The mitochondrial electron transport chain (ETC) ofPlasmodiummalaria parasites is a major antimalarial drug target, but critical cytochrome functions remain unstudied and enigmatic. Parasites express two distinct cyt c homologs (c and c-2) with unusually sparse sequence identity and uncertain fitness contributions.P. falciparumcyt c-2 is the most divergent eukaryotic cyt c homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt c homologs and the related cyt c1 for inducible knockdown. Translational repression of cyt c and cyt c1 was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c-2 knockdown or knock-out had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt c for ETC function. Biochemical and structural studies revealed that both cyt c and c-2 are hemylated by holocytochrome c synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt c-2 has an unusually open active site in which heme is stably coordinated by only a single axial amino-acid ligand and can bind exogenous small molecules. These studies provide a direct dissection of cytochrome functions in the ETC of malaria parasites and identify a highly divergentPlasmodiumcytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution.

The mitochondrial electron transport chain (ETC) of Plasmodium malaria parasites is a major antimalarial drug target, but critical cytochrome (cyt) functions remain unstudied and enigmatic. Parasites express two distinct cyt c homologs ( c and c -2) with unusually sparse sequence identity and uncertain fitness contributions. P. falciparum cyt c -2 is the most divergent eukaryotic cyt c homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt c homologs and the related cyt c 1 for inducible knockdown. Translational repression of cyt c and cyt c 1 was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c -2 knockdown or knockout had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt c for ETC function. Biochemical and structural studies revealed that both cyt c and c -2 are hemylated by holocytochrome c synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt c -2 has an unusually open active site in which heme is stably coordinated by only a single axial amino acid ligand and can bind exogenous small molecules. These studies provide a direct dissection of cytochrome functions in the ETC of malaria parasites and identify a highly divergent Plasmodium cytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution.

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. We used RNA coimmunoprecipitation assays to show that Pet309 interacts with the COX1 mRNA in vivo and that this association is present before processing of the COX1 mRNA from the ATP8/6 polycistronic mRNA. This association was not affected by deletion of 8 of the PPR motifs but was undetectable after deletion of the entire 12-PPR region. However, interaction of the Pet309 protein lacking 12 PPR motifs with the COX1 mRNA was detected after overexpression of the mutated form of the protein, suggesting that deletion of this region decreased the binding affinity for the COX1 mRNA without abolishing it entirely. Moreover, binding of Pet309 to the COX1 mRNA was affected by deletion of Mss51. This work demonstrates an in vivo physical interaction between a yeast mitochondrial translational activator and its target mRNA and shows the cooperativity of the PPR domains of Pet309 in interaction with the COX1 mRNA.

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. BY4742 yeast requires Cbp3 and Cbp6 for efficient mRNA translation, whereas the D273-10b strain synthesizes Cyt at wildtype levels in the absence of Cbp3 and Cbp6. Steady-state levels of Cyt are close to wildtype in mutant D273-10b cells, and Cyt forms non-functional, supercomplex-like species with cytochrome oxidase, in which at least core 1, cytochrome, and Rieske iron-sulfur subunits are present. We demonstrated that Cbp3 interacts with the mitochondrial ribosome and with the mRNA in both BY4742 and D273-10b strains. The polymorphism(s) causing the differential function of Cbp3, Cbp6, and the assembly feedback regulation of Cyt synthesis is of nuclear origin rather than mitochondrial, and Smt1, a mRNA-binding protein, does not seem to be involved in the observed differential phenotype. Our results indicate that the essential role of Cbp3 and Cbp6 is to assist Cyt hemylation and demonstrate that in the absence of heme , Cyt can form non-functional supercomplexes with cytochrome oxidase. Our observations support that an additional protein or proteins are involved in Cyt synthesis in some yeast strains.

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 aI5␤ 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.Cytochrome c oxidase (CcO) 3 is the last electron acceptor of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, this enzyme contains 12 subunits, three of which (Cox1, Cox2, and Cox3) are encoded by the mitochondrial DNA. Assembly of CcO is a complex process regulated by more than 25 factors and chaperones (for reviews, see Ref. 1). The first steps of CcO biogenesis involve the translational activation of the mitochondria-encoded mRNAs COX1, COX2, and COX3 by mRNA-specific proteins. Translational activation of the COX1 mRNA depends on Pet309 and Mss51 (2, 3), whereas COX2 translation depends on Pet111 (4, 5), and COX3 mRNA translation depends on Pet54, Pet122, and Pet494 (6 -9). These proteins act on the target mRNA 5Ј-UTRs to allow translation by the mitochondrial ribosomes. They interact with each other and with the mitochondrial inner membrane and are thought to tether translation initiation close to the assembly sites of CcO in the membrane (for a review, see Ref. 10) (11). The mitochondria-encoded Cox1, Cox2, and Cox3 subunits are proposed to assemble from three different modules, each containing a specific subset of nucleus-encoded subunits (12-14).Cox1, the largest subunit of the CcO, carries the heme aa 3 -Cu B center to reduce oxygen. Synthesis of Cox1 inside mitochondria is highly regulated. If CcO assembly is blocked by mutations on either integral subunits or accessory chaperones, Cox1 synthesis is down-regulated (15, 16). It is proposed that by this mechanism, mitochondria avoids accumulation of pro-oxidant Cox1 intermediates (17). In addition to its role as translational activator of COX1 mRNA, Mss51 also physically inter...