Mitochondrial Transformation in Baker's Yeast to Study Translation and Respiratory Complex Assembly.

Baker´s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about essential, conserved mitochondrial pathways among eukaryotes, and fungi or yeast-specific pathways. One of the many abilities of S.

The ARG8 Reporter for the Study of Yeast Mitochondrial Translation.

Mitochondrial translation is an intricate process involving both general and mRNA-specific factors. In addition, in the yeast Saccharomyces cerevisiae, translation of mitochondrial mRNAs is coupled to assembly of nascent polypeptides into the membrane. ARG8 is a reporter gene widely used to study the mechanisms of yeast mitochondrial translation.

The cytochrome carboxyl terminal region is necessary for mitochondrial complex III assembly.

Mitochondrial complex from yeast has 10 subunits, but only cytochrome (Cyt) subunit is encoded in the mitochondrial genome. Cyt has eight transmembrane helices containing two hemes for electron transfer. Cbp3 and Cbp6 assist Cyt synthesis, and together with Cbp4 induce Cyt hemylation.

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.

Cbp3 and Cbp6 are dispensable for synthesis regulation of cytochrome in yeast mitochondria.

Cytochrome (Cyt) is the only mitochondrial encoded subunit from the complex. Cbp3 and Cbp6 are chaperones necessary for translation of the mRNA and Cyt hemylation. Here we demonstrate that their role in translation is dispensable in some laboratory strains, whereas their role in Cyt hemylation seems to be universally conserved.

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

Cytochrome oxidase (CO) is the last electron acceptor in the respiratory chain. The CO core is formed by mitochondrial DNA-encoded Cox1, Cox2, and Cox3 subunits. Cox1 synthesis is highly regulated; for example, if CO assembly is blocked, Cox1 synthesis decreases.

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 aI5β intron on the COX1 transcript. Here we report a third, novel function of Pet54 on Cox1 synthesis.

The Pet309 pentatricopeptide repeat motifs mediate efficient binding to the mitochondrial COX1 transcript in yeast.

Mitochondrial synthesis of Cox1, the largest subunit of the cytochrome c oxidase complex, is controlled by Mss51 and Pet309, two mRNA-specific translational activators that act via the COX1 mRNA 5'-UTR through an unknown mechanism. Pet309 belongs to the pentatricopeptide repeat (PPR) protein family, which is involved in RNA metabolism in mitochondria and chloroplasts, and its sequence predicts at least 12 PPR motifs in the central portion of the protein. Deletion of these motifs selectively disrupted translation but not accumulation of the COX1 mRNA.

The carboxyl-terminal end of Cox1 is required for feedback assembly regulation of Cox1 synthesis in Saccharomyces cerevisiae mitochondria.

Synthesis of the largest cytochrome c oxidase (CcO) subunit, Cox1, on yeast mitochondrial ribosomes is coupled to assembly of CcO. The translational activator Mss51 is sequestered in early assembly intermediate complexes by an interaction with Cox14 that depends on the presence of newly synthesized Cox1. If CcO assembly is prevented, the level of Mss51 available for translational activation is reduced.

Protein synthesis and assembly in mitochondrial disorders.

Human mitochondrial DNA (mtDNA) codes for 13 polypeptides which constitute the central core of the oxidative phosphorylation (OXPHOS) complexes. The machinery for mitochondrial protein synthesis has a dual origin: a full set of tRNAs, as well as the 12S and 16S rRNAs are encoded in the mitochondrial genome, while most factors necessary for translation are encoded by nuclear genes. The mitochondrial translation apparatus is highly specialized in expressing membrane proteins, and couples the synthesis of proteins to the insertion into the mitochondrial inner membrane.

The pentatricopeptide repeats present in Pet309 are necessary for translation but not for stability of the mitochondrial COX1 mRNA in yeast.

Pet309 is a protein essential for respiratory growth. It is involved in translation of the yeast mitochondrial COX1 gene, which encodes subunit I of the cytochrome c oxidase. Pet309 is also involved in stabilization of the COX1 mRNA.