Abstract
Cytochrome c oxidase (CcO) is the last electron acceptor in the respiratory chain. The CcO core is formed by mitochondrial DNA-encoded Cox1, Cox2, and Cox3 subunits. Cox1 synthesis is highly regulated; for example, if CcO assembly is blocked, Cox1 synthesis decreases. Mss51 activates translation of COX1 mRNA and interacts with Cox1 protein in high-molecular-weight complexes (COA complexes) to form the Cox1 intermediary assembly module. Thus, Mss51 coordinates both Cox1 synthesis and assembly. We previously reported that the last 15 residues of the Cox1 C terminus regulate Cox1 synthesis by modulating an interaction of Mss51 with Cox14, another component of the COA complexes. Here, using site-directed mutagenesis of the mitochondrial COX1 gene from Saccharomyces cerevisiae, we demonstrate that mutations P521A/P522A and V524E disrupt the regulatory role of the Cox1 C terminus. These mutations, as well as C terminus deletion (Cox1ΔC15), reduced binding of Mss51 and Cox14 to COA complexes. Mss51 was enriched in a translationally active form that maintains full Cox1 synthesis even if CcO assembly is blocked in these mutants. Moreover, Cox1ΔC15, but not Cox1-P521A/P522A and Cox1-V524E, promoted formation of aberrant supercomplexes in CcO assembly mutants lacking Cox2 or Cox4 subunits. The aberrant supercomplex formation depended on the presence of cytochrome b and Cox3, supporting the idea that supercomplex assembly factors associate with Cox3 and demonstrating that supercomplexes can be formed even if CcO is inactive and not fully assembled. Our results indicate that the Cox1 C-terminal end is a key regulator of CcO biogenesis and that it is important for supercomplex formation/stability.
Highlights
We previously demonstrated that deletion of the last 11 or 15 C-terminal residues of Cox1 disrupted the assembly-mediated regulation of Cox1 synthesis, but not the assembly of active Cytochrome c oxidase (CcO)
We have examined the effects of altering the sequence of Cox1 C-terminal amino acids on the regulation of its own synthesis, its stability, and the incorporation of CcO into supercomplexes
We previously demonstrated that deletion of the last 15 residues of the yeast Cox1 C terminus does not prevent assembly of active CcO, as judged by respiratory growth of the mutant, despite the fact that this region includes a number of residues that are highly conserved among fungi and mammals [30]
Summary
Cytochrome c oxidase (CcO) is the last redox multisubunit complex of the mitochondrial respiratory chain. This enzyme couples the transference of electrons from cytochrome c to oxygen with the translocation of protons from the matrix to the intermembrane space. In Saccharomyces cerevisiae, CcO contains at least 12 subunits, where Cox, Cox, and Cox are encoded by mitochondrial genes and constitute the catalytic core. Yeast CcO is assembled after formation of three subassembly modules, each containing a mtDNA-encoded subunit and a subset of cytosolic subunits [3,4,5,6]. Assembled Cox may form pro-oxidant intermediaries containing unassembled heme a3-CuB [16]. According to the current model, newly synthesized Cox enters a progressive series of intermediaries named D1–D5 (or COA complexes) in
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