Importance of conserved hydrophobic pocket region in yeast mitoribosomal mL44 protein for mitotranslation and transcript preference

The mitochondrial ribosome (mitoribosome) is responsible for the synthesis of key oxidative phosphorylation subunits encoded by the mitochondrial genome. Defects in mitoribosomal function therefore can have serious consequences for the bioenergetic capacity of the cell. Mutation of the conserved mitoribosomal mL44 protein has been directly linked to childhood cardiomyopathy and progressive neurophysiology issues. To further explore the functional significance of the mL44 protein in supporting mitochondrial protein synthesis, we have performed a mutagenesis study of the yeast mL44 homolog, the MrpL3/mL44 protein. We specifically investigated the conserved hydrophobic pocket region of the MrpL3/mL44 protein, where the known disease-related residue in the human mL44 protein (L156R) is located. While our findings identify a number of residues in this region critical for MrpL3/mL44's ability to support the assembly of translationally active mitoribosomes, the introduction of the disease-related mutation into the equivalent position in the yeast protein (residue A186) was found to not have a major impact on function. The human and yeast mL44 proteins share many similarities in sequence and structure; however results presented here indicate that these two proteins have diverged somewhat in evolution. Finally, we observed that mutation of the MrpL3/mL44 does not impact the translation of all mitochondrial encoded proteins equally, suggesting the mitochondrial translation system may exhibit a transcript hierarchy and prioritization. The mitochondrial ribosome (mitoribosome) is responsible for the synthesis of key oxidative phosphorylation subunits encoded by the mitochondrial genome. Defects in mitoribosomal function therefore can have serious consequences for the bioenergetic capacity of the cell. Mutation of the conserved mitoribosomal mL44 protein has been directly linked to childhood cardiomyopathy and progressive neurophysiology issues. To further explore the functional significance of the mL44 protein in supporting mitochondrial protein synthesis, we have performed a mutagenesis study of the yeast mL44 homolog, the MrpL3/mL44 protein. We specifically investigated the conserved hydrophobic pocket region of the MrpL3/mL44 protein, where the known disease-related residue in the human mL44 protein (L156R) is located. While our findings identify a number of residues in this region critical for MrpL3/mL44's ability to support the assembly of translationally active mitoribosomes, the introduction of the disease-related mutation into the equivalent position in the yeast protein (residue A186) was found to not have a major impact on function. The human and yeast mL44 proteins share many similarities in sequence and structure; however results presented here indicate that these two proteins have diverged somewhat in evolution. Finally, we observed that mutation of the MrpL3/mL44 does not impact the translation of all mitochondrial encoded proteins equally, suggesting the mitochondrial translation system may exhibit a transcript hierarchy and prioritization. ATP, the vital energy source for the cell's diverse metabolic needs, is produced by mitochondria through the process of oxidative phosphorylation (OXPHOS) (1Stuart R.A. Supercomplex organization of the oxidative phosphorylation enzymes in yeast mitochondria.J. Bioenerg. Biomembr. 2008; 40: 411-417Crossref PubMed Scopus (87) Google Scholar, 2Wai T. Langer T. Mitochondrial dynamics and metabolic regulation.Trends Endocrinol. Metab. 2016; 27: 105-117Abstract Full Text Full Text PDF PubMed Scopus (906) Google Scholar, 3Area-Gomez E. Schon E.A. 