Mitochondrial OXPHOS regulation and the Rcf1/Hig1 protein family - a functional link between the ADP/ATP carrier and cytochrome c oxidase enzymes



 DESCRIPTION (provided by applicant): Mitochondria produce ATP for the cell's metabolic needs through an oxygen (O2) consuming process called oxidative phosphorylation (OXPHOS). The survival of many organisms is therefore dependent on the availability of O2, which is directly utilized by the cytochrome c oxidase (COX) enzyme of the OXPHOS system. Understanding how mitochondria regulate the activity of the OXPHOS system, in response to different metabolic environments, or limiting O2 conditions (hypoxia), is therefore of fundamental importance. Reduction in the bioenergetic capacity of mitochondria underlies numerous and diverse neurological and muscular diseases. Mitochondrial O2 dysregulation and oxidative stress are also hallmarks of many conditions such as cancers, ischemic heart disease, diabetes and aging. The OXPHOS system is composed of electron transfer complexes (ETC), referred to as complexes I-IV, and the F1Fo-ATP synthase enzyme, also termed complex V. Electrons from substrates are passed via NADH and FADH2 through the ETCs, to reduce O2 to water, a step catalyzed by the COX enzyme (complex IV). ATP is synthesized in the mitochondrial matrix by the F1Fo-ATP synthase, powered by a pH gradient across the inner membrane (IM) and established by the ETC's. The ADP/ATP carrier (AAC) protein transports the ATP out of the mitochondria for distribution to the rest of the cell and also imports ADP, for recycling back to ATP. The OXPHOS enzymes physically associate with each other to form supercomplexes in the IM, e.g. the cytochrome bc1-COX-AAC supercomplex (III-IV-AAC). The supercomplex organization of OXPHOS enzymes is proposed to enable their co-regulation, however, the molecular details of how this is achieved, are not clear. We have identified a protein, termed Rcf1, which binds to Cox3, a regulatory subunit of the COX enzyme. We found that Rcf1 also exists in close physical proximity to the AAC proteins, and in particular to the H2-L-H3 domain, a dynamic region of the AAC protein, critical for its function. Rcf1 is a member of the hypoxia-induced gene family 1 (Hig1), a highly conserved protein family found through all eukaryotic taxa. The Hig1 protein family includes both constitutively-expressed and stress-induced isoforms (e.g. hypoxic, low glucose stress or carcinomas), suggesting their role in modulating OXPHOS activity under conditions of altered metabolic environments. We demonstrate that Rcf1/Hig1 protein supports the enzyme activity of the COX complex, while at the same time interacts with, and influences the function of, AAC proteins. Our proposed experiments are designed to explore if Rcf1 functions as a bioenergetic conformational sensor/regulator coupling the activities of the COX and the AAC proteins, and within context of the cytochrome bc1-COX-AAC supercomplex.