Importance of S. Cerevisiae Rcf1 and Rcf2 Proteins for the Mitochondrial Protonmotive Force Generation

Mitochondria are the site of oxidative phosphorylation (OXPHOS) pathway, which can supply majority of energy in a eukaryotic cell. OXPHOS enzyme activities generate electrochemical gradient known as mitochondrial protonmotive force (PMF). PMF coordinates OXPHOS enzyme activities and supports essential cell survival functions such as transport of proteins and metabolites in and out of mitochondria. PMF is maintained despite variations in cellular energy demand and oxygen availability.Mitochondrial proteins belonging to the conserved hypoxia induced gene domain (HIGD) family improve cell survival during the hypoxic and hypoglycemic stress. Their molecular function is not fully understood but they seem to act through regulating OXPHOS enzyme cytochrome c oxidase (complex IV). Complex IV activity is important for PMF generation. Using yeast as a model organism, this study addresses function of HIGD proteins Rcf1 and Rcf2. The data presented here indicate that Rcf1 and Rcf2 support complex IV PMF generation and/or prevent proton leak across the inner membrane of the mitochondria. Deletion of Rcf1 (Δrcf1) causes lower complex IV steady state levels and electron transfer activity. Deletion of Rcf2 in Δrcf1 strain (Δrcf1;Δrcf2) does not further decrease complex IV steady state levels and electron transfer activity, yet strongly impairs respiratory growth. Analyses of single mutant strains Δrcf1 and Δrcf2 indicated that deletion of Rcf1 or Rcf2 lowers OXPHOS efficiency and decreases PMF. These defects become more severe when both Rcf1 and Rcf2 are deleted. The inability to maintain PMF and PMF-dependent functions is proposed to underlie the strong respiratory growth deficiency of Δrcf1;Δrcf2 mutant. In addition to this new role of Rcf1 and Rcf2 in PMF maintenance, several pleiotropic phenotypes of Δrcf1;Δrcf2 mutant – such as decreased ATP synthase levels, abnormal mitochondrial morphology – suggest that these proteins may have wider impact on OXPHOS function