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A small, but important, number of the OXPHOS core enzymatic subunits are encoded on the mitochondrial genome (mtDNA) and synthesized by mitochondrial ribosomes (mitoribosomes) located in the mitochondrial matrix and tethered to the inner membrane. In the yeast Saccharomyces cerevisiae (S. cerevisiae), the model organism of this study, the mtDNA encodes components of the cytochrome bc1, cytochrome c oxidase (COX), and F1Fo-ATP synthase enzymes (6Borst P. Grivell L.A. The mitochondrial genome of yeast.Cell. 1978; 15: 705-723Abstract Full Text PDF PubMed Scopus (140) Google Scholar). The biogenesis of these OXPHOS enzymes is therefore dependent on mitochondria having functioning mitoribosomes. Consequently, defects in the mitotranslational process have the potential to negatively impact OXPHOS complex assembly and thus the bioenergetic capacity of the cell (7Ferrari A. Del'Olio S. Barrientos A. 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The molecular analysis of mitoribosomes, their assembly, function, and regulation is therefore crucial to our understanding of eukaryotic cell viability and metabolic regulation. The mitoribosomes are compositionally distinct from their cytosolic counterparts and are composed of rRNA encoded by the mtDNA and proteins which, except for Var1 of the small mitoribosomal subunit in the S. cerevisiae, are encoded by nuclear genes. Mammalian mitoribosomes also contain a structural tRNA component (17Amunts A. Brown A. Bai X.C. Llacer J.L. Hussain T. Emsley P. et al.Structure of the yeast mitochondrial large ribosomal subunit.Science. 2014; 343: 1485-1489Crossref PubMed Scopus (461) Google Scholar, 18Greber B.J. Boehringer D. Leibundgut M. Bieri P. Leitner A. Schmitz N. et al.The complete structure of the large subunit of the mammalian mitochondrial ribosome.Nature. 2014; 515: 283-286Crossref PubMed Scopus (202) Google Scholar, 19Chrzanowska-Lightowlers Z. Rorbach J. Minczuk M. 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Strom T.M. et al.MRPL44 mutations cause a slowly progressive multisystem disease with childhood-onset hypertrophic cardiomyopathy.Neurogenetics. 2015; 16: 319-323Crossref PubMed Google Scholar, 15Yeo J.H. Skinner J.P. Bird M.J. Formosa L.E. Zhang J.G. Kluck R.M. et al.A role for the mitochondrial protein Mrpl44 in maintaining OXPHOS capacity.PLoS One. 2015; 10e0134326Crossref Scopus (12) Google Scholar, 21Mai N. Chrzanowska-Lightowlers Z.M. Lightowlers R.N. The process of mammalian mitochondrial protein synthesis.Cell Tissue Res. 2017; 367: 5-20Crossref PubMed Scopus (99) Google Scholar, 22Lightowlers R.N. Taylor R.W. Turnbull D.M. Mutations causing mitochondrial disease: what is new and what challenges remain?.Science. 2015; 349: 1494-1499Crossref PubMed Scopus (232) Google Scholar, 23Kemp J.P. Smith P.M. Pyle A. Neeve V.C. Tuppen H.A. 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On the other hand, mutations in nuclear-encoded genes can also genetically underlie mitochondrial translation defects, for example, in genes encoding mitochondrial aminoacyl-tRNA synthetases and tRNA-modifying enzymes, the mitoribosomal protein components themselves, and/or nuclear-encoded protein factors involved in mitotranslation initiation, elongation, and termination. The nuclear-encoded mitoribosomal protein mL44 is a component of the large mitoribosomal subunit conserved from fungi through humans. The mL44 protein is termed a mitospecific mitoribosomal protein because the mL44 protein family is found only in mitochondrial ribosomes and not present in their prokaryotic ancestors, the bacterial ribosome (17Amunts A. Brown A. Bai X.C. Llacer J.L. Hussain T. Emsley P. et al.Structure of the yeast mitochondrial large ribosomal subunit.Science. 2014; 343: 1485-1489Crossref PubMed Scopus (461) Google Scholar, 18Greber B.J. Boehringer D. Leibundgut M. Bieri P. Leitner A. Schmitz N. et al.The complete structure of the large subunit of the mammalian mitochondrial ribosome.Nature. 2014; 515: 283-286Crossref PubMed Scopus (202) Google Scholar, 26Amunts A. Brown A. Toots J. Scheres S.H.W. Ramakrishnan V. Ribosome. The structure of the human mitochondrial ribosome.Science. 2015; 348: 95-98Crossref PubMed Scopus (392) Google Scholar, 27Desai N. Brown A. Amunts A. Ramakrishnan V. The structure of the yeast mitochondrial ribosome.Science. 