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 (a plasma-membrane NADH:coenzyme-Q6 reductase), 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 the plasma membrane: its deletion perturbs mitochondrial genome maintenance and respiratory growth, and its predicted polytopic topology is consistent with an internal (likely mitochondrial) membrane. The protein is genuinely membrane-associated, but the specific "plasma membrane" assignment is an over-specific propagation. 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: SGD:S000004594 · PGA3 SUPPORTS SOURCE BUT NOT TARGET PGA3 (AIM33's WGD paralog) is plasma-membrane-associated; AIM33 phenotype and topology suggest an internal/mitochondrial membrane instead. 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 differ (MCR1/CBR1 reduce cytochrome b5; PGA3 reduces coenzyme Q6). 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: 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. |
| 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: 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. |
| 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. |
| 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). |
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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
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