The role of COX4 in the biogenesis of mitochondrial cytochrome c oxidase in Chlamydomonas reinhardtii

The respiratory complexes of the mitochondrial electron transport chain are of dual genetic origin being encoded by both organellar and nuclear genes. Complicated regulatory mechanisms are needed to coordinate gene expression between the two genomes during biogenesis of the complexes. Investigation into the role of the nuclear genome in mitochondrial gene expression was carried out using the eukaryotic alga, Chlamydomonas reinhardtii, as a model organism. Previous analysis of nuclear mutants defective in respiratory function, isolated from an insertional mutagenesis population, identified one mutant, M86, with significantly reduced levels of the mitochondrial cox1 transcript encoding subunit 1 of cytochrome c oxidase (Lown, 2001). Molecular analysis in this thesis revealed that the insertion caused the deletion of COX4, encoding subunit 4 of cytochrome c oxidase. Subunit 4 is a peripheral subunit of cytochrome c oxidase located on the matrix side of the inner mitochondrial membrane – its functional role has not been established. Northern analysis of M86 indicates that, whilst the cox1 transcript is decreased to approximately 1 - 5 % of that in WT cells, other mitochondrial transcript levels are unaffected. These findings suggest that COX4 affects cox1 at the post-transcriptional level. Extremely low levels of the COX4 protein are shown to sustain cytochrome c oxidase activity, hence stoichiometric amounts of the subunit are not required for functioning of the enzyme. Preliminary studies to investigate further the function of the algal COX4 were performed by introducing missense point mutation into the protein, and techniques for screening for cytochrome c oxidase mutants were adapted and employed in C. reinhardtii. Analysis of the cox1 transcript in a yeast ΔCOX4 mutant showed that cox1 mRNA levels are unaffected. To our knowledge, C. reinhardtii illustrates the first example of a nuclear encoded subunit of an organellar complex also being a factor required for expression of an organelle-encoded subunit