2017; 355: 528-531Crossref PubMed Scopus (150) Google Scholar). The mitospecific nature of the mL44 protein family suggests that this protein may play an important structural and/or regulatory role unique to mitochondrial ribosomes, rather than playing an integral part in the protein synthesis peptidyltransferase reaction per se. The mL44 protein is located in the membrane protuberance region of mitoribosomes adjacent to the polypeptide exit site where the nascent chain emerges from the ribosome (Fig. S1A) (17Amunts A. Brown A. Bai X.C. Llacer J.L. Hussain T. Emsley P. et al.Structure of the yeast mitochondrial large ribosomal subunit.Science. 2014; 343: 1485-1489Crossref PubMed Scopus (461) Google Scholar, 18Greber B.J. Boehringer D. Leibundgut M. Bieri P. Leitner A. Schmitz N. et al.The complete structure of the large subunit of the mammalian mitochondrial ribosome.Nature. 2014; 515: 283-286Crossref PubMed Scopus (202) Google Scholar, 20Brown A. Amunts A. Bai X.C. Sugimoto Y. Edwards P.C. Murshudov G. et al.Structure of the large ribosomal subunit from human mitochondria.Science. 2014; 346: 718-722Crossref PubMed Scopus (244) Google Scholar, 26Amunts A. Brown A. Toots J. Scheres S.H.W. Ramakrishnan V. Ribosome. The structure of the human mitochondrial ribosome.Science. 2015; 348: 95-98Crossref PubMed Scopus (392) Google Scholar, 27Desai N. Brown A. Amunts A. Ramakrishnan V. The structure of the yeast mitochondrial ribosome.Science. 2017; 355: 528-531Crossref PubMed Scopus (150) Google Scholar, 28Greber B.J. Bieri P. Leibundgut M. Leitner A. Aebersold R. Boehringer D. et al.Ribosome. The complete structure of the 55S mammalian mitochondrial ribosome.Science. 2015; 348: 303-308Crossref PubMed Scopus (313) Google Scholar, 29Greber B.J. Boehringer D. Leitner A. Bieri P. Voigts-Hoffmann F. Erzberger J.P. et al.Architecture of the large subunit of the mammalian mitochondrial ribosome.Nature. 2014; 505: 515-519Crossref PubMed Scopus (185) Google Scholar). The membrane protuberance region has been proposed to support the membrane anchoring of mitoribosomes, which enables the cotranslational insertion of the resulting nascent chains into the mitochondrial inner membrane (20Brown A. Amunts A. Bai X.C. Sugimoto Y. Edwards P.C. Murshudov G. et al.Structure of the large ribosomal subunit from human mitochondria.Science. 2014; 346: 718-722Crossref PubMed Scopus (244) Google Scholar). Mitochondrial ribosomes are assembled directly and are permanently tethered to the membrane and unlike bacterial ribosomes, do not cycle on and off the membrane (12Nolden M. Ehses S. Koppen M. Bernacchia A. Rugarli E.I. Langer T. The m-AAA protease defective in hereditary spastic paraplegia controls ribosome assembly in mitochondria.Cell. 2005; 123: 277-289Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 30Kaur J. Stuart R.A. Truncation of the Mrp20 protein reveals new ribosome-assembly subcomplex in mitochondria.EMBO Rep. 2011; 12: 950-955Crossref PubMed Scopus (25) Google Scholar, 31Zeng R. Smith E. Barrientos A. Yeast mitoribosome large subunit assembly proceeds by hierarchical incorporation of protein clusters and modules on the inner membrane.Cell Metab. 2018; 27: 645-656.e647Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Mutation of the human MRPL44/mL44 gene has been reported to be directly linked to childhood-onset hypertrophic cardiomyopathy and progressive multi-system disease (13Carroll C.J. Isohanni P. Poyhonen R. Euro L. Richter U. Brilhante V. et al.Whole-exome sequencing identifies a mutation in the mitochondrial ribosome protein MRPL44 to underlie mitochondrial infantile cardiomyopathy.J. Med. Genet. 2013; 50: 151-159Crossref PubMed Scopus (81) Google Scholar, 14Distelmaier F. Haack T.B. Catarino C.B. Gallenmuller C. Rodenburg R.J. Strom T.M. et al.MRPL44 mutations cause a slowly progressive multisystem disease with childhood-onset hypertrophic cardiomyopathy.Neurogenetics. 2015; 16: 319-323Crossref PubMed Google Scholar, 15Yeo J.H. Skinner J.P. Bird M.J. Formosa L.E. Zhang J.G. Kluck R.M. et al.A role for the mitochondrial protein Mrpl44 in maintaining OXPHOS capacity.PLoS One. 2015; 10e0134326Crossref Scopus (12) Google Scholar, 32Friederich M.W. Geddes G.C. Wortmann S.B. Punnoose A. Wartchow E. Knight K.M. et al.Pathogenic variants in MRPL44 cause infantile cardiomyopathy due to a mitochondrial translation defect.Mol. Genet. Metab. 