Abstract
We have characterized Cox16p, a new cytochrome oxidase (COX) assembly factor. This protein is encoded by COX16, corresponding to the previously uncharacterized open reading frame YJL003w of the yeast genome. COX16 was identified in studies of COX-deficient mutants previously assigned to complementation group G22 of a collection of yeast pet mutants. To determine its location, Cox16p was tagged with a Myc epitope at the C terminus. The fusion protein, when expressed from a low-copy plasmid, complements the mutant and is detected solely in mitochondria. Cox16p-myc is an integral component of the mitochondrial inner membrane, with its C terminus exposed to the intermembrane space. Cox16 homologues are found in both the human and murine genomes, although human COX16 does not complement the yeast mutant. Cox16p does not appear to be involved in maturation of subunit 2, copper recruitment, or heme A biosynthesis. Cox16p is thus a new protein in the growing family of eukaryotic COX assembly factors for which there are as yet no specific functions known. Like other recently described nuclear gene products involved in expression of cytochrome oxidase, COX16 is a candidate for screening in inherited human COX deficiencies.
Highlights
Cytochrome oxidase (COX),1 the terminal enzyme of the mitochondrial respiratory chain, catalyzes the transfer of electrons derived from sugars, fats, and amino acids to molecular oxygen
Like many of the other cytochrome oxidase (COX) assembly factors, Cox16p is an integral component of the mitochondrial inner membrane and appears to exist in a higher molecular weight complex
The absence of cytochrome oxidase in cox16 mutants is accompanied by a loss of the cytochrome aa3 spectral signal and a marked reduction in the steady state concentrations of the mitochondrial-encoded but not nuclear-encoded subunits of COX
Summary
Cloning and Disruption of COX16 —The yeast genomic library used to clone COX16 was constructed from partial Sau3A fragments of nuclear DNA of the respiratory-competent haploid strain S. cerevisiae D273–10B/A1. C25/U1, a mutant from complementation group G22 [8], was transformed with the yeast genomic library as described previously [11], and the COX16 gene identified by isolating subclones capable of conferring respiratory competence on C25/U1. The COX16 gene was disrupted by insertion of a 1.1-kb URA3 fragment at the internal HindIII site and transformation of the respiratory-competent haploid strain, W303–1A, with the linear fragment containing the disrupted gene and flanking sequences. The transformation yielded the cox mutant aW303⌬COX16.
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