Mammalian mitochondrial DNA (mtDNA) is inherited uniparentally through the female germline without undergoing recombination. This poses a major problem as deleterious mtDNA mutations must be ...eliminated to avoid a mutational meltdown over generations. At least two mechanisms that can decrease the mutation load during maternal transmission are operational: a stochastic bottleneck for mtDNA transmission from mother to child, and a directed purifying selection against transmission of deleterious mtDNA mutations. However, the molecular mechanisms controlling these processes remain unknown. In this study, we systematically tested whether decreased autophagy contributes to purifying selection by crossing the C5024T mouse model harbouring a single pathogenic heteroplasmic mutation in the tRNAAla gene of the mtDNA with different autophagy-deficient mouse models, including knockouts of Parkin, Bcl2l13, Ulk1, and Ulk2. Our study reveals a statistically robust effect of knockout of Bcl2l13 on the selection process, and weaker evidence for the effect of Ulk1 and potentially Ulk2, while no statistically significant impact is seen for knockout of Parkin. This points at distinctive roles of these players in germline purifying selection. Overall, our approach provides a framework for investigating the roles of other important factors involved in the enigmatic process of purifying selection and guides further investigations for the role of BCL2L13 in the elimination of non-synonymous mutations in protein-coding genes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Although the mammalian mtDNA transcription machinery is simple and resembles bacteriophage systems, there are many reports that nuclear transcription regulators, as exemplified by MEF2D, MOF, PGC-1α, ...and hormone receptors, are imported into mammalian mitochondria and directly interact with the mtDNA transcription machinery. However, the supporting experimental evidence for this concept is open to alternate interpretations, and a main issue is the difficulty in distinguishing indirect regulation of mtDNA transcription, caused by altered nuclear gene expression, from direct intramitochondrial effects. We provide a critical discussion and experimental guidelines to stringently assess roles of intramitochondrial factors implicated in direct regulation of mammalian mtDNA transcription.
The machinery for mammalian mtDNA transcription is distinct from the ones in the nucleus. Nevertheless, several controversial reports suggest that nuclear transcription regulators also localize to mitochondria to directly modulate mtDNA transcription. Rubalcava-Gracia et al. revisit the roles of such factors and discuss experimental hurdles associated with assigning intramitochondrial functions.
The constraints of allotopic expression Nieto-Panqueva, Felipe; Rubalcava-Gracia, Diana; Hamel, Patrice P. ...
Mitochondrion,
11/2023, Letnik:
73
Journal Article
Recenzirano
•We describe the natural transfer of mitochondrial OXPHOS genes to the nucleus and critically review claims of their allotopic expression.•We discuss three constraints that should be considered when ...modifying a mitochondrial gene for allotopic expression.•The TIM23 translocator sorts alpha-helices through two different routes that eventually determine the final topology of membrane proteins.•We assigned a color code to the transmembrane stretches (TMS) of OXPHOS proteins to predict which are likely to be internalized into mitochondria.•We propose that the design of proteins for allotopic expression must make allowance for μΔGapp maximization of highly hydrophobic TMS.
Allotopic expression is the functional transfer of an organellar gene to the nucleus, followed by synthesis of the gene product in the cytosol and import into the appropriate organellar sub compartment. Here, we focus on mitochondrial genes encoding OXPHOS subunits that were naturally transferred to the nucleus, and critically review experimental evidence that claim their allotopic expression. We emphasize aspects that may have been overlooked before, i.e., when modifying a mitochondrial gene for allotopic expression━besides adapting the codon usage and including sequences encoding mitochondrial targeting signals━three additional constraints should be considered: (i) the average apparent free energy of membrane insertion (μΔGapp) of the transmembrane stretches (TMS) in proteins earmarked for the inner mitochondrial membrane, (ii) the final, functional topology attained by each membrane-bound OXPHOS subunit; and (iii) the defined mechanism by which the protein translocator TIM23 sorts cytosol-synthesized precursors. The mechanistic constraints imposed by TIM23 dictate the operation of two pathways through which alpha-helices in TMS are sorted, that eventually determine the final topology of membrane proteins. We used the biological hydrophobicity scale to assign an average apparent free energy of membrane insertion (μΔGapp) and a “traffic light” color code to all TMS of OXPHOS membrane proteins, thereby predicting which are more likely to be internalized into mitochondria if allotopically produced. We propose that the design of proteins for allotopic expression must make allowance for μΔGapp maximization of highly hydrophobic TMS in polypeptides whose corresponding genes have not been transferred to the nucleus in some organisms.
