classified as a zinc-binding protein in a proteome-wide zinc-proteome analysis. Zinc coordination is the best-supported molecular property of the protein. |
| GO:0005575 cellular_component | ND GO_REF:0000015 | ACCEPT | Summary: Root cellular_component term with the ND (No biological Data available) evidence code, correctly signalling that LEE1's subcellular localization is not known. This is the appropriate placeholder for an uncharacterized protein and should be retained until localization data exist. Reason: ND root term accurately records that no localization data are available for LEE1; standard practice is to keep it. |
| GO:0008150 biological_process | ND GO_REF:0000015 | ACCEPT | Summary: Root biological_process term with the ND evidence code, correctly signalling that LEE1's biological role is unknown. Existing deletion phenotypes are pleiotropic high-throughput readouts that do not define a pathway, so the ND placeholder remains appropriate. Reason: ND root term accurately records the absence of biological-process data; retained as the honest placeholder for a dark gene. |
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Download this section (compressed HTML)Q: Do the two CCCH zinc fingers of LEE1 bind RNA (or DNA) in vivo, and if so what are the target transcripts or sites?
Q: Given the absence of a RING domain, does LEE1 have any role in ubiquitination (e.g. as a substrate-recruiting adaptor), or is the makorin-family ubiquitin-ligase annotation a family-level over-annotation for this member?
Q: Where in the cell does LEE1 act, and which biological process explains its deletion/overexpression phenotypes (chemical resistance, competitive fitness, sporulation, invasive growth)?
Experiment: Perform UV-crosslinking RNA interactome capture / CLIP-seq (or RIP-seq) on tagged LEE1 to test for RNA binding and identify bound transcripts; complement with in vitro EMSA using recombinant LEE1 and candidate RNAs, and mutate the zinc-coordinating Cys/His residues to confirm zinc-finger dependence.
Hypothesis: LEE1 is an RNA-binding protein via its tandem CCCH zinc fingers.
Type: RNA-protein interaction mapping
Experiment: Reconstitute in vitro ubiquitination assays (E1, a panel of E2s, ubiquitin) with recombinant LEE1 and assay for autoubiquitination or substrate ubiquitination; in parallel, monitor bulk/target ubiquitin-conjugate levels in LEE1 deletion vs wild-type cells.
Hypothesis: LEE1 does not act as an E3 ubiquitin ligase because it lacks a RING domain.
Type: in vitro/in vivo ubiquitination assay
Experiment: Determine LEE1 subcellular localization by fluorescent tagging under native and stress conditions; validate top high-throughput interactors by co-immunoprecipitation; run condition-matched phenotypic assays to connect the screen phenotypes to a pathway.
Hypothesis: LEE1 localizes to a specific compartment consistent with a defined biological process.
Type: localization and interaction validation
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The molecular function of LEE1 beyond metal (zinc) coordination is unknown. No catalytic activity or specific binding activity has been demonstrated experimentally. In particular, whether the two CCCH zinc fingers bind nucleic acid — and, if so, whether the target is RNA or DNA and what its sequence or identity is — has not been tested.
OPEN BIOLOGY MF_DARK
What is known: Firmly established: LEE1 is a 301-aa protein with two CCCH-type (C3H1) zinc fingers (residues 87-114 and 123-145) that coordinate zinc, and it is a predicted member of the yeast zinc proteome. CCCH tandem zinc fingers are canonically nucleic-acid-binding (frequently RNA-binding) modules, making RNA/nucleic-acid binding a reasonable but unproven hypothesis.
Significance: A defined molecular activity would convert LEE1 from a fully uncharacterized zinc-finger protein into an assignable factor (e.g. an RNA-binding protein), enabling downstream mechanistic and pathway placement.
What would resolve it: Direct binding assays for the CCCH fingers: RNA interactome capture / CLIP or RIP-seq to test for and identify RNA targets, and EMSA / protein-binding microarray for possible DNA binding; structural or biophysical confirmation of zinc-dependent fold.
Provenance (the field's own admissions):
Gap: Whether LEE1 has any role in protein ubiquitination is unresolved. The makorin-family (PTHR11224 / MKRN-like) ubiquitin-ligase annotations are propagated from RING-domain-containing metazoan makorins, but LEE1 lacks the catalytic RING domain, so it is unknown whether LEE1 participates in ubiquitination through any non-canonical mechanism or not at all.
OPEN BIOLOGY MF_DARK
What is known: Firmly established: LEE1 is classified in the makorin-related family by its CCCH zinc fingers, yet direct InterPro analysis of Q02799 shows no RING (zf-C3HC4) domain, the module that carries makorin E3 ligase catalysis.
Significance: Resolving this determines whether the widely propagated ubiquitin-ligase annotation is a genuine (RING-independent) function or a family-level over-annotation to be discounted for this member.
What would resolve it: In vitro ubiquitination (E1/E2/E3) assays with LEE1 and candidate E2s; in vivo tests for ubiquitin-conjugate accumulation dependent on LEE1; assessment of any adaptor role bringing substrates to a separate RING/E3.
Gap: The biological process(es) in which LEE1 acts are unknown. Reported deletion and overexpression phenotypes (altered chemical resistance/accumulation, decreased competitive fitness, increased sporulation efficiency, altered invasive/vegetative growth, and a hit in a minisatellite-stability screen) come from high-throughput screens and do not define a pathway; and its nitrogen-source-responsive (nitrogen catabolite repression) expression is a regulatory observation, not a mechanism.
OPEN BIOLOGY WHOLLY_DARK
What is known: Firmly established: LEE1 is non-essential (viable null); it is transcriptionally responsive to nitrogen source and is reported cytoplasmic in global GFP studies; its GO biological process is recorded with the ND evidence code; it has numerous high-throughput physical/genetic interactors but no curated functional GO annotation.
Significance: Placing LEE1 in a pathway and compartment is prerequisite to interpreting its pleiotropic screen phenotypes and to any mechanistic model of its function.
What would resolve it: GFP/fluorescent-tag localization; condition-resolved phenotyping to identify the process(es) underlying the screen phenotypes; validation of high-confidence interactors by co-IP; epistasis analysis.
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