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. 2009 Jan 2;284(1):696-707.
doi: 10.1074/jbc.M804766200. Epub 2008 Oct 21.

COMMD1 forms oligomeric complexes targeted to the endocytic membranes via specific interactions with phosphatidylinositol 4,5-bisphosphate

Jason L Burkhead  1 , Clinton T Morgan  1 , Ujwal Shinde  1 , Gabrielle Haddock  1 , Svetlana Lutsenko  2
Affiliations

COMMD1 forms oligomeric complexes targeted to the endocytic membranes via specific interactions with phosphatidylinositol 4,5-bisphosphate

Jason L Burkhead et al. J Biol Chem. .

Abstract

Copper metabolism Murr1 domain 1 (COMMD1) is a 21-kDa protein involved in copper export from the liver, NF-kappaB signaling, HIV infection, and sodium transport. The precise function of COMMD and the mechanism through which COMMD1 performs its multiple roles are not understood. Recombinant COMMD1 is a soluble protein, yet in cells COMMD1 is largely seen as targeted to cellular membranes. Using co-localization with organelle markers and cell fractionation, we determined that COMMD1 is located in the vesicles of the endocytic pathway, whereas little COMMD1 is detected in either the trans-Golgi network or lysosomes. The mechanism of COMMD1 recruitment to cell membranes was investigated using lipid-spotted arrays and liposomes. COMMD1 specifically binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in the absence of other proteins and does not bind structural lipids; the phosphorylation of PtdIns at position 4 is essential for COMMD1 binding. Proteolytic sensitivity and molecular modeling experiments identified two distinct domains in the structure of COMMD1. The C-terminal domain appears sufficient for lipid binding, because both the full-length and C-terminal domain proteins bind to PtdIns(4,5)P2. In native conditions, endogenous COMMD1 forms large oligomeric complexes both in the cytosol and at the membrane; interaction with PtdIns(4,5)P2 increases the stability of oligomers. Altogether, our results suggest that COMMD1 is a scaffold protein in a distinct sub-compartment of endocytic pathway and offer first clues to its role as a regulator of structurally unrelated membrane transporters.

