M BoC | ARTICLE COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A Christine A. Phillips-Krawczaka,*, Amika Singlab,*, Petro Starokadomskyyb, Zhihui Denga,c, Douglas G. Osbornea, Haiying Lib, Christopher J. Dicka, Timothy S. Gomeza, Megan Koeneckeb, Jin-San Zhanga,d, Haiming Daie, Luis F. Sifuentes-Dominguezb, Linda N. Gengb, Scott H. Kaufmanne, Marco Y. Heinf, Mathew Wallisg, Julie McGaughrang,h, Jozef Geczi,j, Bart van de Sluisk, Daniel D. Billadeaua,l, and Ezra Bursteinb,m aDepartment of Immunology, eDepartment of Molecular Pharmacology and Experimental Therapeutics, and lDepartment of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN 55905; bDepartment of Internal Medicine and mDepartment of Molecular Biology, UT Southwestern Medical Center, Dallas, TX 75390-9151; cDepartment of Pathophysiology, Qiqihar Medical University, Qiqihar, Heilongjiang 161006, China; dSchool of Pharmaceutical Sciences and Key Laboratory of Biotechnology and Pharmaceutical Engineering, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China; fMax Planck Institute of Biochemistry, 82152 Martinsried, Germany; gGenetic Health Queensland at the Royal Brisbane and Women’s Hospital, Herston, Queensland 4029, Australia; hSchool of Medicine, University of Queensland, Brisbane, Queensland 4072, Australia; iRobinson Institute and jDepartment of Paediatrics, University of Adelaide, Adelaide, South Australia 5005, Australia; kSection of Molecular Genetics at the Department of Pediatrics, University Medical Center Groningen, University of Groningen, 9713 Groningen, Netherlands ABSTRACT COMMD1 deficiency results in defective copper homeostasis, but the mecha- Monitoring Editor nism for this has remained elusive. Here we report that COMMD1 is directly linked to early Jean E. Gruenberg endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and University of Geneva C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex interacts with the multisubunit Received: Jun 11, 2014 WASH complex, an evolutionarily conserved system, which is required for endosomal deposi- Revised: Oct 21, 2014 tion of F-actin and cargo trafficking in conjunction with the retromer. Interactions between Accepted: Oct 24, 2014 the WASH complex subunit FAM21, and the carboxyl-terminal ends of CCDC22 and CCDC93 are responsible for CCC complex recruitment to endosomes. We show that depletion of CCC complex components leads to lack of copper-dependent movement of the copper trans- porter ATP7A from endosomes, resulting in intracellular copper accumulation and modest alterations in copper homeostasis in humans with CCDC22 mutations. This work provides a mechanistic explanation for the role of COMMD1 in copper homeostasis and uncovers addi- tional genes involved in the regulation of copper transporter recycling. This article was published online ahead of print in MBoC in Press (http://www INTRODUCTION .molbiolcell.org/cgi/doi/10.1091/mbc.E14-06-1073) on October 29, 2014. *These authors contributed equally to this work. Copper is an essential transition metal that is required for the enzy- Address correspondence to: Daniel D. Billadeau ([email protected]), matic activity of several vital proteins and pathways. However, given Ezra Burstein ([email protected]). the high reactivity of copper, its levels are carefully regulated, and Abbreviations used: CCC complex, COMMD/CCDC22/CCDC93 complex; NN- consequently, alterations in pathways that handle copper can result CH, NDC80 and NUF2-calponin homology domain. in disease states. In humans, mutations in the copper transporters © 2015 Phillips-Krawczak, Singla, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publi- are the main causes of disorders of copper metabolism (Wang et al., cation it is available to the public under an Attribution–Noncommercial–Share 2011), including copper deficiency, due to mutations inATP7A , and Alike 3.0 Unported Creative Commons License (http://creativecommons.org/ copper accumulation, also known as Wilson’s disease, which is licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of due to mutations in the closely related transporter ATP7B. A key the Cell®” are registered trademarks of The American Society for Cell Biology. functional aspect of ATP7A and ATP7B is their copper-responsive Supplemental Material can be found at: Volume 26 January 1, 2015 http://www.molbiolcell.org/content/suppl/2014/10/28/mbc.E14-06-1073v1.DC1.html 91 trafficking between the trans-Golgi network (TGN) and vesicles in endosomes. In the absence of the CCC complex or its recruitment the cell periphery, which is the subject of significant study (Wang to endosomes, ATP7A is mislocalized and does