Allotopic expression of elucidates Atco-driven co-assembly of cytochrome oxidase and ATP synthase.

The Cox6 subunit of cytochrome oxidase (COX) and the Atp9 subunit of the ATP synthase are encoded in nuclear and mitochondrial DNA, respectively. The two proteins interact to form Atco complexes that serve as the source of Atp9 for ATP synthase assembly. To determine if Atco is also a precursor of COX, we pulse-labeled Cox6 in isolated mitochondria of a nuclear mutant with in mitochondrial DNA.

Atco, a yeast mitochondrial complex of Atp9 and Cox6, is an assembly intermediate of the ATP synthase.

Mitochondrial oxidative phosphorylation (oxphos) is the process by which the ATP synthase conserves the energy released during the oxidation of different nutrients as ATP. The yeast ATP synthase consists of three assembly modules, one of which is a ring consisting of 10 copies of the Atp9 subunit. We previously reported the existence in yeast mitochondria of high molecular weight complexes composed of mitochondrially encoded Atp9 and of Cox6, an imported structural subunit of cytochrome oxidase (COX).

Modular assembly of yeast mitochondrial ATP synthase and cytochrome oxidase.

The respiratory pathway of mitochondria is composed of four electron transfer complexes and the ATP synthase. In this article, we review evidence from studies of Saccharomyces cerevisiae that both ATP synthase and cytochrome oxidase (COX) are assembled from independent modules that correspond to structurally and functionally identifiable components of each complex. Biogenesis of the respiratory chain requires a coordinate and balanced expression of gene products that become partner subunits of the same complex, but are encoded in the two physically separated genomes.

5' processing of Saccharomyces cerevisiae mitochondrial tRNAs requires expression of multiple genes.

Mitochondrial tRNAs are processed at their 5'ends by highly divergent but ubiquitous RNase P. In Saccharomyces cerevisiae, Rpm2p is the protein component of RNase P. Here, we identify four novel genes MTA1, MTA2, GEP5 and PET130 of the Saccharomycetaceae family that are necessary for an efficient processing of mitochondrial tRNAs.

Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules.

Cytochrome oxidase (COX) is a hetero-oligomeric complex of the mitochondrial inner membrane that reduces molecular oxygen to water, a reaction coupled to proton transfer from the mitochondrial matrix to the intermembrane space. In the yeast , COX is composed of 11-13 different polypeptide subunits. Here, using pulse labeling of mitochondrial gene products in isolated yeast mitochondria, combined with purification of tagged COX subunits and ancillary factors, we studied the Cox2p assembly intermediates.

Cox16 protein is physically associated with Cox1p assembly intermediates and with cytochrome oxidase.

Mitochondrial cytochrome oxidase (COX) catalyzes the last step in the respiratory pathway. In the yeast , this inner membrane complex is composed of 11 protein subunits. Expression of COX is assisted by some two dozen ancillary proteins that intercede at different stages of the assembly pathway.

Aep3p-dependent translation of yeast mitochondrial .

Translation of mitochondrial gene products in depends on mRNA-specific activators that bind to the 5' untranslated regions and promote translation on mitochondrial ribosomes. Here we find that Aep3p, previously shown to stabilize the bicistronic mRNA and facilitate initiation of translation from unformylated methionine, also activates specifically translation of This is supported by several lines of evidence. Temperature-sensitive mutants are selectively blocked in incorporating [S]methionine into Atp8p at nonpermissive but not at the permissive temperature.

Mitochondrial cytochrome c oxidase deficiency.

As with other mitochondrial respiratory chain components, marked clinical and genetic heterogeneity is observed in patients with a cytochrome c oxidase deficiency. This constitutes a considerable diagnostic challenge and raises a number of puzzling questions. So far, pathological mutations have been reported in more than 30 genes, in both mitochondrial and nuclear DNA, affecting either structural subunits of the enzyme or proteins involved in its biogenesis.

Regulation of mitochondrial translation of the ATP8/ATP6 mRNA by Smt1p.

