PAINT phylogenetic inference placing MIM complex membership and outer-membrane protein insertion at node PANTHER:PTN002000670 of the MITOCHONDRIAL IMPORT PROTEIN 1 family (PTHR28241). S. cerevisiae MIM1 (SGD:S000005386) appears in the WITH/FROM field because its own experimental annotations are among the descendant evidences used to place the IBD.

Inter-ontology logical inference generating GO:0055085 "transmembrane transport" from the molecular function GO:0022832 (the WITH/FROM field is literally GO:0022832). The inference is mechanically valid but inherits the substrate imprecision of the source MF annotation.

Automated cellular-component assignment of GO:0005741 from InterPro:IPR013262 (OMP_MIM1/TOM13_mt) and UniProtKB-SubCell:SL-0172. Fully concordant with three independent IDA annotations on the same gene.

YOL026C, named Tom13 in this study, encodes an integral protein of the mitochondrial outer membrane, identified in a survey of essential yeast genes of unknown function.

Protease treatment and alkaline carbonate extraction of isolated mitochondria show Tom13 to be an integral outer-membrane protein, behaving like Tom70 and Tim23 and unlike the peripheral protein Tom38/Sam35.

Depletion of Tom13 markedly retards assembly of newly imported Tom40 into the mature TOM40 complex, while earlier assembly intermediates still form.

The defect is specific to Tom40: import and insertion of the other beta-barrel protein porin is unaffected by Tom13 depletion, although it is impaired by depletion of Tom38/Sam35. This distinguishes Tom13/Mim1 from a general beta-barrel assembly factor.

Depletion of Tom13 does not impair translocation of matrix or inner-membrane precursors in vitro, showing that the phenotype is not a general collapse of mitochondrial import.

Using C-terminally tagged Tom13-HA, the authors concluded that the protein exposes its COOH terminus to the cytosol. This specific topological inference did not survive: three later studies place the C-terminus in the intermembrane space and the N-terminus in the cytosol.

Mim1 is an integral protein of the mitochondrial outer membrane: it is degraded by low proteinase K concentrations applied to intact mitochondria (with the intermembrane-space marker cytochrome b2 protected) and resists alkaline carbonate extraction.

Carbonate extraction places Mim1 in the membrane fraction alongside the known integral outer-membrane proteins Tom40 and Tom70.

An N-terminally His-tagged allele, which supports wild-type growth, loses anti-His immunoreactivity on protease shaving of intact mitochondria, establishing that the N-terminus faces the cytosol.

Mim1 resides in a high-molecular-mass complex of about 400-450 kDa that is chromatographically distinct from both TOM and TOB/SAM; Mim1 is therefore not a subunit of either.

Conditional depletion of Mim1 abrogates assembly of the TOM complex, with Tom40 accumulating as a low-molecular-mass species.

The Mim1-requiring steps lie downstream of TOB/SAM-mediated insertion of the Tom40 beta-barrel into the outer membrane, so Mim1 acts late in TOM maturation rather than at beta-barrel insertion itself.

The requirement is specific: biogenesis of the TOB/SAM and porin complexes proceeds normally in the absence of Mim1.

Sequence conservation within the Mim1 family is high among fungi and is concentrated in the predicted transmembrane region.

Mim1 is required for efficient membrane insertion and assembly of the signal-anchored TOM receptors Tom20 and Tom70, but not of the tail-anchored Tom22 - a substrate-resolved specificity that points to insertion of N-terminal alpha-helical anchors as the underlying activity.

Mim1 associates with SAM(core) components in a large SAM complex, which accounts for its involvement in the late steps of Tom40 assembly.

The combined requirement for beta-barrel Tom40 maturation and for insertion of the signal-anchored receptors makes Mim1 central to efficient TOM assembly.

Mim2 interacts physically and genetically with Mim1, and the two proteins constitute the MIM complex. This is the primary evidence behind both the GO:0140595 IPI annotations and the GO:0005515 IPI.

Some single-span and all known multiple-span mitochondrial outer-membrane proteins are inserted by a Mim1-dependent pathway, establishing the substrate scope of the MIM route as it stood in 2012.

Recombinant full-length Mim1 reconstituted into planar lipid bilayers shows a characteristic channel activity that is abolished by Mim1-specific antibodies.

A second, independent expression system gives the same conductance, strengthening the assignment of the activity to Mim1 itself rather than to a contaminant.

The pore closes at high positive or negative membrane potentials - the voltage-dependence on which the GO:0022832 annotation rests, and the same behaviour exhibited by the other protein-conducting channels of this membrane.

The Mim1 channel is strongly cation selective, with PK+/PCl- of 23.5:1 at a reversal potential of 53 mV.

Co-reconstitution with the physiological partner Mim2 attenuates the conductance and lowers selectivity to PK+/PCl- of 11:1, showing the measured pore is a property of the MIM complex rather than an artefact of isolated Mim1.

