No role for nuclear transcription regulators in mammalian mitochondria?

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.

High levels of TFAM repress mammalian mitochondrial DNA transcription in vivo.

Mitochondrial transcription factor A (TFAM) is compacting mitochondrial DNA (dmtDNA) into nucleoids and directly controls mtDNA copy number. Here, we show that the TFAM-to-mtDNA ratio is critical for maintaining normal mtDNA expression in different mouse tissues. Moderately increased TFAM protein levels increase mtDNA copy number but a normal TFAM-to-mtDNA ratio is maintained resulting in unaltered mtDNA expression and normal whole animal metabolism. Mice ubiquitously expressing very high TFAM levels develop pathology leading to deficient oxidative phosphorylation (OXPHOS) and early postnatal lethality. The TFAM-to-mtDNA ratio varies widely between tissues in these mice and is very high in skeletal muscle leading to strong repression of mtDNA expression and OXPHOS deficiency. In the heart, increased mtDNA copy number results in a near normal TFAM-to-mtDNA ratio and maintained OXPHOS capacity. In liver, induction of LONP1 protease and mitochondrial RNA polymerase expression counteracts the silencing effect of high TFAM levels. TFAM thus acts as a general repressor of mtDNA expression and this effect can be counterbalanced by tissue-specific expression of regulatory factors.

The mitoribosome-specific protein mS38 is preferentially required for synthesis of cytochrome c oxidase subunits.

Message-specific translational regulation mechanisms shape the biogenesis of multimeric oxidative phosphorylation (OXPHOS) enzyme in mitochondria from the yeast Saccharomyces cerevisiae. These mechanisms, driven mainly by the action of mRNA-specific translational activators, help to coordinate synthesis of OXPHOS catalytic subunits by the mitoribosomes with both the import of their nucleus-encoded partners and their assembly to form the holocomplexes. However, little is known regarding the role that the mitoribosome itself may play in mRNA-specific translational regulation. Here, we show that the mitoribosome small subunit protein Cox24/mS38, known to be necessary for mitoribosome-specific intersubunit bridge formation and 15S rRNA H44 stabilization, is required for efficient mitoribogenesis. Consequently, mS38 is necessary to sustain the overall mitochondrial protein synthesis rate, despite an adaptive ∼2-fold increase in mitoribosome abundance in mS38-deleted cells. Additionally, the absence of mS38 preferentially disturbs translation initiation of COX1, COX2, and COX3 mRNAs, without affecting the levels of mRNA-specific translational activators. We propose that mS38 confers the mitochondrial ribosome an intrinsic capacity of translational regulation, probably acquired during evolution from bacterial ribosomes to facilitate the translation of mitochondrial mRNAs, which lack typical anti-Shine-Dalgarno sequences.

The Cox1 C-terminal domain is a central regulator of cytochrome c oxidase biogenesis in yeast mitochondria.

Cytochrome c oxidase (CcO) is the last electron acceptor in the respiratory chain. The CcO core is formed by mitochondrial DNA-encoded Cox1, Cox2, and Cox3 subunits. Cox1 synthesis is highly regulated; for example, if CcO assembly is blocked, Cox1 synthesis decreases. Mss51 activates translation of COX1 mRNA and interacts with Cox1 protein in high-molecular-weight complexes (COA complexes) to form the Cox1 intermediary assembly module. Thus, Mss51 coordinates both Cox1 synthesis and assembly. We previously reported that the last 15 residues of the Cox1 C terminus regulate Cox1 synthesis by modulating an interaction of Mss51 with Cox14, another component of the COA complexes. Here, using site-directed mutagenesis of the mitochondrial COX1 gene from Saccharomyces cerevisiae, we demonstrate that mutations P521A/P522A and V524E disrupt the regulatory role of the Cox1 C terminus. These mutations, as well as C terminus deletion (Cox1ΔC15), reduced binding of Mss51 and Cox14 to COA complexes. Mss51 was enriched in a translationally active form that maintains full Cox1 synthesis even if CcO assembly is blocked in these mutants. Moreover, Cox1ΔC15, but not Cox1-P521A/P522A and Cox1-V524E, promoted formation of aberrant supercomplexes in CcO assembly mutants lacking Cox2 or Cox4 subunits. The aberrant supercomplex formation depended on the presence of cytochrome b and Cox3, supporting the idea that supercomplex assembly factors associate with Cox3 and demonstrating that supercomplexes can be formed even if CcO is inactive and not fully assembled. Our results indicate that the Cox1 C-terminal end is a key regulator of CcO biogenesis and that it is important for supercomplex formation/stability.

