Cytochrome oxidase (COX), the terminal complex of the respiratory chain, consists of 11 different subunit polypeptides. The mitochondrially encoded Cox1p, Cox2p and Cox3p subunits constitute the catalytic core while eight other structural subunits are products of nuclear genes. Because of its genetic and compositional complexity, biogenesis of COX is a highly integrated process that is assisted by numerous accessory proteins and regulatory factors for coordinating a balanced output of compartmentally separated genes. Pulse-chase studies with isolated mitochondria have shown that prior to their assimilation into COX, Cox1p and Cox3p, each, transitions through a series of intermediates differentiated by their compositions of accessory factors and structural subunits. This finding has led us to propose a biogenesis model involving three separate pathways with modular products that ultimately combine to form the holoenzyme. This proposal has four objectives, each related to an important facet of COX biogenesis. 1) We wish to consolidate the modular assembly model with direct experimental evidence that Cox2p also preassembles as an independent module. This will be addressed by identifying and characterizing Cox2p intermediates in pulse-chase labeled mitochondria. 2) We have indirect evidence that mitochondrial gene products of COX and the bc1 complex, both of which exist in a supercomplex, are inserted in the same or neighboring subcompartments of the membrane during their biogenesis. This suggests that assembly of the COX/bc1 supercomplex may be coordinated both physically and temporally. We will test this hypothesis by genetic and biochemical means. 3) Some recent results suggest that the proper stoichiometry of COX and ATP synthase may be achieved via a complex of the rotor subunit Atp9p and the COX peripheral subunit Cox6p. We will further test this interesting possibility by studying assembly of the ATP synthase in cox6 null mutants and conversely of COX assembly in atp9 mutants. 4) Much of our current knowledge of COX biogenesis has come from functional studies of accessory factors that intervene at different stages of the Cox1p-Cox3p assembly pathways. Many but not all of these factors were identified through studies of yeast mutants displaying a specific deficiency in COX. The fourth and last objective of this proposal is to continue mining a collection of nuclear respiratory defective yeast pet mutants for still undiscovered genes essential for COX biogenesis. These studies will enlarge our understanding of the mechanisms and the factors that govern assembly of an important mitochondrial respiratory complex derived from genetic information residing in two spatially distinct compartments of the cell.

Public Health Relevance

Cytochrome oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the reduction of oxygen to water using electrons extracted from different food sources. Biogenesis of this complex enzyme depends on some three dozen genes that regulate and assist this process. The studies embodied in this application are intended to solidify our modular model of COX assembly, to elucidate how this process is regulated to achieve a proper stoichiometry of COX with respect to the energy-conserving ATP synthase, to screen our collection of yeast mutants for new COX assembly factors and in collaborations with other laboratories to continue using the yeast model to better understand the genetic and biochemical basis of human COX deficiencies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing (MBPP)
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Barski, Oleg
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Columbia University (N.Y.)
Other Domestic Higher Education
New York
United States
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Franco, Leticia Veloso R; Su, Chen-Hsien; McStay, Gavin P et al. (2018) Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules. J Biol Chem 293:16899-16911
Su, Chen-Hsien; Tzagoloff, Alexander (2017) Cox16 protein is physically associated with Cox1p assembly intermediates and with cytochrome oxidase. J Biol Chem 292:16277-16283
Rak, Malgorzata; Bénit, Paule; Chrétien, Dominique et al. (2016) Mitochondrial cytochrome c oxidase deficiency. Clin Sci (Lond) 130:393-407