AltMIEF1 (also called AltMiD51 or MIEF1-MP; UniProt L0R8F8) is a 70-amino-acid protein translated from an upstream open reading frame located in exon 2 of the bicistronic human MIEF1 gene, in the 5' leader of the mRNA that also encodes MIEF1/MiD51. It is encoded in a different reading frame and shares no sequence with MiD51 (Q9NQG6), the outer-mitochondrial-membrane DRP1 adaptor of mitochondrial fission; the two are distinct proteins that happen to share a transcript and a gene symbol. In cultured human cells and colon tissue AltMIEF1 is two- to six-fold more abundant than MiD51. AltMIEF1 is a member of the LYR-motif (complex I LYR / LYRM) family, consisting of three alpha-helices with an N-terminal LYR tripeptide, and it resides in the mitochondrial matrix. Through its LYR motif it binds the 4'-phosphopantetheine arm of the mitochondrial acyl carrier protein NDUFAB1 (mt-ACP), and together with MALSU1 and mt-ACP it forms a heterotrimeric module that sits on the intersubunit face of late-stage mitoribosomal large-subunit (mt-LSU) assembly intermediates next to uL14m and bL19m. This MALSU1-AltMIEF1-mt-ACP module persists through multiple maturation steps alongside factors such as NSUN4-MTERF4, GTPBP5, GTPBP7 and GTPBP10, and sterically blocks premature joining of the small subunit until maturation is complete. Knockout of AltMIEF1 impairs mt-LSU assembly, and depletion with rescue experiments indicates that it supports the overall rate of mitochondrial protein synthesis. Overexpression in HeLa cells causes DRP1-dependent mitochondrial fragmentation, but this phenotype has not been linked to a loss-of-function defect.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000266 mitochondrial fission | IMP PMID:29083303 Deep transcriptome annotation enables the discovery and func... | MARK AS OVER ANNOTATED | Summary: Based on overexpression of Flag- or GFP-tagged AltMiD51 in HeLa cells, which fragmented the mitochondrial network in an LYR-motif- and DRP1-dependent manner. Reason: The full text of PMID:29083303 shows that the only evidence is a gain-of-function phenotype: overexpression fragments the network, the LYR->AAA mutant does not, and DRP1 knockdown or dominant-negative DRP1 prevents it. No loss-of-function fission defect was reported. A matrix-resident LYR protein has no plausible direct access to the outer-membrane DRP1 machinery, and the authors themselves suggest the effect may be indirect. All later work places AltMIEF1 in the MALSU1-mt-ACP module on assembling mt-LSU (PMID:28892042, PMID:34135318, PMID:38042949, PMID:35177605). The fragmentation is best read as a secondary consequence of overexpression in the matrix rather than participation in the fission process. The annotation is also easily confused with the genuine fission role of MiD51, the protein from the downstream ORF of the same gene. It should not be propagated as a core function. Supporting Evidence: PMID:29083303 Remarkably, we found that altMiD51 also localizes at the mitochondria (Figure 12c; Figure 12—figure supplement 3) and that its overexpression results in mitochondrial fission (Figure 12d). PMID:29083303 It remains possible that altMiD51 promotes mitochondrial fission independently of Drp1 and is able to reverse the hyperfusion induced by Drp1 inactivation. PMID:28892042 We conclude that L0R8F8 and mt-ACP are assembly factors for the human mitoribosome. |
| GO:0005515 protein binding | IPI PMID:28892042 Structures of the human mitochondrial ribosome in native sta... | REMOVE | Summary: Interaction with NDUFAB1 (mt-ACP), observed directly in the cryo-EM structures of mt-LSU assembly intermediates, where the 4'-phosphopantetheine arm of mt-ACP inserts into a hydrophobic pocket of AltMIEF1. Reason: The interaction is real and well-resolved structurally, but bare protein binding is uninformative. No GO molecular function term captures LYRM binding of the acyl-pantetheine arm of ACP, and the functional output of the MALSU1-AltMIEF1-mt-ACP module is already captured by GO:0140978 mitochondrial large ribosomal subunit binding and GO:1902775 mitochondrial large ribosomal subunit assembly. Supporting Evidence: PMID:28892042 The 4-PP modification adopts a “flipped-out” conformation29 and inserts into a hydrophobic pocket of L0R8F8 |
