COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A

Abstract

COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex interacts with the multisubunit WASH complex, an evolutionarily conserved system, which is required for endosomal deposition of F-actin and cargo trafficking in conjunction with the retromer. Interactions between 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 transporter 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 additional genes involved in the regulation of copper transporter recycling.

FIGURE 1:

Regulation of ATP7A trafficking by CCDC22 and COMMD1. (A, B) ATP7A localization in response to copper was assessed in control and CCDC22 T17A fibroblasts via immuno­fluorescence staining for endogenous ATP7A (red) and GM130 (green). (A) Representative images. (B) Quantification of ATP7A distribution pattern in >50 cells/group. The ATP7A distribution observed in CCDC22 T17A fibroblasts was statistically different from the control line (p < 0.001). Scale bar, 20 μm. (C) ATP7A localization in control dermal fibroblasts was examined as before. Cells were transfected with either control siRNA duplexes or siRNA targeting COMMD1. After copper treatments, cells were stained for ATP7A (red) and nuclei (blue) and imaged by confocal microscopy. (D) Increased levels of cellular Cu were noted after siRNA silencing of CCDC22. (E) Pedigree of a kindred affected by a CCDC22 (c.49A>G/p.T17A) mutation. (F) The concentration of copper (Cu) in serum and urine was determined in selected probands of this kindred. Similarly, serum ceruloplasmin concentration (CP) and urinary excretion of copper over 24 h (Cu/24h) were also ascertained when possible. Normal value ranges are indicated at the top of the table. Abnormal values are marked in red.

FIGURE 2:

Discovery of the COMMD1–CCDC22 interactome. (A) Coimmunoprecipitation of endogenous CCDC22, CCDC93, and C16orf62 in HEK293T cell lysates confirm their interactions. (B) Endogenous CCDC93 coimmunoprecipitates with COMMD1 and COMMD6 in HEK293T cell lysates. (C) Endogenous COMMD1 coimmunoprecipitates with C16orf62 and CCDC93 in HEK 293 cell lysates. (D) Expression of CCC complex subunits was examined by immunoblotting in CCDC22 T17A fibroblasts. (E) Expression of CCC complex subunits was examined by immunoblotting in Commd1-deficient fibroblasts (Commd1^−/−) and its isogenic control (Commd1^F/F). (F, G) Domain organization of CCDC93 and CCDC22. Truncation mutants of CCDC93 (F) or CCDC22 (G) were expressed in HEK293T cells and subsequently immunoprecipitated from cell lysates to assess their ability to interact with CCDC22, CCDC93, COMMD1, and C16orf62, as indicated. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and NEMO served as specificity controls. The asterisk in G denotes bands resulting from the hemagglutinin (HA) immunoblot and are not the GAPDH band, which migrates faster.

FIGURE 3:

Interaction and colocalization of the CCC and WASH complexes. (A) Interaction map between CCC complex components and other cellular complexes were drawn using data extracted from NCBI. Intracomplex interactions, where useful, are also indicated. Human studies are depicted, and interactions identified in this study are depicted in red. (B) Coimmunoprecipitation of endogenous CCC subunits (CCDC22, CCDC93, and C16orf62) with the WASH components (FAM21 and WASH) was noted using HeLa cell lysates. (C) Colocalization of endogenous COMMD1 (green) and CCDC93 (red) or C16orf62 (red) was examined by immunostaining in HeLa cells. Scale bar, 5 μm. (D) Colocalization of endogenous COMMD1 (green) with WASH (red, top), and the retromer subunit VPS35 (red, bottom) was examined by immunofluorescence staining of HeLa cells. Scale bar, 5 μm.