2021; 133: 362-371Crossref PubMed Scopus (9) Google Scholar, 33Horga A. Manole A. Mitchell A.L. Bugiardini E. Hargreaves I.P. Mowafi W. et al.Uniparental isodisomy of chromosome 2 causing MRPL44-related multisystem mitochondrial disease.Mol. Biol. Rep. 2021; 48: 2093-2104Crossref PubMed Scopus (3) Google Scholar). The MRPL44/mL44 mutation underlying this pathophysiological presentation was shown to be a homozygous missense mutation in codon 156 encoding the hydrophobic residue Leu(L)156 and where it was mutated to encode the polar Arg(R) residue and termed the L156R mutation (13Carroll C.J. Isohanni P. Poyhonen R. Euro L. Richter U. Brilhante V. et al.Whole-exome sequencing identifies a mutation in the mitochondrial ribosome protein MRPL44 to underlie mitochondrial infantile cardiomyopathy.J. Med. Genet. 2013; 50: 151-159Crossref PubMed Scopus (81) Google Scholar, 14Distelmaier F. Haack T.B. Catarino C.B. Gallenmuller C. Rodenburg R.J. Strom T.M. et al.MRPL44 mutations cause a slowly progressive multisystem disease with childhood-onset hypertrophic cardiomyopathy.Neurogenetics. 2015; 16: 319-323Crossref PubMed Google Scholar). Measurements of OXPHOS levels in fibroblasts isolated from patients bearing the homozygous L156R mutation indicated they displayed strongly decreased OXPHOS capacity, in particular levels of the terminal COX enzyme (complex IV, COX) (13Carroll C.J. Isohanni P. Poyhonen R. Euro L. Richter U. Brilhante V. et al.Whole-exome sequencing identifies a mutation in the mitochondrial ribosome protein MRPL44 to underlie mitochondrial infantile cardiomyopathy.J. Med. Genet. 2013; 50: 151-159Crossref PubMed Scopus (81) Google Scholar, 15Yeo J.H. Skinner J.P. Bird M.J. Formosa L.E. Zhang J.G. Kluck R.M. et al.A role for the mitochondrial protein Mrpl44 in maintaining OXPHOS capacity.PLoS One. 2015; 10e0134326Crossref Scopus (12) Google Scholar, 32Friederich M.W. Geddes G.C. Wortmann S.B. Punnoose A. Wartchow E. Knight K.M. et al.Pathogenic variants in MRPL44 cause infantile cardiomyopathy due to a mitochondrial translation defect.Mol. Genet. Metab. 2021; 133: 362-371Crossref PubMed Scopus (9) Google Scholar). A reduction in mitochondrial translation output levels was also reported for cells harboring the MRPL44/mL44 L156R mutation (15Yeo J.H. Skinner J.P. Bird M.J. Formosa L.E. Zhang J.G. Kluck R.M. et al.A role for the mitochondrial protein Mrpl44 in maintaining OXPHOS capacity.PLoS One. 2015; 10e0134326Crossref Scopus (12) Google Scholar). Although these clinical studies highlight the importance of MRPL44/mL44 protein, the molecular basis for the defect underlying the measured loss of OXPHOS activity caused by the L156R mutation, remains unknown. In this study, we have analyzed the yeast S. cerevisiae mL44 protein, which is termed MrpL3/mL44. The MrpL3/mL44 protein has been previously demonstrated to be required for mitotranslation and thus for cellular respiration in yeast (31Zeng R. Smith E. Barrientos A. Yeast mitoribosome large subunit assembly proceeds by hierarchical incorporation of protein clusters and modules on the inner membrane.Cell Metab. 2018; 27: 645-656.e647Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The yeast and human mL44 proteins share sequence similarity, and available cryoEM analysis of both yeast and mammalian mitoribosomes indicate that they also share considerable structural similarity. The pathogenic mL44 mutation L156R is located in a hydrophobic pocket region conserved between all mL44 proteins (17Amunts A. Brown A. Bai X.C. Llacer J.L. Hussain T. Emsley P. et al.Structure of the yeast mitochondrial large ribosomal subunit.Science. 2014; 343: 1485-1489Crossref PubMed Scopus (461) Google Scholar, 18Greber B.J. Boehringer D. Leibundgut M. Bieri P. Leitner A. Schmitz N. et al.The complete structure of the large subunit of the mammalian mitochondrial ribosome.Nature. 