A Novel Function of Pet54 in Regulation of Cox1 Synthesis in Saccharomyces cerevisiae Mitochondria.

Cytochrome c oxidase assembly requires the synthesis of the mitochondria-encoded core subunits, Cox1, Cox2, and Cox3. In yeast, Pet54 protein is required to activate translation of the COX3 mRNA and to process the aI5ß intron on the COX1 transcript. Here we report a third, novel function of Pet54 on Cox1 synthesis. We observed that Pet54 is necessary to achieve an efficient Cox1 synthesis. Translation of the COX1 mRNA is coupled to the assembly of cytochrome c oxidase by a mechanism that involves Mss51. This protein activates translation of the COX1 mRNA by acting on the COX1 5'-UTR, and, in addition, it interacts with the newly synthesized Cox1 protein in high molecular weight complexes that include the factors Coa3 and Cox14. Deletion of Pet54 decreased Cox1 synthesis, and, in contrast to what is commonly observed for other assembly mutants, double deletion of cox14 or coa3 did not recover Cox1 synthesis. Our results show that Pet54 is a positive regulator of Cox1 synthesis that renders Mss51 competent as a translational activator of the COX1 mRNA and that this role is independent of the assembly feedback regulatory loop of Cox1 synthesis. Pet54 may play a role in Mss51 hemylation/conformational change necessary for translational activity. Moreover, Pet54 physically interacts with the COX1 mRNA, and this binding was independent of the presence of Mss51.

The cytosol-synthesized subunit II (Cox2) precursor with the point mutation W56R is correctly processed in yeast mitochondria to rescue cytochrome oxidase.

Deletion of the yeast mitochondrial gene COX2 encoding subunit 2 (Cox2) of cytochrome c oxidase (CcO) results in loss of respiration (Δcox2 strain). Supekova et al. (2010) [1] transformed a Δcox2 strain with a vector expressing Cox2 with a mitochondrial targeting sequence (MTS) and the point mutation W56R (Cox2(W56R)), restoring respiratory growth. Here, the CcO carrying the allotopically-expressed Cox2(W56R) was characterized. Yeast mitochondria from the wild-type (WT) and the Δcox2+Cox2(W56R) strains were subjected to Blue Native electrophoresis. In-gel activity of CcO and spectroscopic quantitation of cytochromes revealed that only 60% of CcO is present in the complemented strain, and that less CcO is found associated in supercomplexes as compared to WT. CcOs from the WT and the mutant exhibited similar subunit composition, although activity was 20-25% lower in the enzyme containing Cox2(W56R) than in the one with Cox2(WT). Tandem mass spectrometry confirmed that W(56) was substituted by R(56) in Cox2(W56R). In addition, Cox2(W56R) exhibited the same N-terminus than Cox2(WT), indicating that the MTS of Oxa1 and the leader sequence of 15 residues were removed from Cox2(W56R) during maturation. Thus, Cox2(W56R) is identical to Cox2(WT) except for the point mutation W56R. Mitochondrial Cox1 synthesis is strongly reduced in Δcox2 mutants, but the Cox2(W56R) complemented strain led to full restoration of Cox1 synthesis. We conclude that the cytosol-synthesized Cox2(W56R) follows a rate-limiting process of import, maturation or assembly that yields lower steady-state levels of CcO. Still, the allotopically-expressed Cox2(W56R) restores CcO activity and allows mitochondrial Cox1 synthesis to advance at WT levels.