AIM33 (YML087C) is a poorly characterized Saccharomyces cerevisiae protein of the flavoprotein pyridine nucleotide cytochrome reductase (cytochrome-b5 reductase, CYB5R) superfamily. It carries the diagnostic two-domain flavin/pyridine-nucleotide reductase module — an FAD-binding FR-type domain (residues ~70-173) followed by an NAD(P)H-binding domain — and is predicted to bind FAD, so it is expected to act as an NAD(P)H-dependent flavin oxidoreductase. Unlike most family members, which are soluble or single-pass tail-anchored, AIM33 is predicted to be a polytopic (multi-pass) membrane protein. Its specific catalytic activity, physiological electron acceptor, and site of action have not been experimentally established. Deletion of AIM33 alters the frequency of spontaneous mitochondrial genome loss (petite frequency) and impairs growth on non-fermentable carbon sources, linking the protein to mitochondrial genome maintenance and respiratory competence, and giving it the "Altered Inheritance of Mitochondria" (AIM) name. AIM33 has a whole-genome-duplication paralog, PGA3, and is related at the reductase-domain level to the mitochondrial/ER cytochrome-b5 reductases MCR1 and CBR1 and to human CYB5R-family enzymes.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | MODIFY | Summary: Phylogenetically-propagated (IBA) plasma-membrane location. AIM33 is a membrane protein, but the "plasma membrane" specificity is not supported for AIM33 itself. Reason: The IBA is transferred across the cytochrome-b5-reductase family (PANTHER PTHR19370), whose members occupy several membranes: the paralog PGA3 is plasma-membrane-associated, but MCR1 is at the mitochondrial outer membrane / intermembrane space and CBR1 is in the ER. AIM33's own experimental data point away from a simple PGA3-like plasma-membrane transfer: its deletion perturbs mitochondrial genome maintenance and respiratory growth. PAINT also carries a negated mitochondrial IRD at the AIM33/PGA3 node, showing a clade-specific decision that AIM33/PGA3 should not inherit the upstream mitochondrial site, so that location cannot be asserted either. The protein is genuinely membrane-associated, but the specific "plasma membrane" assignment rests on PGA3 while AIM33 remains experimentally unlocalized. Generalize to GO:0016020 (membrane) until the true membrane is determined experimentally. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: COMPARTMENT OR COMPLEX MISMATCH WRONG ORTHOLOG OR PARALOG Sources checked: PANTHER:PTN001064672 · PTN001064672 SUPPORTS SOURCE BUT NOT TARGET Current PTHR19370 PAINT places the plasma-membrane IBD at the AIM33/PGA3 Saccharomycetaceae node and adds a negated GO:0005739 IRD at the same node, showing a clade-specific decision that the broader mitochondrial PTN000452207 localization should not propagate to AIM33/PGA3. The plasma-membrane site still rests on PGA3, while AIM33's own petite-frequency and respiratory-growth phenotypes point elsewhere, so AIM33 should be kept at generic membrane until directly localized. SGD:S000004594 · PGA3 SUPPORTS SOURCE BUT NOT TARGET PGA3 (AIM33's WGD paralog) is the direct plasma-membrane seed, but the AIM33 membrane remains unresolved. Proposed replacements: membrane |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phylogenetically-propagated cytochrome-b5 reductase activity. Domain-plausible but not demonstrated for AIM33, and the specific cytochrome-b5 electron acceptor is unproven for this paralog. Reason: AIM33 has the FAD-binding FR-type + NAD(P)H-binding module diagnostic of the cytochrome-b5 reductase superfamily, so an NAD(P)H:acceptor flavin-oxidoreductase activity is reasonable in principle. However, no enzyme assay has demonstrated cytochrome-b5 reductase activity for AIM33, and the physiological electron acceptor (cytochrome b5 vs coenzyme Q vs a P450 vs an unknown partner) is not established. The activity is transferred from characterized paralogs (MCR1, CBR1, PGA3) whose acceptors are not demonstrably identical across the family: MCR1/CBR1 reduce cytochrome b5, while the PGA3 PAINT donor is in a PANTHER subfamily named for a plasma-membrane coenzyme-Q6 reductase. Asserting the specific "cytochrome-b5" acceptor for AIM33 is an over-annotation; the defensible MF is the more general oxidoreductase activity (see GO:0016491, retained). Keep the reductase idea on record but flagged, pending direct biochemistry. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000452207 · PTN000452207 SUPPORTS SOURCE BUT NOT TARGET Current PTHR19370 PAINT retains a cytochrome-b5 reductase IBD at this broad eukaryotic node, seeded by MCR1 and PGA3; the node supports an inherited flavin reductase fold, but not the specific electron acceptor for AIM33. SGD:S000001633 · MCR1 SUPPORTS SOURCE BUT NOT TARGET MCR1 is an experimentally validated NADH-cytochrome b5 reductase; AIM33 shares the reductase fold but its acceptor is unverified. SGD:S000004594 · PGA3 SUPPORTS SOURCE BUT NOT TARGET PGA3 seeds the same PAINT cytochrome-b5 reductase node even though its own PANTHER subfamily is named plasma membrane-associated coenzyme Q6 reductase; the acceptor is therefore not settled even on the AIM33/PGA3 branch. |
| GO:0006696 ergosterol biosynthetic process | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phylogenetically-propagated ergosterol-biosynthesis role. No AIM33-specific evidence; transferred from paralogs and contradicted by AIM33's mitochondrial-genome phenotype. Reason: An ergosterol-biosynthesis role is well established for the related cytochrome-b5 reductases that donate electrons to sterol-biosynthetic cytochrome P450s (e.g. MCR1 to Erg11/Erg5/Erg1), and this is the basis for the IBA transfer. AIM33 itself has no experimental link to sterol biosynthesis; instead its characterized phenotype is altered mitochondrial genome maintenance and loss of respiratory growth. Treating AIM33 as an ergosterol-biosynthesis enzyme on the strength of paralog function is an over-annotation. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000452208 · PTN000452208 SUPPORTS SOURCE BUT NOT TARGET Current PTHR19370 PAINT still carries ergosterol biosynthesis from the MCR1 seed at a very broad ancestral node; that assignment should not be propagated unchanged to the AIM33/PGA3 paralog branch. SGD:S000001633 · MCR1 SUPPORTS SOURCE BUT NOT TARGET Ergosterol-pathway electron donation is documented for MCR1, not for AIM33. |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA machine-learning electronic annotation asserting the same specific cytochrome-b5 reductase activity; same over-specificity concern as the IBA above. Reason: This ARBA rule fires on family/domain features shared across the CYB5R superfamily and assigns the specific cytochrome-b5 acceptor. As with the IBA, the reductase fold is present but the cytochrome-b5 acceptor is not verified for AIM33; the defensible electronic MF is the general oxidoreductase activity (GO:0016491), which is separately annotated and retained. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: ARBA:ARBA00027922 · ARBA rule (cytochrome-b5 reductase) SOURCE WEAK OR INFERRED Rule assigns a specific acceptor not established for this protein. |
| GO:0016020 membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic membrane localization from the UniProt SubCell mapping; well supported by predicted transmembrane helices and the global membrane-proteome dataset. Reason: AIM33 has multiple predicted transmembrane helices (UniProt TRANSMEM 15-35, 42-62, 180-200) and is included in the experimentally-constrained membrane-proteome topology map (PMID:16847258). "Membrane" is the correct, appropriately general cellular-component term; the specific membrane is not yet determined. Supporting Evidence: PMID:16847258 we report the cloning and expression of 617 S. cerevisiae membrane proteins as fusions to a C-terminal topology reporter and present experimentally constrained topology models for 546 proteins. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO electronic annotation of general oxidoreductase activity — the best-supported, appropriately general molecular-function call for AIM33. Reason: AIM33 carries the FAD-binding FR-type domain and the NAD_binding_1 domain characteristic of flavin/pyridine-nucleotide oxidoreductases, and is predicted to bind FAD. Oxidoreductase activity (GO:0016491) is strongly supported by the domain architecture while remaining agnostic about the specific electron acceptor, which is unknown. This is the honest core molecular-function term for this protein. |