2014; 515: 283-286Crossref PubMed Scopus (202) Google Scholar, 20Brown A. Amunts A. Bai X.C. Sugimoto Y. Edwards P.C. Murshudov G. et al.Structure of the large ribosomal subunit from human mitochondria.Science. 2014; 346: 718-722Crossref PubMed Scopus (244) Google Scholar, 27Desai N. Brown A. Amunts A. Ramakrishnan V. The structure of the yeast mitochondrial ribosome.Science. 2017; 355: 528-531Crossref PubMed Scopus (150) Google Scholar). The yeast system provides genetic and biochemical advantages to probe the molecular consequences of disease-related mitoribosomal mutations. Due to their ability to thrive under fermentation conditions when grown on suitable carbon sources (e.g., glucose or galactose), respiratory-deficient yeast mutants caused by impaired mitoribosomal function remain viable when grown under fermentation conditions, enabling their further biochemical analysis. In this study, we have created a genetic system to analyze the yeast MrpL3/mL44 protein and to model the L156R mutation of the human MRPL44/mL44 protein in its yeast counterpart. We report here that while yeast MrpL3/mL44 and human MRPL44/mL44 proteins share many similarities, the corresponding disease-related mutation, L156R, does not greatly impact the function of the yeast MrpL3/mL44 protein. Furthermore, we have used the yeast MrpL3/mL44 genetic system to further probe conserved aspects of the hydrophobic pocket of the mL44 proteins where the disease-related mutation is located. We report here on the isolation of a number of temperature-sensitive mutations in the MrpL3/mL44 protein, which significantly impair the respiratory viability of the cell at stress temperatures. Our results demonstrate the importance of the conserved hydrophobic pocket region of the mL44 protein for supporting the assembly and hence translational activity of the mitoribosome. Our analysis also uncovered mutations of the MrpL3/mL44 that result in a novel phenotype, elevated levels of Cox2 synthesis, and COX complex accumulation, which was correlated with decreased cellular viability under fermentation conditions. Finally, our findings shed important light on how the human and yeast mitoribosomes may have evolutionarily diverged with respect to their mL44 proteins and membrane protuberances. In the human MRPL44/mL44, the disease-related residue L156 is located in a highly hydrophobic pocket environment, largely composed of two adjacent alpha (α)-helical segments, with hydrophobic aspects of the mL44 protein (13Carroll C.J. Isohanni P. Poyhonen R. Euro L. Richter U. Brilhante V. et al.Whole-exome sequencing identifies a mutation in the mitochondrial ribosome protein MRPL44 to underlie mitochondrial infantile cardiomyopathy.J. Med. Genet. 2013; 50: 151-159Crossref PubMed Scopus (81) Google Scholar, 20Brown A. Amunts A. Bai X.C. Sugimoto Y. Edwards P.C. Murshudov G. et al.Structure of the large ribosomal subunit from human mitochondria.Science. 2014; 346: 718-722Crossref PubMed Scopus (244) Google Scholar). The α-helical segment arrangement (termed here "α-helix #1-turn-α-helix #2") with its formed hydrophobic pocket are elements conserved between the mammalian mL44 proteins and their yeast homolog MrpL3/mL44 (Fig. S1B). The introduction of a charged residue through the disease related L156R mutation has been viewed as incompatible with the hydrophobic nature of this mL44 pocket and predicted to cause a perturbation in the overall structure of the MRPL44/mL44 protein (13Carroll C.J. Isohanni P. Poyhonen R. Euro L. Richter U. Brilhante V. et al.Whole-exome sequencing identifies a mutation in the mitochondrial ribosome protein MRPL44 to underlie mitochondrial infantile cardiomyopathy.J. Med. Genet. 2013; 50: 151-159Crossref PubMed Scopus (81) Google Scholar, 20Brown A. Amunts A. Bai X.C. Sugimoto Y. Edwards P.C. Murshudov G. et al.Structure of the large ribosomal subunit from human mitochondria.Science. 