Evidence That Translation Reinitiation Leads to a Partially Functional Menkes Protein Containing Two Copper-Binding Sites

Menkes disease (MD) is an X-linked recessive disorder of copper metabolism. It is caused by mutations in the ATP7A gene encoding a copper-translocating P-type ATPase, which contains six N-terminal copper-binding sites (CBS1–CBS6). Most patients die in early childhood. We investigated the functional effect of a large frameshift deletion in ATP7A (including exons 3 and 4) identified in a patient with MD with unexpectedly mild symptoms and long survival. The mutated transcript, ATP7AΔex3+ex4, contains a premature termination codon after 46 codons. Although such transcripts are generally degraded by nonsense-mediated mRNA decay (NMD), it was established by real-time PCR quantification that the ATP7AΔex3+ex4 transcript was protected from degradation. A combination of in vitro translation, recombinant expression, and immunocytochemical analysis provided evidence that the ATP7AΔex3+ex4 transcript was protected from degradation because of reinitiation of protein translation. Our findings suggest that reinitiation takes place at two downstream internal codons. The putative N-terminally truncated proteins contain only CBS5 and CBS6. Cellular localization and copper-dependent trafficking of the major part of endogenous and recombinant ATP7AΔex3+ex4 proteins were similar to the wild-type ATP7A protein. Furthermore, the ATP7AΔex3+ex4 cDNA was able to rescue a yeast strain lacking the homologous gene, CCC2. In summary, we propose that reinitiation of the NMD-resistant ATP7AΔex3+ex4 transcript leads to the synthesis of N-terminally truncated and at-least-partially functional Menkes proteins missing CBS1–CBS4. This finding—that a mutation that would have been assumed to be null is not—highlights the need to examine the biochemical phenotype of patients to deduce the efficacy of copper therapy.