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Mitochondrial methionyl N-formylation affects steady-state levels of oxidative phosphorylation complexes and their organization into supercomplexes.

ABSTRACT: N-Formylation of the Met-tRNA^Met by the nuclearly encoded mitochondrial methionyl-tRNA formyltransferase (MTFMT) has been found to be a key determinant of protein synthesis initiation in mitochondria. In humans, mutations in the MTFMT gene result in Leigh syndrome, a progressive and severe neurometabolic disorder. However, the absolute requirement of formylation of Met-tRNA^Met for protein synthesis in mammalian mitochondria is still debated. Here, we generated a Mtfmt-KO mouse fibroblast cell line and demonstrated that N-formylation of the first methionine via fMet-tRNA^Met by MTFMT is not an absolute requirement for initiation of protein synthesis. However, it differentially affected the efficiency of synthesis of mtDNA-coded polypeptides. Lack of methionine N-formylation did not compromise the stability of these individual subunits but had a marked effect on the assembly and stability of the OXPHOS complexes I and IV and on their supercomplexes. In summary, N-formylation is not essential for mitochondrial protein synthesis but is critical for efficient synthesis of several mitochondrially encoded peptides and for OXPHOS complex stability and assembly into supercomplexes.

SUBMITTER: Arguello T

PROVIDER: S-EPMC6166738 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Mitochondrial methionyl N-formylation affects steady-state levels of oxidative phosphorylation complexes and their organization into supercomplexes.

Arguello Tania T Köhrer Caroline C RajBhandary Uttam L UL Moraes Carlos T CT

The Journal of biological chemistry 20180807 39

N-Formylation of the Met-tRNAMet by the nuclearly encoded mitochondrial methionyl-tRNA formyltransferase (MTFMT) has been found to be a key determinant of protein synthesis initiation in mitochondria. In humans, mutations in the MTFMT gene result in Leigh syndrome, a progressive and severe neurometabolic disorder. However, the absolute requirement of formylation of Met-tRNAMet for protein synthesis in mammalian mitochondria is still debated. Here, we generated ...[more]

PMID: 30087118

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Project description:Mitochondrial protein phosphorylation is a well-recognized metabolic control mechanism, with the classical example of pyruvate dehydrogenase (PDH) regulation by specific kinases and phosphatases of bacterial origin. However, despite the growing number of reported mitochondrial phosphoproteins, the identity of the protein kinases mediating these phosphorylation events remains largely unknown. The detection of mitochondrial protein kinases is complicated by the low concentration of kinase relative to that of the target protein, the lack of specific antibodies, and contamination from associated, but nonmatrix, proteins. In this study, we use blue native gel electrophoresis (BN-PAGE) to isolate rat and porcine heart mitochondrial complexes for screening of protein kinase activity. To detect kinase activity, one-dimensional BN-PAGE gels were exposed to [?-(32)P]ATP and then followed by sodium dodecyl sulfate gel electrophoresis. Dozens of mitochondrial proteins were labeled with (32)P in this setting, including all five complexes of oxidative phosphorylation and several citric acid cycle enzymes. The nearly ubiquitous (32)P protein labeling demonstrates protein kinase activity within each mitochondrial protein complex. The validity of this two-dimensional BN-PAGE method was demonstrated by detecting the known PDH kinases and phosphatases within the PDH complex band using Western blots and mass spectrometry. Surprisingly, these same approaches detected only a few additional conventional protein kinases, suggesting a major role for autophosphorylation in mitochondrial proteins. Studies on purified Complex V and creatine kinase confirmed that these proteins undergo autophosphorylation and, to a lesser degree, tenacious (32)P-metabolite association. In-gel Complex IV activity was shown to be inhibited by ATP, and partially reversed by phosphatase activity, consistent with an inhibitory role for protein phosphorylation in this complex. Collectively, this study proposes that many of the mitochondrial complexes contain an autophosphorylation mechanism, which may play a functional role in the regulation of these multiprotein units.

Dataset Information

Mitochondrial methionyl N-formylation affects steady-state levels of oxidative phosphorylation complexes and their organization into supercomplexes.

Publications

Mitochondrial methionyl <i>N</i>-formylation affects steady-state levels of oxidative phosphorylation complexes and their organization into supercomplexes.

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