2014; 346: 718-722Crossref PubMed Scopus (244) Google Scholar). In the yeast MrpL3/mL44 protein, the α-helix #1-turn-α-helix #2 region is comprised of residues 151-202 (Fig. 1, A and B). Sequence alignment of yeast MrpL3/mL44 and human MRPL44/mL44 indicates that residue Ala(A)186 of MrpL3/mL44, a small hydrophobic residue, is present in the equivalent position of L156 from the human MRPL44/mL44 protein (Fig. 1, A and B). The functional significance of the α-helix #1-turn-α-helix #2 region of mL44 was addressed in this study using the yeast MrpL3/mL44 protein as a model system. The disease-equivalent residue located in this region, that is, A186 of yeast MrpL3/mL44, was initially targeted for site-directed mutagenesis. We created three A186 mutant mrpL3 derivatives, where the codon encoding A186 was mutated either to one encoding a positively charged residue Arg(R) or Lys(K) residue (to mimic the L156R disease mutation of MRPL44/mL44) or exchanged with one encoding another hydrophobic residue, Leu(L) (to resemble the WT human MRPL44/mL44 residue L156, thereby retaining the hydrophobic nature of the pocket). The A186 mutant mrpL3 derivatives were expressed in yeast and tested for their ability to functionally substitute for the WT MrpL3/mL44 protein (Fig. 2). The resulting strains, mrpL3A186R, mrpL3A186K, and mrpL3A186L were first grown on the nonfermentable carbon source glycerol to test for their ability to support oxidative phosphorylation-based cellular metabolism. Consistent with previous reports (31Zeng R. Smith E. Barrientos A. Yeast mitoribosome large subunit assembly proceeds by hierarchical incorporation of protein clusters and modules on the inner membrane.Cell Metab. 2018; 27: 645-656.e647Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), the presence of MrpL3/mL44 was found to be required for yeast respiratory-based growth (Fig. 2A). A serial dilution-based growth analysis indicated that all three mrpL3 mutant strains appeared fully capable of supporting respiratory-based yeast growth at the optimal and stress growth temperatures for yeast, 30 °C and 37 °C, respectively (Fig. 2A). Fermentation-based growth (glucose media) also appeared normal for the mrpL3 mutant strains and closely mirrored that of the corresponding MrpL3/mL44 WT control analyzed in parallel. Mitochondrial translation capacities of the mrpL3 A186 strains were next directly analyzed through in vivo radiolabeling in the presence of [35S]methionine (and additional cycloheximide to inhibit cytosolic ribosomal activity) (Fig. 2B). Analysis was performed on cells grown in glycerol/ethanol media (OXPHOS-demanding conditions) and at 37 °C, an elevated temperature to stress the cells with the hope of teasing out a translational phenotype if it existed in the mrpL3 A186 mutants. The synthesis of each of the mtDNA-encoded proteins, Var1 (a component of the small ribosomal subunit), Cox1, Cox2, and Cox3 of the COX complex, cytochrome b of the cytochrome bc1 enzyme, and Fo-ATP synthase subunits Atp6, Atp8, and Atp9, was observed in the three mrpL3 A186 mutants analyzed. Both the levels and profiles of radiolabeled proteins synthesized in the mrpL3A186R, mrpL3A186K, and mrpL3A186L strains were found to be similar to those of the control WT MrpL3/mL44 cells, suggesting that mitotranslation was not limiting for OXPHOS assembly in these mutants (Fig. 2B, upper panel and Fig. 2C). Western blotting and immunedecoration of the resulting cell extracts with antibodies against key marker subunits of the OXPHOS enzymes confirmed this. The steady state levels of Cox2 (COX complex), Qcr7 (cytochrome bc1 complex), and the Fo-subunit Atp9 (F1Fo-ATP synthase) indicated that the cellular levels of the OXPHOS enzymes in all three of the mrpL3 A186 mutants appeared similar to those of the MrpL3/mL44 WT control (Fig. 2B, lower panel and Fig. S2). The cellular level of these specific OXPHOS subunits was used as an indicator of OXPHOS complex levels in this study, as each of the marker proteins analyzed are proteolytically turned over if the assembly of their respective OXPHOS complex is defective (34Anderson J.M. Box J.M. Stuart R.A. The N-terminal region of yeast mitoribosomal Mrp7/bL27m protein serves to optimize translation of nascent chains competent for OXPHOS complex assembly.FEBS Lett. 2023; 597: 1579-1594Crossref PubMed Scopus (1) Google Scholar, 35Box J.M. Anderson J.M. Stuart R.A. Mutation of the PEBP-like domain of the