Characterization of the RCF1 Q-X3-R-X-R-X3-Q Motif and Its Role in Supporting Oxidative Phosphorylation Enzyme Complexes

Mitochondrial respiratory complexes are critical components of cellular energy production that require tight regulation to ensure optimal function. Rcf1 and Rcf2 are mitochondrial proteins that can physically associate with the yeast respiratory complexes III and IV, and the higher-ordered III-IV respiratory supercomplex that also contains the ADP/ATP translocase, AAC. Rcf1 can physically associate with both complex III and IV independently, and can be chemically crosslinked to AAC, indicating a close physical proximity to a predominant regulator of energy flux within the cell. It was therefore hypothesized that Rcf1, through its physical association with complexes III and IV, and its close proximity to AAC, may possess the capacity to communicate with multiple components of supercomplexes, a feature which may be important for coordinated regulation of the respiratory enzymes. The goal of this dissertation was to characterize the importance of an evolutionarily conserved motif, referred to here as the Q-X3-R-X-R-X3-Q (QRRQ) motif, within Rcf1 that defines it as a member of the Hypoxia-induced gene 1 (Hig1) protein family. To investigate the functional significance of the QRRQ motif, His-tagged Rcf1 proteins harboring mutations in the QRRQ motif were generated and expressed these in a yeast strain devoid of Hig1 proteins (Δrcf1Δrcf2). The importance of the QRRQ motif for the function of yeast Rcf1 was explored. I was found that mutations in conserved residues of the QRRQ motif affect the organization of the III-IV supercomplex and lead to the assembly of a novel Rcf1-complex IV subpopulation that displays altered enzymatic properties. The QRRQ motif impacts the ability of Rcf1 to associate with assembled complex IV and newly-synthesized unassembled Cox3, a core subunit of complex IV. Additionally, it was determined that Rcf1 exists in close physical proximity to Cox2, a catalytically important subunit of complex IV. It was found that the QRRQ motif influences the molecular environment of Rcf1 including its relationship to AAC proteins and possibly the cardiolipin-remodeling enzyme TAZ. Based on these findings, a model of the Rcf1-complex IV binding site is proposed and it is speculated that Rcf1 functions to modulate the complex IV enzyme activity and supercomplex organization