Expression of the mitochondrially encoded ATP6 and ATP8 genes is translationally regulated by F1 ATPase. We report a translational repressor (Smt1p) of the ATP6/8 mRNA that, when mutated, restores translation of the encoded Atp6p and Atp8p subunits of the ATP synthase. Heterozygous smt1 mutants fail to rescue the translation defect, indicating that the mutations are recessive.

Assembly of the rotor component of yeast mitochondrial ATP synthase is enhanced when Atp9p is supplied by Atp9p-Cox6p complexes.

The Atp9p ring is one of several assembly modules of yeast mitochondrial ATP synthase. The ring, composed of 10 copies of Atp9p, is part of the rotor that couples proton translocation to synthesis or hydrolysis of ATP. We present evidence that before its assembly with other ATP synthase modules, most of Atp9p is present in at least three complexes with masses of 200-400 kDa that co-immunopurify with Cox6p.

The Cox3p assembly module of yeast cytochrome oxidase.

Yeast cytochrome oxidase (COX) was previously inferred to assemble from three modules, each containing one of the three mitochondrially encoded subunits and a different subset of the eight nuclear gene products that make up this respiratory complex. Pull-down assays of pulse-labeled mitochondria enabled us to characterize Cox3p subassemblies that behave as COX precursors and contain Cox4p, Cox7p, and Cox13p. Surprisingly, Cox4p is a constituent of two other complexes, one of which was previously proposed to be an intermediate of Cox1p biogenesis.

Mutations in CYC1, encoding cytochrome c1 subunit of respiratory chain complex III, cause insulin-responsive hyperglycemia.

Many individuals with abnormalities of mitochondrial respiratory chain complex III remain genetically undefined. Here, we report mutations (c.288G>T [p.

Characterization of assembly intermediates containing subunit 1 of yeast cytochrome oxidase.

Mitochondrial-encoded Cox1p, one of the three core subunits of yeast cytochrome oxidase (COX), was previously shown to associate with regulatory proteins and nuclear-encoded subunits into five high molecular weight complexes that were proposed to constitute the pathway for biogenesis of the Cox1p assembly module. One of the intermediates (D5) was inferred, but not directly shown to exist. In the present study mitochondria of strains expressing C-terminal-tagged subunits of COX that had not been looked at previously were pulse-labeled and analyzed for the presence of newly translated Cox1p in the immunoprecipitates.

Stabilization of Cox1p intermediates by the Cox14p-Coa3p complex.

Cox14p and Coa3p have been shown to regulate translation of the mitochondrial COX1 mRNA and to be required for assembly of cytochrome oxidase. We present evidence that Cox14p and Coa3p stabilize previously identified Cox1p intermediates and that in the absence of either protein, Cox1p aggregates with itself and other mitochondrial gene products, including cytochrome b, Var1p and Cox2p. Our evidence suggests that Cox1p assembly intermediates are in close proximity to other mitochondrially translated proteins and that an important function of Cox14p and Coa3p is to prevent Cox1 from entering into unproductive aggregation pathways.

Modular assembly of yeast cytochrome oxidase.

Previous studies of yeast cytochrome oxidase (COX) biogenesis identified Cox1p, one of the three mitochondrially encoded core subunits, in two high-molecular weight complexes combined with regulatory/assembly factors essential for expression of this subunit. In the present study we use pulse-chase labeling experiments in conjunction with isolated mitochondria to identify new Cox1p intermediates and place them in an ordered pathway. Our results indicate that before its assimilation into COX, Cox1p transitions through five intermediates that are differentiated by their compositions of accessory factors and of two of the eight imported subunits.

The putative GTPase encoded by MTG3 functions in a novel pathway for regulating assembly of the small subunit of yeast mitochondrial ribosomes.

Very little is known about biogenesis of mitochondrial ribosomes. The GTPases encoded by the nuclear MTG1 and MTG2 genes of Saccharomyces cerevisiae have been reported to play a role in assembly of the ribosomal 54 S subunit. In the present study biochemical screens of a collection of respiratory deficient yeast mutants have enabled us to identify a third gene essential for expression of mitochondrial ribosomes.

