None of the cell lines used in this study belong to the Database of Cross-Contaminated or Misidentified Cell Lines defined by the International Cell Collection Authentication Committee. Immunoprecipitation of ATP7A To assess interactions of ATP7A in the presence and absence of copper, we performed cross-linking in intact cells with dithiobis(succinimidylpropionate) (DSP) followed by immunoprecipitation as previously described but with the following modifications (Gokhale et al., 2015a). in Supplementary file 1 to give origin to the ATP7A interactome in the Supplementary file 3 Tab (BCS+Cu Hits).DOI: http://dx.doi.org/10.7554/eLife.24722.014 elife-24722-supp2.xlsx (52K) DOI:?10.7554/eLife.24722.014 Supplementary file 3: Curated LF3 proteins defining the ATP7A interactome and their analysis by bioinformatics. Selected LF3 hits from BCS treated cells and copper treated cell immunoisolated ATP7A complexes. Tab with the sum of these hits (BCS+Cu Hits) was utilized for bioinformatics (Tabs A-C). Crapome lists hits from one of the CRAPome datasets and the proteins shared by the ATP7A interactome and the CRAPome. Tabs (A), (B), and (C) contain DAVID, ENRICHR and GDA bioinformatic analyses, respectively, which are graphically depicted in Figures 2 and ?and33.DOI: http://dx.doi.org/10.7554/eLife.24722.015 elife-24722-supp3.xlsx (648K) DOI:?10.7554/eLife.24722.015 Abstract Genetic and environmental factors, such as metals, interact to determine neurological traits. We reasoned that interactomes of molecules handling metals in neurons should include novel metal homeostasis pathways. We focused on copper and its transporter ATP7A because ATP7A null mutations cause neurodegeneration. We performed ATP7A immunoaffinity chromatography and recognized 541 proteins co-isolating with ATP7A. The ATP7A interactome concentrated gene products implicated in neurodegeneration and neurodevelopmental disorders, including subunits of the Golgi-localized conserved oligomeric Golgi (COG) complex. COG null cells possess altered content and subcellular localization of ATP7A and CTR1 (SLC31A1), the transporter required for copper uptake, as well as decreased total cellular copper, and impaired copper-dependent metabolic responses. Changes in the expression of ATP7A and COG subunits in neurons altered synapse development in larvae and copper-induced mortality of adult flies. We conclude that this ATP7A interactome encompasses a novel COG-dependent mechanism to specify neuronal development and survival. DOI: http://dx.doi.org/10.7554/eLife.24722.001 ATP7A and COG complex subunits genetically interact to specify synapse morphology in the developing neuromuscular junction of the third instar larva (Figure 9). We overexpressed ATP7A in neurons using the pan-neuronal GAL4 driver (C155) (Lin and Goodman, 1994). Overexpression of ATP7A reduced the cumulative synapse branch length; thus, inducing a collapse of the synapse as measured as an increased synaptic bouton density (Physique 9A image increase cumulative synapse branch length while maintaining wild type synaptic bouton density (Physique 9ACC, column 3). As predicted by our hypothesis, overexpression of Itgb1 ATP7A in flies restored synaptic bouton density to wild type levels (Physique 9A and B, compare columns 4 and 5). These results demonstrate that a component of the ATP7A interactome, the COG complex, genetically interact with ATP7A to specify a neurodevelopmental synapse phenotype. Open in a separate window Physique 9. Drosophila ATP7A and COG1 genetically interact to specify synapse development.Third instar larvae neuromuscular junction synapses were stained with anti HRP antibodies (A) imaged and their morphology assessed using as parameters branch length (B) and bouton density (C). Scoring was carried out blind to the animal genotype. Control animals (C155 outcross, column 1; or UAS-ATP7A outcross, column 2), animals carrying one copy of the null allele (cog1outcrossed, column 3), flies overexpressing ATP7A in neuronal cells (c155 UAS-ATP7A; column 4), and animals overexpressing ATP7A and mutant for (C155 UAS-ATP7A x adult nervous system (Physique 10). We controlled the expression of ATP7A in adult dopaminergic neurons, a group of cells frequently used to model Parkinsons disease in (Feany and Bender, 2000; Haass and Kahle, 2000; Li et al., 2000; Yang et al., 2003; Lin et al., 2010). We drove the expression of UAS-ATP7A selectively in dopaminergic and serotoninergic neurons with the (driver (Feany and Bender, 2000). We LF3 reasoned that overexpression of ATP7A, which decreases cellular levels of copper (Hwang et al., 2014; Lye et al., 2011), should reduce the toxicity to copper diet exposure. We previously observed a high sensitivity to copper in the diet of wild type animals (Gokhale et al., 2015a). Copper feeding progressively increased mortality in wild type male (Physique 10A) and female adults (Physique 10B) over a period of three days. Overexpression of ATP7A in adult dopaminergic neurons was sufficient to significantly safeguard males and female adult animals from the harmful effect of copper feed at 48 hr (Physique 10ACB, (Ddc UAS-ATP7A)). Similarly, mutation of the COG complex subunit protected animals from copper diet induced death (Physique 10ACB, (Ddc x cog1e02840)). In contrast, the mortality phenotype observed in animals overexpressing ATP7A was restored to the levels of wild type lethality by adding in trans a genetic defect in (Physique 10ACB, (UAS-ATP7A; Ddc x cog1e02840)). Importantly,.