FIGURE 4:

Depletion of CCC complex components impairs trafficking of ATP7A and TGN46. (A) The indicated HeLa control and CCDC93- or VPS35-knockdown cell lines (sh, short hairpin against the indicated target) were maintained in high-copper conditions or transitioned to low copper as indicated in Materials and Methods. The cells were subsequently stained for ATP7A (red) and DNA (blue) and imaged by confocal fluorescence microscopy. Scale bar, 20 μm. (B) Quantification of ATP7A localization in the indicated knockdown cell lines was examined (>50 cells/group were scored). The ATP7A distribution observed in shCCDC93 and shVPS35 was statistically different from the control line (p < 0.001). (C) Plasma membrane expression of ATP7A was determined in the same cell lines after high- and low-copper conditions using biotinylation as described in Materials and Methods. N-Cadherin served as a loading control. (D) The indicated HeLa knockdown cell lines were stained for GM130 (green), TGN46 (red), F-actin (dark blue), and DNA (light blue) and imaged by confocal microscopy. Scale bar, 20 μm. (E) TGN46 localization was quantified in the indicated HeLa knockdown lines (>50 cells/group were scored). The TGN46 distribution observed in shCCDC22, shC16orf62, and shVPS35 was statistically different from that in the control line (p < 0.001).

FIGURE 5:

FAM21 recruits the CCC complex to endosomes, and this is necessary for ATP7A trafficking. (A) Formation of cytosolic puncti of FAM21, COMMD1, CCDC93, and C16orf62 was assessed by immunofluorescence staining after stable silencing of FAM21 (shFAM21). Average number of puncta per cell (of ∼75 imaged cells) for the indicated CCC complex member. Representative images, including EEA1 staining to show the localization of early endosomes. Scale bar, 5 μm. (B) Endogenous COMMD1 (red) and FAM21 (green) localization was evaluated by immunofluorescence staining in control fibroblasts and fibroblasts derived from patients with the CCDC22 T17A mutation, as indicated. Scale bar, 5 μm. (C) Localization of ATP7A (red) in control and CCDC93 deleted cells (CRISPR 93). Localization of retromer (VPS35, green) and the CCC complex (COMMD1, purple) in cytosolic vesicles and their relative colocalization are also depicted. Scale bar, 10 μm. (D, E) The indicated HA-tagged CCDC93 or CCDC22 construct was transfected into HEK293T cells and subsequently immunoprecipitated with anti-FAM21 or control immunoglobulin G (IgG). Associated proteins were identified by immunoblotting with anti-HA. Note that the CCDC93 1–430 fragment was also detected in the IgG immunoprecipitation, indicating that this interaction with FAM21 is likely nonspecific (marked by an arrowhead).

FIGURE 6:

CCDC93 interacts directly with the FAM21 tail. (A) Diagram of the fragments of FAM21 tail used in the in vitro binding studies. Black bars in the diagram indicate the location of leucine–phenylalanine acidic motifs in the tail of FAM21. (B) The indicated HA-YFP-FAM21 suppression/reexpression vectors were transfected into HEK293T cells and subsequently immunoprecipitated with anti-HA. Associated CCC complex components were detected by immunoblotting. (C) The indicated GST-FAM21 fragments were incubated with MBP-CCDC93 and stained for Coomassie or immunoblotted for MBP after GSH-agarose pull down. MBP-CCDC93 is denoted by an arrowhead. (D) The GST-FAM21 356–600 protein was incubated with the indicated MBP-CCDC93 fragments, and interactions were detected by Coomassie staining after GSH-agarose pull down. (E) The indicated concentrations of MBP-CCDC93 were assessed for binding with GST-FAM21 306-600 using surface plasmon resonance.

FIGURE 7:

Endosomal targeting of the CCC complex is required for ATP7A regulation. (A, B) ATP7A localization (red) under low-copper conditions was examined in CCDC93-deleted cells (CRISPR 93), which were rescued with an empty vector (YFP), full-length CCDC93 (WT), or a truncation mutant unable to bind FAM21 (1–438). In addition to ATP7A, cells were stained for COMMD1 (purple). (A) Representative images. (B) Quantitation of ATP7A distribution (in 30 transfected cells from each construct). Scale bar, 10 μm. (C) Model depicting the trafficking of ATP7A, the organization of CCC complex components, and its interaction with the WASH complex through FAM21.