not traffic in re- et al., 2011). sponse to copper availability, resulting in altered copper handling. In addition to these genetic disorders in humans, several Mende- lian copper metabolic disorders in animals have provided critical RESULTS clues regarding copper homeostatic mechanisms. Among these is Loss of CCDC22 or COMMD1 affects copper-dependent an autosomal recessive form of copper accumulation affecting some ATP7A trafficking purebred dogs, most notably Bedlington terriers (van de Sluis et al., ATP7A is dynamically recycled between the TGN, where it is present 2002), which is characterized by chronic hepatitis and cirrhosis, mim- when copper availability is low, and cytosolic vesicles, where it is icking several aspects of Wilson’s disease. This disorder is due to the redistributed under high copper conditions. From these cytosolic deletion of exon 2 of the copper metabolism MURR1 domain–con- vesicles, ATP7A then reaches the plasma membrane and is respon- taining 1 (COMMD1) gene. COMMD1 is the founding member sible for cellular copper efflux (Wang et al., 2011). Whereas of the COMMD protein family, which includes 10 highly conserved COMMD1 deficiency leads to copper accumulation (van de Sluis factors present in most eukaryotes (Burstein et al., 2005) that are et al., 2002; Burstein et al., 2004; Vonk et al., 2011; Materia et al., also linked to proinflammatory signaling (Maine et al., 2007; 2012), a possible role for CCDC22 in copper handling has not been Starokadomskyy et al., 2013; Li et al., 2014a), hypoxia adaptation previously studied. (van de Sluis et al., 2007, 2010), and electrolyte transport (Biasio We first examined the subcellular distribution of ATP7A using fi- et al., 2004; Drevillon et al., 2011). With regard to copper metabo- broblasts derived from a patient with a recently described point mu- lism, COMMD1 has been reported to bind to both ATP7A and tation in CCDC22 (c.49A>G/p.T17A). This mutation results in abnor- ATP7B, but the specific regulation that it provides has not been con- mal mRNA splicing, reduced protein expression, and impaired clusively elucidated (Tao et al., 2003; Vonk et al., 2011; Materia et al., CCDC22–COMMD1 protein interactions (Voineagu et al., 2012; 2012). According to one report, the retrograde transport of ATP7B Starokadomskyy et al., 2013). Whereas control fibroblasts demon- from peripheral endosomal vesicles back to the TGN in response to strated dramatic redistribution of ATP7A in response to copper copper deprivation is deregulated in the absence of COMMD1 availability, CCDC22 T17A fibroblasts had lost the normal dynamic (Miyayama et al., 2010). However, the precise mechanism by which redistribution upon changes in copper availability (Figure 1A and COMMD1 may affect ATP7A/7B trafficking has remained elusive Supplemental Figure S1). These cells displayed a peripheral distri- (Weiss et al., 2008; Miyayama et al., 2010; Materia et al., 2012). bution of ATP7A in large vesicles irrespective of copper levels and Recent studies have shown that movement of the copper trans- lacked the TGN localization of ATP7A upon copper deprivation porter ATP7A from endosomal vesicles to the plasma membrane (Figure 1, A and B). These alterations in ATP7A distribution were mir- (Steinberg et al., 2013) is dependent on sorting nexin 27 (SNX27) rored by COMMD1 deficiency in control fibroblasts after small inter- acting in conjunction with the retromer complex. This multisubunit fering RNA (siRNA)–mediated silencing (Figure 1C). Consistent with protein complex was initially identified in yeast (Seamanet al., 1998) these alterations in ATP7A trafficking, CCDC22 deficiency led to -in and has been subsequently identified in a wide range of organisms, creased intracellular copper levels (Figure 1D), akin to what has been including mammals (Haft et al., 2000). Retromer consists of a cargo reported for COMMD1 deficiency (Bursteinet al., 2004; Spee et al., selection complex comprising a trimer of VPS26, VPS29, and VPS35, 2007). However, patients with the CCDC22 T17A mutation did not which acts in concert with other cellular proteins to orchestrate the display overt clinical signs of copper toxicosis, nor did they display movement of selected endosomal cargos. This is accomplished first biochemical evidence of copper overload, which is primarily deter- through the recruitment of sorting nexins (SNX1, 2, 5, and 6), which mined in clinical settings by increased urinary copper excretion over facilitate membrane deformation and tubulation for the generation 24 h (Figure 1, E and F). Instead, some affected individuals had ele- of the nascent cargo-loaded vesicles (Cullen and