| GO:0005515 protein binding | IPI PMID:28892042 Structures of the human mitochondrial ribosome in native sta... | REMOVE | Summary: Interaction with MALSU1, the RsfS-family anti-association factor bound to uL14m, seen in the same cryo-EM structures as part of the MALSU1-L0R8F8-mt-ACP module. Reason: A genuine direct contact within the assembly module, but bare protein binding adds nothing. The function of the module is represented by mitochondrial large ribosomal subunit binding and assembly terms. Supporting Evidence: PMID:28892042 However, one functional consequence of the MALSU1–L0R8F8–mt-ACP module is that it would sterically obstruct the binding of the mt-SSU. |
| GO:0005515 protein binding | IPI PMID:30215512 MIEF1 Microprotein Regulates Mitochondrial Translation. | REMOVE | Summary: Co-immunoprecipitation and APEX2 proximity labelling of overexpressed MIEF1-MP-FLAG enriched NDUFAB1, and the interaction required the LYR motif and F39. Reason: Uninformative protein binding term. The interaction is consistent with the structural data, but the authors found no functional consequence for complex I activity or lipid synthesis and judged the overexpression interaction not functionally relevant. The biologically meaningful context, the MALSU1-AltMIEF1-mt-ACP module on mt-LSU, is captured by other terms. Supporting Evidence: PMID:30215512 Together the data confirm that overexpressed MIEF1-MP interacts with the ACPM/NDUFAB1 protein via its LYR motif. PMID:30215512 However, we observed no effects of MIEF1-MP using a mitochondrial complex 1 activity assay (Figure S2A) or lipid biosynthesis (Figure S2B) suggesting that MIEF1-MP: ACPM/NDUFAB1 interaction is not functionally relevant. |
| GO:0005515 protein binding | IPI PMID:30215512 MIEF1 Microprotein Regulates Mitochondrial Translation. | REMOVE | Summary: Co-immunoprecipitation of MIEF1-MP with an antibody against MRPS27, a protein of the mitoribosomal small subunit. Reason: Bare protein binding is uninformative. A more specific term is not appropriate here. The structures place AltMIEF1 only on mt-LSU intermediates, in a module that sterically excludes the small subunit, so co-precipitation with an mt-SSU protein is most likely indirect, through monosomes or lysate-level association. It does not support small-subunit binding. Supporting Evidence: PMID:30215512 We validated the MRPL4 and MRPS27 interaction by Western blot (Figure 4C and Figure S3). PMID:28892042 However, one functional consequence of the MALSU1–L0R8F8–mt-ACP module is that it would sterically obstruct the binding of the mt-SSU. |
| GO:0005515 protein binding | IPI PMID:30215512 MIEF1 Microprotein Regulates Mitochondrial Translation. | MODIFY | Summary: Co-immunoprecipitation of MIEF1-MP with MRPL4 (uL4m), a large-subunit mitoribosomal protein. BioPlex pulldowns of MRPL4 and other mt-LSU proteins also recovered MIEF1-MP. Reason: The informative content is association with the mitoribosomal large subunit, which is better expressed as GO:0140978 mitochondrial large ribosomal subunit binding (already annotated from this paper and from PMID:28892042). Proposed replacements: mitochondrial large ribosomal subunit binding Supporting Evidence: PMID:30215512 The analysis of ACPM/NDUFAB1 detected only a single spectral count from MIEF1-MP and while the mitoribosome proteins MRPL4, MRPL10, MRPL12, MRPL21, and MRPL39 had many spectral counts for MIEF1-MP (Table 1). |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: High-throughput mitochondrial proteome (MitoCoP) detection of the protein. Reason: Correct and consistent with all targeted studies, although less specific than the mitochondrial matrix annotations. |
| GO:0005739 mitochondrion | IDA PMID:28892042 Structures of the human mitochondrial ribosome in native sta... | ACCEPT | Summary: Identified as a component of native mitoribosomal large-subunit assembly intermediates isolated from human mitochondria. Reason: Correct; the more specific matrix location is also annotated. Supporting Evidence: PMID:28892042 We conclude that L0R8F8 and mt-ACP are assembly factors for the human mitoribosome. |
| GO:0005739 mitochondrion | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the Complex1_LYR domain (IPR008011). Reason: LYR-motif proteins are characteristically mitochondrial, and the prediction is confirmed experimentally for this protein. |
| GO:0005759 mitochondrial matrix | EXP PMID:28892042 Structures of the human mitochondrial ribosome in native sta... | ACCEPT | Summary: Inferred from its presence, together with matrix-resident MALSU1 and mt-ACP, on mitoribosomal large-subunit intermediates. Reason: Mitoribosome assembly takes place in the matrix, and protease-protection and quenching data from other studies independently confirm a matrix location. Supporting Evidence: PMID:28892042 We conclude that L0R8F8 and mt-ACP are assembly factors for the human mitoribosome. |