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Figures

FIGURE 1.
FIGURE 1.
Subcellular fractionation of COMMD1 in hepatic cells. A, immunodetection of COMMD1 in sub-cellular fractions of HepG2 grown under copper-limiting (BCS) and 50 μm copper (Cu) conditions. Fractions are: N, nuclei; S, soluble cytosol; M, pelleted membranes. B, immunodetection of Golgin-97, CHMP2B, and COMMD1 in fractions from Percoll density gradient.
FIGURE 2.
FIGURE 2.
Endogenous COMMD1 is present in endocytic/MVB pathway. Confocal immunofluorescence microscopy of COMMD1 with markers for EEA1 (early endosomes), RAB9 (late endosomes), CHMP2B (late endosome/multivesicular bodies), LAMP1 (lysosomes), Golgin-97 (Golgi), and ATP7B (trans-Golgi network).
FIGURE 3.
FIGURE 3.
Intracellular localization of COMMD1 and ATP7B in HepG2 cells following BCS or CuCl2 treatment. Confocal immunofluorescence microscopy of COMMD1 and ATP7B was performed after HepG2 cells are treated for 8 h with either 200 μm BCS or 50 μm CuCl2. Non-polarized HepG2 cells are shown; the pattern of COMMD1 in polarized cells was very similar.
FIGURE 4.
FIGURE 4.
COMMD1 binds phospholipids. A, PIP strip assay for the full-length HisTag-COMMD1 and a negative control HisTag-ATP-BD. Immunodetection with anti-His6 antibody is shown. B, liposome binding to 0.5 μg of each immobilized protein: COMMD1; PH-phospholipase Cδ is positive control, ATP-BD and ADH are negative controls. COMMD1 or control proteins were bound to a 96-well plate and incubated with biotinylated liposomes containing PtdIns(4,5)P2 or non-phosphorylated PtdIns. Liposome retention is determined by fluorescence of IRDye-conjugated streptavidin.
FIGURE 5.
FIGURE 5.
Equilibrium binding of COMMD1 to liposomes demonstrates preference to the bis-phosphorylated phosphatidylinositol. In A: Left, schematic of liposome floatation assay, liposomes and proteins are loaded in the 30% sucrose layer and liposomes with bound protein are recovered from the 15% sucrose/buffer interface. Unbound protein remains in the 30% sucrose layer. Right, SDS-PAGE of COMMD1 recovered with liposomes containing 2% of indicated bis- and tri-phosphorylated liposomes. Vard is the stable degradation product of COMMD1 (residues 1-121). In B: Left, COMMD1 recovered with liposomes containing increasing concentrations of PtdIns(4)P and PtdIns(4,5)P2. Right, increasing amounts of COMMD1 recovered with 2% PtdIns(4)P or PtdIns(4,5)P2.
FIGURE 6.
FIGURE 6.
COMMD1 proteolytic sensitivity and domain organization. A, COMMD1 degrades to a 15-kDa cleavage product (VARD, lower band) yielding three possible masses depending on cleavage site. B, amino acid sequence of COMMD1 indicating domain organization; the experimentally determined VARD is italicized. C, full-length COMMD1, but not the N-terminal domain is recruited to liposomes. COMMD1, V-121, and V-108 binding to liposomes containing 10% phosphatidic acid (PA), phosphatidylserine (PS), or PtdIns(4,5)P2. D, comparison of the COMMD1 and CTD binding to liposomes containing 10% PtdIns(4,5)P2 demonstrates that CTD alone is sufficient for interactions with the lipid. Immunodetection with anti-COMMD1 Ab is shown to confirm the identity of oligomeric bands in the CTD sample.
FIGURE 7.
FIGURE 7.
Endogenous and recombinant COMMD1 form large oligomeric complexes. A, BN-PAGE of 2.0 μg and 1.0 μg of purified recombinant COMMD1. B, BN-PAGE of soluble (S) and membrane (M) fractions from HepG2 cells. Immunodetection with the anti-COMMD1 Ab is shown. The COMMD1-containing complexes are indicated by the arrows.
FIGURE 8.
FIGURE 8.
CTD is sufficient for oligomerization. VARD and CTD were separated on a 15% acrylamide Laemmli gel and transferred to nitrocellulose for immunodetection. The samples were loaded with or without 20 mm dithiothreitol, as indicated. COMMD1 monomer, dimer, and tetramer sizes are labeled with the letters “m,” “d,” and “t”(left), respectively. CTD species are indicated with as “mCTD,” “dCTD,” and “tCTD”(right). B, redox regulation of COMMD1 oligomerization with indicated treatments.
FIGURE 9.
FIGURE 9.
Structural model of COMMD1. A, ab initio model with experimental VARD in green and the empirically determined CTD in orange. B, NMR solution structure of residues 1-108. C, overlay of Ab initio modeled COMMD1 residues 1-108 (green) and the NMR solution structure (cyan). D, COMMD1 model with solution structure of residues 1-108 as initial constraint. Residues 1-108 are shown in cyan, 109-129 in green, and the remainder of the empirically determined CTD is orange. The proteolytic site is indicated by the arrow.
FIGURE 10.
FIGURE 10.
Schematic of COMMD1 complex formation at the membrane. COMMD1 is recruited to the membrane by PtdIns(4,5)P2. Complex formation with the membrane and intrinsic membrane proteins signals recruitment of additional effectors.
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References

    1. Maine, G. N., and Burstein, E. (2007) Cell Mol. Life Sci. 64 1997-2005 - PMC - PubMed
    1. van De Sluis, B., Rothuizen, J., Pearson, P. L., van Oost, B. A., and Wijmenga, C. (2002) Hum. Mol. Genet. 11 165-173 - PubMed
    1. Klomp, A. E., van de Sluis, B., Klomp, L. W., and Wijmenga, C. (2003) J. Hepatol. 39 703-709 - PubMed
    1. Kawamura, M., Takahashi, I., and Kaneko, J. J. (2002) Vet. Pathol. 39 747-750 - PubMed
    1. Narindrasorasak, S., Kulkarni, P., Deschamps, P., She, Y. M., and Sarkar, B. (2007) Biochemistry 46 3116-3128 - PubMed

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