Characterization of Drosophila Ctr1a: New Roles for Ctr1 Proteins and Copper in Physiology and Cell Signaling Pathways

Abstract

Copper is an essential trace element required by all aerobic organisms as a co-factor for enzymes involved in normal growth, development and physiology. Ctr1 proteins are members of a highly conserved family of copper importers responsible for copper uptake across the plasma membrane. Mice lacking Ctr1 die during embryogenesis from widespread developmental defects, demonstrating the need for adequate copper acquisition in the development of metazoan organisms via as yet uncharacterized mechanisms. The early lethality of the Ctr1 knockout mouse has made it difficult to study the functions of copper and Ctr1 proteins in metazoan development and physiology. Drosophila melanogaster, a genetically tractable system expresses three Ctr1 genes, Ctr1A, Ctr1B and Ctr1C, and may help to further understand the roles of copper and Ctr1 proteins in metazoan development and physiology. Described here is the characterization of Drosophila Ctr1A. Localization studies using an affinity purified anti-Ctr1A peptide antibody show Ctr1A is predominantly expressed at the plasma membrane in whole embryos and in larval tissues. Ctr1A is an essential gene in Drosophila as loss-of-function mutants, generated by imprecise p-element excision arrest at early larval stages of development. Inductively coupled plasma mass spectroscopy (ICP-MS) demonstrated that whole body copper levels are reduced in Ctr1A mutants and consequently, a number of copper-dependent enzyme deficiencies were detected by in vitro enzyme and cell biological assays. Ctr1A maternal and zygotic mutants have a more severe developmental phenotype and also showed reductions in heart rate, which could be partially rescued by dietary copper supplementation. Heart-specific Ctr1A knockdown flies were subsequently examined for heart rate defects using optical coherence tomography (OCT) and while they did have reduced heart rate measurements, heart contractility was compromised. While investigating tissue-specific requirements for Ctr1A in the development of Drosophila, a genetic interaction between Ctr1A and Ras was observed. Genetic experiments in Drosophila and cell culture experiments in both Drosophila and mammalian cell lines demonstrate a conserved role for Ctr1 proteins and copper as positive modulators of Ras/MAPK pathway signaling. Immunoblot analysis shows that signal transduction is intact until the point at which MEK1/2 phosphorylates ERK1/2. MEK2 protein levels are reduced in copper deficient cells, while MEK1 is able to bind copper-chelated beads, suggesting that these two proteins may be copper-binding proteins. In summary, this work demonstrates that Ctr1A is an essential gene in Drosophila and through characterization studies of Ctr1A, has uncovered conserved roles for Ctr1 proteins and copper in physiological processes and in an important signaling pathway that controls a number of fundamental biological processes.