The authors interpret the cation preference functionally as suited to polypeptide translocation, not to ion flux: MIM substrates carry positively charged residues flanking their transmembrane segments.

Mim1 oligomers are the major constituent of the MIM complex, with Mim2 present at one to two copies per complex and sharing the same topology.

Mim1 spans the membrane once via a predicted central alpha-helix, with the N-terminal domain cytosolic and the C-terminal domain in the intermembrane space.

The MIM complex inserts precursors of multi-spanning alpha-helical outer-membrane proteins, complementing TOM-SAM for the beta-barrel class.

MIM also inserts single-spanning precursors with either N-terminal (signal-anchored) or C-terminal (tail-anchored) membrane anchors, extending the substrate scope well beyond multi-span proteins.

Free MIM complexes insert single-spanning proteins independently of Tom70, while other precursors are handed over from the TOM receptor Tom70, defining two routes into the complex.

Coupling of MIM to SAM promotes early assembly steps of TOM subunits, providing the mechanistic link between MIM and TOM biogenesis.

The overall conclusion is that MIM is a major and versatile protein translocase of the mitochondrial outer membrane - the statement that most directly warrants a membrane insertase molecular function.

Genome-scale promoter-shutoff screen of essential yeast genes. MIM1 was one of the genes whose phenotypic signature assigned it to mitochondrial import, which is the origin of the gene name; UniProt cites this work with ECO:0000269 for the FUNCTION statement on Q08176.

States the membrane topology of Mim1 directly - cytosolic N-terminal domain, central transmembrane segment, intermembrane-space C-terminal domain. This is the primary source for the topology that PMID:28916712 later restates, and it resolves the discrepancy with the C-terminally tagged construct used in PMID:15326197.

Mim1 is required for integration of the import receptor Tom20 into the outer membrane, corroborating the independent finding of PMID:17974559.

Mechanism of oligomerisation: Mim1 self-associates through two GXXXG helix-dimerisation motifs in its transmembrane segment, and GXXXG mutants are neither oligomeric nor active. This is the mechanistic basis for the "Mim1 oligomers" that constitute the bulk of the MIM complex, and it explains the unusual concentration of sequence conservation in the transmembrane region noted in PMID:15608614.

The transmembrane segment alone is the minimal functional domain; the N- and C-terminal domains are dispensable for both function and biogenesis.

Organism confirmed from the full text: the study is done in Saccharomyces cerevisiae strain YPH499, with a mim1 null strain made by replacing the MIM1 open reading frame with a HIS3 cassette. Note that mim1-delta is viable, which qualifies the "essential gene" designation MIM1 carried from the early deletion-project and promoter-shutoff screens.

The GXXXG motifs are mapped and functionally tested. A variant mutating both motifs fails to complement, forms no dimer, and gives no detectable Mim1-containing complex on BN-PAGE, while phenocopying mim1-delta for reduced Tom20 and the low-molecular-mass Tom40 species.

Oligomerisation is necessary for function, not merely correlated with it - the direct warrant for treating the oligomer rather than the monomer as the functional unit in core_functions.

Cross-linking and dual-tag co-isolation establish a minimum stoichiometry of two Mim1 copies per complex.

Cross-species complementation: the Neurospora crassa orthologue rescues mim1-delta only partially and only when overexpressed, whereas the Schizosaccharomyces pombe orthologue complements fully. This is the only direct functional evidence bearing on the N. crassa protein and is a caution against treating the phylogenetic propagation to that orthologue as equivalence of function.

The soluble domains tolerate wide sequence variation, consistent with the conservation being concentrated in the transmembrane segment.

Independent laboratory (Lithgow group) reaching the same conclusion as PMID:17974559 and PMID:18177669 by a different route: Mim1/Tom13 catalyses docking of the Tom20 transmembrane segment into the TOM complex.

Loss of Mim1 prevents Tom20 from functioning as an import receptor, linking the insertion defect to a functional consequence for protein import.

Organism confirmed from the full text: S. cerevisiae, using the BY4743 deletion-consortium background and W303 strains, with mim1-delta cells.

Cysteine-scanning cross-linking shows Mim1 is not itself a stable neighbour of docked Tom20; the authors' reading is that Mim1 acts catalytically on the assembly reaction rather than forming part of the product. This is the cleanest experimental support for an insertase/assembly-factor molecular function as opposed to a structural subunit role.

Independent evidence that the Tom40 requirement is indirect, predating the Becker 2010 result cited via PMID:21825073 and proposing a different route: mim1-delta mitochondria import less Tom40 because they have less functional Tom20, but Tom40 that does arrive progresses normally through the 250K and 100K assembly intermediates. Note this is in tension with PMID:15608614, which reported the second intermediate reduced on Mim1 depletion.

The mechanistic explanation offered for the general import defect of mim1-delta cells - it is downstream of Tom20 mispositioning, not a direct effect on translocation.