| GO:0005759 mitochondrial matrix | IDA PMID:29083303 Deep transcriptome annotation enables the discovery and func... | ACCEPT | Summary: Matrix localisation shown by a fluorescence-quenching assay (no quenching relative to an intermembrane-space Venus control). Reason: Direct experimental evidence consistent with the other studies. Supporting Evidence: PMID:29083303 The absence of quenching of the fluorescence compared to IMS-Venus indicates the matricial localization of altMiD51. |
| GO:0005759 mitochondrial matrix | IDA PMID:30215512 MIEF1 Microprotein Regulates Mitochondrial Translation. | ACCEPT | Summary: Proteinase K protection of isolated mitochondria: MIEF1-MP-FLAG behaved like the matrix marker HSP60. Reason: Clear direct evidence for matrix localisation. Supporting Evidence: PMID:30215512 MIEF1-MP-FLAG degradation in the proteinase K assay had an identical pattern to that of HSP60, indicating that MIEF1-MP is a mitochondrial matrix microprotein. |
| GO:0005759 mitochondrial matrix | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (Mitochondrion matrix). Reason: Mapped from the experimentally supported UniProt location; correct. |
| GO:0032981 mitochondrial respiratory chain complex I assembly | IMP PMID:31666358 The Mitochondrial Acyl-carrier Protein Interaction Network H... | UNDECIDED | Summary: From the NDUFAB1 interaction-network study of Dibley et al., in which AltMiD51 knockout cells were analysed. UniProt states that AltMIEF1 is required for complete complex I assembly, citing this paper. Reason: The cached record is abstract-only and full text could not be retrieved. The abstract reports only that AltMiD51 knockout impairs mt-LSU assembly, and attributes the complex I (N-module) assembly defect to LYRM2. A complex I deficit in AltMiD51 knockout cells is plausible, but it would be expected as a downstream consequence of reduced synthesis of the mtDNA-encoded ND subunits. That would not show direct participation in complex I assembly, unlike the dedicated LYRM assembly factors. Overexpression did not change complex I activity (PMID:30215512). Without the full data, the curator's call cannot be assessed, so the annotation is neither accepted as core nor removed. Supporting Evidence: PMID:31666358 Subsequent knockout and interaction network studies in human cells revealed the LYRM member AltMiD51 to be important for optimal assembly of the large mitoribosome subunit, consistent with recent structural studies. PMID:31666358 we used proteomics coupled with topographical heat-mapping to reveal that knockout of LYRM2 impairs assembly of the NADH-dehydrogenase module of complex I, leading to defects in cellular respiration. |
| GO:0070131 positive regulation of mitochondrial translation | IDA PMID:30215512 MIEF1 Microprotein Regulates Mitochondrial Translation. | KEEP AS NON CORE | Summary: BONCAT/AHA labelling showed that overexpression of MIEF1-MP increased mtDNA-encoded protein synthesis by about 54%. Knockdown decreased it by about 32%, and the decrease was rescued by siRNA-resistant MIEF1-MP. Reason: The data are sound and the rescue experiment controls for co-depletion of MiD51. Mechanistically, however, the effect on translation rate reflects the role of AltMIEF1 in mt-LSU biogenesis rather than a regulatory activity on translation itself. The authors interpret it the same way, as support for an assembly-factor role. The annotation is retained as a valid downstream consequence, not as a core function. Supporting Evidence: PMID:30215512 We expressed a siRNA resistant MIEF1-MP in cells treated with siRNA against the MIEF1-MP transcript and found that expression of MIEF1-MP rescued the loss of protein translation (Figure 6) supporting a role for MIEF1-MP and not MIEF1 in the mitochondrial translation. PMID:30215512 We tested the hypothesis that MIEF1-MP might be a functional component of the mitoribosome and obtained data from overexpression and knockdown studies that indicates that MIEF1-MP can regulate mitochondrial translation, which supports the hypothesis that MIEF1-MP is a mitoribosome assembly factor. |