Abstract In mammals, the leucine-rich pentatricopeptide repeat protein (LRPPRC) and the stem-loop interacting RNA-binding protein (SLIRP) form a complex in the mitochondrial matrix that is required ...throughout the life cycle of most mitochondrial mRNAs. Although pathogenic mutations in the LRPPRC and SLIRP genes cause devastating human mitochondrial diseases, the in vivo function of the corresponding proteins is incompletely understood. We show here that loss of SLIRP in mice causes a decrease of complex I levels whereas other OXPHOS complexes are unaffected. We generated knock-in mice to study the in vivo interdependency of SLIRP and LRPPRC by mutating specific amino acids necessary for protein complex formation. When protein complex formation is disrupted, LRPPRC is partially degraded and SLIRP disappears. Livers from Lrpprc knock-in mice had impaired mitochondrial translation except for a marked increase in the synthesis of ATP8. Furthermore, the introduction of a heteroplasmic pathogenic mtDNA mutation (m.C5024T of the tRNAAla gene) into Slirp knockout mice causes an additive effect on mitochondrial translation leading to embryonic lethality and reduced growth of mouse embryonic fibroblasts. To summarize, we report that the LRPPRC/SLIRP protein complex is critical for maintaining normal complex I levels and that it also coordinates mitochondrial translation in a tissue-specific manner.
Experimentally relocating mitochondrial genes to the nucleus for functional expression (allotopic expression) is a challenging process. The high hydrophobicity of mitochondria-encoded proteins seems ...to be one of the main factors preventing this allotopic expression. We focused on subunit II of cytochrome
oxidase (Cox2) to study which modifications may enable or improve its allotopic expression in yeast. Cox2 can be imported from the cytosol into mitochondria in the presence of the W56R substitution, which decreases the protein hydrophobicity and allows partial respiratory rescue of a
-null strain. We show that the inclusion of a positive charge is more favorable than substitutions that only decrease the hydrophobicity. We also searched for other determinants enabling allotopic expression in yeast by examining the
gene in organisms where it was transferred to the nucleus during evolution. We found that naturally occurring variations at within-membrane residues in the legume
Cox2 could enable yeast
allotopic expression. We also evidence that directing high doses of allotopically synthesized Cox2 to mitochondria seems to be counterproductive because the subunit aggregates at the mitochondrial surface. Our findings are relevant to the design of allotopic expression strategies and contribute to the understanding of gene retention in organellar genomes.
Deletion of the yeast mitochondrial gene
, encoding subunit 2 (mtCox2) of cytochrome
oxidase (C
O), results in a respiratory-incompetent
strain. For a cytosol-synthesized Cox2 to restore respiratory ...growth, it must carry the W56R mutation (cCox2
). Nevertheless, only a fraction of cCox2
is matured in mitochondria, allowing ∼60% steady-state accumulation of C
O. This can be attributed either to the point mutation or to an inefficient biogenesis of cCox2
. We generated a strain expressing the mutant protein mtCox2
inside mitochondria which should follow the canonical biogenesis of mitochondria-encoded Cox2. This strain exhibited growth rates, C
O steady-state levels, and C
O activity similar to those of the wild type; therefore, the efficiency of Cox2 biogenesis is the limiting step for successful allotopic expression. Upon coexpression of cCox2
and mtCox2, each protein assembled into C
O independently from its genetic origin, resulting in a mixed population of C
O with most complexes containing the mtCox2 version. Notably, the presence of the mtCox2 enhances cCox2
incorporation. We provide proof of principle that an allotopically expressed Cox2 may complement a phenotype due to a mutant mitochondrial
gene. These results are relevant to developing a rational design of genes for allotopic expression intended to treat human mitochondrial diseases.
The cox3 gene, encoding subunit III of cytochrome c oxidase (Cox3) is in mitochondrial genomes except in chlorophycean algae, where it is localized in the nucleus. Therefore, algae like Chlamydomonas ...reinhardtii, Polytomella sp. and Volvox carteri, synthesize the Cox3 polypeptide in the cytosol, import it into mitochondria, and integrate it into the cytochrome c oxidase complex. In this work, we followed the in vitro internalization of the Cox3 precursor by isolated, import-competent mitochondria of Polytomella sp. In this colorless alga, the precursor Cox3 protein is synthesized with a long, cleavable, N-terminal mitochondrial targeting sequence (MTS) of 98 residues. In an import time course, a transient Cox3 intermediate was identified, suggesting that the long MTS is processed more than once. The first processing step is sensitive to the metalo-protease inhibitor 1,10-ortophenantroline, suggesting that it is probably carried out by the matrix-located Mitochondrial Processing Protease. Cox3 is readily imported through an energy-dependent import pathway and integrated into the inner mitochondrial membrane, becoming resistant to carbonate extraction. Furthermore, the imported Cox3 protein was assembled into cytochrome c oxidase, as judged by the presence of a labeled band co-migrating with complex IV in Blue Native Electrophoresis. A model for the biogenesis of Cox3 in chlorophycean algae is proposed. This is the first time that the in vitro mitochondrial import of a cytosol-synthesized Cox3 subunit is described.
PDF
