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.
Highlights
Copper metabolism Murr1 domain 1 (COMMD1) was shown to bind Cu(II) in vitro (5), but whether this binding represents an in vivo property is uncertain
It was proposed that COMMD1 controls copper export by regulating the intracellular localization of the copper-transporting ATPase ATP7B, which is responsible for the removal of excess copper from the liver into the bile
Down-regulation of COMMD1 has no apparent effect on the ability of ATP7B to traffic from the transGolgi network (TGN) to exocytic vesicles (Ref. 9 and our data, not shown), suggesting that the effect of COMMD1 is downstream of the TGN
Summary
COMMD1 was shown to bind Cu(II) in vitro (5), but whether this binding represents an in vivo property is uncertain. In support of this hypothesis, recombinant COMMD1 was shown to interact with the N-terminal domain of ATP7B in vitro and in cell lysates (8). To determine whether COMMD1 can interact with membrane lipids in the absence of other proteins, we first utilized lipid spotted arrays (PIPstrips), whereby COMMD1 reproducibly showed binding to several lipids (Fig. 4A, left).
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