| GO:0003674 molecular_function | ND GO_REF:0000015 | ACCEPT | Summary: Root-term ND placeholder recording that a specific molecular function was not determined. Reason: Standard ND root annotation (GO_REF:0000015). Appropriate as a historical placeholder; the InterPro-based oxidoreductase MF now provides more specific (if still general) information. Supporting Evidence: file:yeast/AIM33/AIM33-notes.md Searched newer `AIM33` / `YML087C` / `Q04516` literature after the refresh. No direct paper was file:yeast/AIM33/AIM33-notes.md No enzyme assay demonstrates cytochrome-b5 reductase |
| GO:0005575 cellular_component | ND GO_REF:0000015 | ACCEPT | Summary: Root-term ND placeholder for undetermined cellular component. Reason: Standard ND root annotation. The membrane annotations now supersede this with more specific (if still general) localization information. Supporting Evidence: file:yeast/AIM33/AIM33-notes.md Searched newer `AIM33` / `YML087C` / `Q04516` literature after the refresh. No direct paper was file:yeast/AIM33/AIM33-notes.md its true membrane (mitochondrial vs plasma vs ER) is unresolved. |
| GO:0008150 biological_process | ND GO_REF:0000015 | ACCEPT | Summary: Root-term ND placeholder for undetermined biological process. Reason: Standard ND root annotation. The disruption phenotype implicates AIM33 in mitochondrial genome maintenance / respiratory competence, but no specific BP term has been experimentally validated for AIM33 (see knowledge_gaps and core_functions). Supporting Evidence: file:yeast/AIM33/AIM33-notes.md Searched newer `AIM33` / `YML087C` / `Q04516` literature after the refresh. No direct paper was file:yeast/AIM33/AIM33-notes.md Mechanism behind the AIM (mitochondrial genome maintenance) phenotype |
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Download this section (compressed HTML)Q: What is the physiological electron acceptor of AIM33 (cytochrome b5, coenzyme Q6, a cytochrome P450, or another partner), and is NADH or NADPH the electron donor?
Q: In which membrane does AIM33 reside endogenously — mitochondrial, ER, or plasma membrane — and does its polytopic topology differ from the mostly tail-anchored/soluble family members?
Q: Does AIM33 promote or antagonize petite formation, and does it act on mitochondrial genome maintenance directly (e.g. via redox homeostasis or membrane potential) or indirectly?
Experiment: Express and purify recombinant AIM33 (full-length in detergent/nanodiscs and the isolated soluble reductase domain), confirm bound FAD spectroscopically, and measure steady-state NADH- and NADPH-dependent reduction of candidate acceptors (cytochrome b5, coenzyme Q6/CoQ analogs, ferricyanide, cytochrome c, methemoglobin).
Hypothesis: AIM33 is an NAD(P)H-dependent flavin oxidoreductase whose physiological acceptor is one of cytochrome b5, coenzyme Q6, or a P450.
Type: enzyme kinetics / spectroscopy
Experiment: Determine endogenous localization of a functional C-terminally tagged AIM33 by live-cell fluorescence colocalization with mitochondrial, ER, and plasma-membrane markers, and by subcellular and submitochondrial fractionation; verify functionality of the tagged allele by complementation of the petite phenotype.
Hypothesis: AIM33 localizes to a mitochondrial membrane consistent with its mitochondrial-genome maintenance phenotype rather than to the plasma membrane.
Type: localization / cell biology
Experiment: In a clean aim33-delta strain, quantify petite frequency against isogenic wild type, measure mtDNA copy number and integrity, mitochondrial membrane potential, and respiratory-complex assembly; perform targeted genetic-interaction tests with genes for mtDNA maintenance and with the paralog PGA3 and relatives MCR1/CBR1.
Hypothesis: Loss of AIM33 alters mitochondrial genome stability through a defined mechanism.
Type: yeast genetics / functional genomics
Protein of unknown function, highly conserved across species; homolog of human CYB5R4; null mutant displays reduced frequency of mitochondrial genome loss; AIM33 has a paralog, PGA3, that arose from the whole genome duplication
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