Specificity control: porin assembly, which needs both TOM and SAM, proceeds without Mim1, confirming those machineries are intact.

A third value for the size of the Mim1 complex, ~300 kDa by BN-PAGE, against 400-450 kDa by gel filtration and ~180 kDa by glycerol gradient. The paper notes the complex also needs more digitonin to solubilise than TOM does, which is a plausible methodological source of the spread.

Establishes the multispanning alpha-helical substrate class by loss-of-function and gain-of-function together, which is stronger than depletion alone.

The Mim1 complex cooperates with Tom70 in binding precursors and promotes their insertion and assembly - the sentence that most directly matches both halves of the GO:0032977 membrane insertase definition.

Direct physical interaction between Mim1 and its precursor substrates. This is the first report of such an interaction and supplies the "binds transmembrane domain-containing proteins" half of GO:0032977, which had previously been inferred from phenotype rather than observed.

Important mechanistic correction to the early literature: the Tom40 defect in Mim1-deficient mitochondria is indirect, arising from failure to import the small Tom proteins that Tom40 assembly requires, rather than from Mim1 acting on the beta-barrel itself.

Organism explicitly stated as Saccharomyces cerevisiae.

Companion paper to PMID:21825073 from an independent laboratory, reaching the same conclusion for the model multispan substrate Ugo1.

Organism explicitly stated as yeast.

Second independent primary source for the cytosolic-N / intermembrane-space-C topology, published a year after PMID:18177669. PMID:28916712 cites the two together, so with both now in the review the topology rests on two laboratories rather than one.

Assigns discrete roles to the soluble domains: the N-terminal domain regulates the SAM-mediated early reaction of Tom40 assembly, and Mim1 also influences a Sam37-dependent late reaction.

Concludes that Mim1 acts at more than one step of TOM assembly, which is the framing that makes the Tom40 requirement partly separable from the Tom20/Tom70 insertase role.

This is the primary source for the "Becker et al., 2010" result that PMID:21825073 cites second-hand and that the review used to record the Tom40 requirement as indirect. Mim1 inserts the small alpha-helical Tom subunit Tom6, which the SAM-Tom5/Tom40 module then acts on; Mim1 is therefore upstream of Tom40 maturation via the small Tom proteins rather than acting on the beta-barrel itself.

The parallel SAM-Mdm10 module handles Tom22, which is consistent with PMID:17974559 finding Tom22 to be the one TOM subunit whose insertion does not require Mim1.

Establishes tail-anchored proteins as MIM substrates, independently of and slightly before the ComplexPortal-annotated PMID:32348752.

Important qualification of the substrate class: MIM dependence is partial and substrate-specific rather than universal among single-span outer-membrane proteins.

Further qualification of substrate dependence, for a multi-span client: Mim1 supports but is not strictly required for Om14 integration, and membrane properties contribute alongside proteinaceous factors.

Concerns the biogenesis of Mim1 itself rather than its activity: the cytosolic Hsp40 cochaperone Djp1 and the receptors Tom70/Tom71 are needed for Mim1 to reach the outer membrane, and without Djp1 overexpressed GFP-Mim1 is massively mislocalised to the ER.

Newly synthesised Mim1 interacts directly with Tom70. Note the direction: here Tom70 acts on Mim1 as a client, which is the converse of the Mim1-Tom70 cooperation in substrate handover reported by PMID:21825073.

States the taxonomic restriction directly: the Mim1/Mim2 complex is fungi-specific, which is the phylogenetic context for the narrow PANTHER family PTHR28241 and for the absence of a metazoan orthologue.

Demonstrates convergent evolution experimentally rather than by inference: the trypanosome protein pATOM36 is a functional analogue of Mim1/Mim2 with neither sequence nor topological similarity, shown by reciprocal complementation in both directions.

pATOM36 rescues nearly all mim1 and mim2 deletion phenotypes in yeast, including the mitochondrial morphology defects.

Reports a second, non-import function for the MIM complex: the lipid metabolism enzyme Ayr1 captures MIM and thereby nucleates mitochondria-lipid droplet contact sites.

The contact-site claim itself, and the mechanism of capture.

A functional consequence at the cellular level - lipid droplet number depends on MIM and Ayr1 - which is what would justify a lipid droplet organization biological process annotation.

Mechanistically elegant and important for how the two functions relate: Ayr1 engages the same site as a precursor would, via a single hydrophobic segment, but is never released. The second function is therefore a subverted insertase reaction, not a separate activity of the same protein.

The two functions are carried by distinct complex populations rather than by one complex switching roles.

Quantifies the scope of the import function, higher than any earlier estimate in this review.

Current review-level statement of the MIM complex's composition and function, including the cooperation with Tom20 and Tom70.

Places the yeast MIM complex, trypanosome pATOM36 and mammalian MTCH1/MTCH2 as convergently evolved solutions to the same problem.