| GO:0140978 mitochondrial large ribosomal subunit binding | IDA PMID:28892042 Structures of the human mitochondrial ribosome in native sta... | ACCEPT | Summary: Built de novo into cryo-EM density of native mt-LSU assembly intermediates, as part of the MALSU1-L0R8F8-mt-ACP module bound adjacent to uL14m. Reason: Direct structural evidence, reproduced in many later structures (PMID:34135318, PMID:38042949, PMID:35177605). This is the best available MF term for the protein. Supporting Evidence: PMID:28892042 The structures reveal that binding of bL36m and accommodation of interfacial rRNA are among the concluding steps of mitoribosomal biogenesis, and that L0R8F8 and mt-ACP are mitoribosomal assembly factors. PMID:35177605 The MALSU1: L0R8F8 :mtACP anti-association module is clearly present in all states |
| GO:0140978 mitochondrial large ribosomal subunit binding | IDA PMID:30215512 MIEF1 Microprotein Regulates Mitochondrial Translation. | ACCEPT | Summary: APEX2 proximity labelling, BioPlex reciprocal pulldowns of mt-LSU proteins and co-immunoprecipitation with MRPL4 show association with the mitoribosomal large subunit. Reason: Independent biochemical evidence consistent with the structural data. Supporting Evidence: PMID:30215512 Overall, the mitoribosome proteins were the only cluster with substantial evidence of MIEF1-MP binding indicating that under endogenous conditions (i.e., no overexpression of MIEF1-MP) MIEF1-MP interacts primarily with the mitoribosome. |
| GO:1902775 mitochondrial large ribosomal subunit assembly | IDA PMID:34135318 Structural basis for late maturation steps of the human mito... | ACCEPT | Summary: Cryo-EM of mt-LSU intermediates accumulating in GTPBP5 knockout cells or bound to GTPBP5 shows the MALSU1:L0R8F8:mt-ACP module among the trapped assembly factors. Reason: Places AltMIEF1 directly on late assembly intermediates as part of the anti-association module that is released before subunit joining. The module contributes structural (steric-block) work to the assembly pathway, so this counts as participation. Supporting Evidence: PMID:34135318 Both the GTPBP5KO and GTPBP5IP mt-LSU assembly intermediates reveal several trapped assembly factors: the MTERF4-NSUN4 complex, MRM2, MTG1, and the MALSU1:L0R8F8:mt-ACP module |
| GO:1902775 mitochondrial large ribosomal subunit assembly | IDA PMID:38042949 Structural insights into the role of GTPBP10 in the RNA matu... | ACCEPT | Summary: Cryo-EM of GTPBP10- and GTPBP7-bound mt-LSU intermediates shows the MALSU1-L0R8F8-mtACP module binding early and persisting over multiple maturation steps. Reason: Consistent direct structural evidence for participation in mt-LSU maturation. Supporting Evidence: PMID:38042949 Both, NSUN4—MTERF4 and MALSU1—L0R8F8—mtACP bind early in the mtLSU maturation process and persist over multiple maturation steps. |
| GO:1902775 mitochondrial large ribosomal subunit assembly | IMP PMID:31666358 The Mitochondrial Acyl-carrier Protein Interaction Network H... | ACCEPT | Summary: AltMiD51 knockout cells show suboptimal assembly of the large mitoribosomal subunit. Reason: The loss-of-function evidence complements the structural data. The abstract states the finding explicitly, although the full text is not cached. Supporting Evidence: PMID:31666358 Subsequent knockout and interaction network studies in human cells revealed the LYRM member AltMiD51 to be important for optimal assembly of the large mitoribosome subunit, consistent with recent structural studies. |
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Download this section (compressed HTML)Q: Is the complex I assembly defect reported for AltMiD51 knockout cells (PMID:31666358) distinguishable from the secondary effect of reduced mitochondrial translation of ND subunits? For example, are nuclear-encoded complex I modules affected differently from mtDNA-encoded ones, and is the defect rescued by restoring translation?
Q: Does the mitochondrial fragmentation caused by AltMIEF1 overexpression (PMID:29083303) have any loss-of-function counterpart, or is it a secondary stress response to matrix overexpression?
Q: What does AltMIEF1 contribute that MALSU1 and mt-ACP alone cannot? For example, is it required for mt-ACP recruitment to the module, or for module release before subunit joining?
Experiment: In AltMIEF1 knockout cells made by frameshifting only the uORF, so that MiD51 is preserved (as in PMID:30181344), perform cryo-EM or sucrose-gradient analysis of mt-LSU particles. This would test whether MALSU1 and mt-ACP still bind and whether premature subunit association occurs.
Experiment: Run complexome profiling of complex I assembly intermediates in uORF-specific knockout cells, with and without re-expression of AltMIEF1 or of an LYR->AAA mutant. This would separate a direct assembly-factor role from a translation-dependent effect.
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