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

Mutational Analysis of the QRRQ Motif in the Yeast Hig1 Type 2 Protein Rcf1 Reveals a Regulatory Role for the Cytochrome \u3cem\u3ec\u3c/em\u3e Oxidase Complex

The yeast Rcf1 protein is a member of the conserved family of proteins termed the hypoxia-induced gene (domain) 1 (Hig1 or HIGD1) family. Rcf1 interacts with components of the mitochondrial oxidative phosphorylation system, in particular the cytochrome bc1(complex III)-cytochrome c oxidase (complex IV) supercomplex (termed III-IV) and the ADP/ATP carrier proteins. Rcf1 plays a role in the assembly and modulation of the activity of complex IV; however, the molecular basis for how Rcf1 influences the activity of complex IV is currently unknown. Hig1 type 2 isoforms, which include the Rcf1 protein, are characterized in part by the presence of a conserved motif, (Q/I)X3(R/H)XRX3Q, termed here the QRRQ motif. We show that mutation of conserved residues within the Rcf1 QRRQ motif alters the interactions between Rcf1 and partner proteins and results in the destabilization of complex IV and alteration of its enzymatic properties. Our findings indicate that Rcf1 does not serve as a stoichiometric component, i.e. as a subunit of complex IV, to support its activity. Rather, we propose that Rcf1 serves to dynamically interact with complex IV during its assembly process and, in doing so, regulates a late maturation step of complex IV. We speculate that the Rcf1/Hig1 proteins play a role in the incorporation and/or remodeling of lipids, in particular cardiolipin, into complex IV and. possibly, other mitochondrial proteins such as ADP/ATP carrier proteins

Rcf1 and Rcf2, Members of the Hypoxia-induced Gene 1 Protein Family, Are Critical Components of the Mitochondrial Cytochrome bc1-cytochrome Oxidase Supercomplex

We report that Rcf1 (formerly Aim31), a member of the conserved hypoxia-induced gene 1 (Hig1) protein family, represents a novel component of the yeast cytochrome bc1-cytochrome c oxidase (COX) supercomplex. Rcf1 (respiratory supercomplex factor 1) partitions with the COX complex, and evidence that it may act as a bridge to the cytochrome bc1 complex is presented. Rcf1 interacts with the Cox3 subunit and can do so prior to their assembly into the COX complex. A close proximity of Rcf1 and members of the ADP/ATP carrier (AAC) family was also established. Rcf1 displays overlapping function with another Hig1-related protein, Rcf2 (formerly Aim38), and their joint presence is required for optimal COX enzyme activity and the correct assembly of the cytochrome bc1-COX supercomplex. Rcf1 and Rcf2 can independently associate with the cytochrome bc1-COX supercomplex, indicating that at least two forms of this supercomplex exist within mitochondria. We provide evidence that the association with the cytochrome bc1-COX supercomplex and regulation of the COX complex are a conserved feature of Hig1 family members. Based on our findings, we propose a model where the Hig1 proteins regulate the COX enzyme activity through Cox3 and associated Cox12 protein, in a manner that may be influenced by the neighboring AAC proteins