Characterization of Gtf1p, the connector subunit of yeast mitochondrial tRNA-dependent amidotransferase.

The bacterial GatCAB operon for tRNA-dependent amidotransferase (AdT) catalyzes the transamidation of mischarged glutamyl-tRNA(Gln) to glutaminyl-tRNA(Gln). Here we describe the phenotype of temperature-sensitive (ts) mutants of GTF1, a gene proposed to code for subunit F of mitochondrial AdT in Saccharomyces cerevisiae. The ts gtf1 mutants accumulate an electrophoretic variant of the mitochondrially encoded Cox2p subunit of cytochrome oxidase and an unstable form of the Atp8p subunit of the F(1)-F(0) ATP synthase that is degraded, thereby preventing assembly of the F(0) sector.

Turnover of ATP synthase subunits in F1-depleted HeLa and yeast cells.

Mitochondrial translation of the Saccharomyces cerevisiae Atp6p subunit of F(1)-F(0) ATP synthase is regulated by the F(1) ATPase. Here we show normal expression of Atp6p in HeLa cells depleted of the F(1) β subunit. Instead of being translationally down-regulated, HeLa cells lacking F(1) degrade Atp6p, thereby preventing proton leakage across the inner membrane.

Modular assembly of yeast mitochondrial ATP synthase.

The mitochondrial ATP synthase (F(1)-F(0) complex) of Saccharomces cerevisiae is a composite of different structural and functional units that jointly couple ATP synthesis and hydrolysis to proton transfer across the inner membrane. In organello, pulse labelling and pulse-chase experiments have enabled us to track the mitochondrially encoded Atp6p, Atp8p and Atp9p subunits of F(0) and to identify different assembly intermediates into which they are assimilated. Surprisingly, these core subunits of F(0) segregated into two different assembly intermediates one of which is composed of Atp6p, Atp8p, at least two stator subunits, and the Atp10p chaperone while the second consists of the F(1) ATPase and Atp9p ring.

Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration.

COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q(2). Rescue of respiration by Q(2) is a characteristic of mutants blocked in coenzyme Q(6) synthesis. Unlike Q(6) deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q(6).

Saccharomyces cerevisiae coq10 null mutants are responsive to antimycin A.

Deletion of COQ10 in Saccharomyces cerevisiae elicits a respiratory defect characterized by the absence of cytochrome c reduction, which is correctable by the addition of exogenous diffusible coenzyme Q(2). Unlike other coq mutants with hampered coenzyme Q(6) (Q(6) ) synthesis, coq10 mutants have near wild-type concentrations of Q(6). In the present study, we used Q-cycle inhibitors of the coenzyme QH(2)-cytochrome c reductase complex to assess the electron transfer properties of coq10 cells.

SDHA is a tumor suppressor gene causing paraganglioma.

Mitochondrial succinate-coenzyme Q reductase (complex II) consists of four subunits, SDHA, SDHB, SDHC and SDHD. Heterozygous germline mutations in SDHB, SDHC, SDHD and SDHAF2 [encoding for succinate dehydrogenase (SDH) complex assembly factor 2] cause hereditary paragangliomas and pheochromocytomas. Surprisingly, no genetic link between SDHA and paraganglioma/pheochromocytoma syndrome has ever been established.

F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase.

The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F(1) ATPase (or assembly intermediates thereof).

Assembly of F0 in Saccharomyces cerevisiae.

Respiratory deficient mutants of Saccharomyces cerevisiae have been instrumental in identifying an increasing number of nuclear gene products that promote pre- and post-translational steps of the pathway responsible for biogenesis of the mitochondrial ATP synthase. In this article we have attempted to marshal current information about the functions of such accessory factors and the roles they play in expression and assembly of the mitochondrially encoded subunits of the ATP synthase. We also discuss evidence that the ATP synthase may be built up from three separate modules corresponding to the F1 ATPase, the stator and F0.