Akio Takehara, Tadashi Baba, Jun-Ichi Hayashi, Hidenori Arai, Hiroko Kodama, Koichiro Kako, Kazuto Nakada, Yoshinori Takahashi, Shin-ichi Kashiwabara, Yoshiko Inada, and Eisuke Munekata
Copper is an essential trace element for aerobic organisms, acting as a cofactor for mitochondrial, cytosolic, and vesicular enzymes (27). In the past few years, three independent pathways for intracellular copper trafficking have been identified in yeast. Generally, copper in the form of Cu(I) is transported across the plasma membrane by the high-affinity Cu transporter Ctr1 and is then picked up and transported to each target organelle by cytosolic small proteins (18). Cox17p is one of these small proteins and is considered to be involved in copper recruitment to mitochondria and in the functional assembly of cytochrome c oxidase (CCO), the terminal enzyme of the mitochondrial respiratory chain (5, 6). It was reported that a proline-rich 62-mer polypeptide was purified from the gel filtration fraction of a porcine heart extract (26). A structural analysis showed that it was a mammalian homologue of yeast Cox17p (yCox17p). Along with the porcine protein (4, 26), human (1), rat (12), and mouse (12, 17) Cox17p homologues have been identified to date. Although it was thought that yCox17p forms a homooligomer (8) and guides Cu to mitochondria for incorporation into CCO, its physiological role in mammals is unclear. Recently, it was reported that expression levels of Cox17p mRNA were high in the mouse heart, kidneys, and brain as well as in some endocrine cell lines but quite low in the small intestine, liver, and some fibroblast cell lines (12). Furthermore, COX17 genomic DNA has been isolated, and its genetic structure and the 5′-promoter function were determinated. Mouse COX17 is a single gene that spans ∼6 kb, consists of three exons, and is mapped to the center of chromosome 16 (25). Transcription factors Sp1 and NRF-1 (nuclear respiration factor 1) drive the basal transcription of this gene (24). The transcriptional mechanism of COX17 is similar to that of other COX subunits (21), which indirectly implies that the Cox17p is also involved in cellular respiration. Since our goal is to determine the physiological function of Cox17p in the mammalian system, we first examined the copper-binding activity of mammalian Cox17p in vitro and then generated mice carrying a null mutation of COX17 and analyzed them. Next, we present genetic evidence that COX17 is required for the transport of copper to the mitochondria and CCO activity. Several case of specific deficiencies of CCO in humans have been reported, with most of them being associated with severe neonatal or infantile lactic acidosis and early death. For example, patients with a fatal cardioencephalomyopathy or hypertrophic cardiomyopathy, marked by a severe CCO deficiency, harbor mutations in the SCO2 gene, which is a related CCO assembly gene that is thought to cooperate with Cox17p (10, 11, 20). A COX17-deficient mouse underwent embryonic death with severe reduction in CCO activity, which is consistent with the previous clinical evidence as in the case of the SCO2 mutation. Furthermore, we show here that Cox17p is not only indispensable for the activation of CCO but also essential for embryonic growth and development. We also report a marked reduction in CCO activity in 6.5-day viable embryos (E6.5), indicating that CCO-independent embryogenesis progressed up to this stage. Most recently, gene disruption of Ctr1 was also reported to result in embryonic death (13, 14). The relationship between this molecule and Cox17p is also discussed in light of the developmental mechanism.