Abstract
Copper–zinc superoxide dismutase (Sod1) is a critical antioxidant enzyme that rids the cell of reactive oxygen through the redox cycling of a catalytic copper ion provided by its copper chaperone (Ccs). Ccs must first acquire this copper ion, directly or indirectly, from the influx copper transporter, Ctr1. The three proteins of this transport pathway ensure careful trafficking of copper ions from cell entry to target delivery, but the intricacies remain undefined. Biochemical examination of each step in the pathway determined that the activation of the target (Sod1) regulates the Ccs·Ctr1 interaction. Ccs stably interacts with the cytosolic C-terminal tail of Ctr1 (Ctr1c) in a copper-dependent manner. This interaction becomes tripartite upon the addition of an engineered immature form of Sod1 creating a stable Cu(I)-Ctr1c·Ccs·Sod1 heterotrimer in solution. This heterotrimer can also be made by the addition of a preformed Sod1·Ccs heterodimer to Cu(I)-Ctr1c, suggestive of multiple routes to the same destination. Only complete Sod1 activation (i.e. active site copper delivery and intra-subunit disulfide bond formation) breaks the Sod1·Ccs·Ctr1c complex. The results provide a new and extended view of the Sod1 activation pathway(s) originating at cellular copper import.
Highlights
Copper is a critical cofactor for many enzymes that take advantage of its redox activity to catalyze a wide range of chemical reactions
Apo-Ctr1c488 did not bind to copper chaperone for Sod1 (Ccs), but Cu(I)-Ctr1c488 formed a stable complex that ran as induced coupled plasma mass spectrometry (ICP-MS) results
A considerable amount of work has been completed on the Ccs-mediated Sod1 activation process, yet details of copper transfer to Ccs were still unclear
Summary
Copper is a critical cofactor for many enzymes that take advantage of its redox activity to catalyze a wide range of chemical reactions. Copper ion import is facilitated by the Ctr family of integral membrane transporters that bring copper into the cytoplasm in an ATP-independent fashion (Dancis and Haile 1994; Lee et al 2002; Kim et al 2008). Trimerization of Ctr monomers forms a pore through which Cu(I) (i.e. reduced/cuprous copper) is transported into the cell (De Feo et al 2009). Cu(I) is simultaneously coordinated by three Ctr1c tails, each containing an HCH copper binding motif. (Pickering et al 1993) By holding the Cu(I) between multiple tails, Ctr can mimic the more favorable Cu(I) coordinating motifs. Two ATP-dependent pumps ATP7A and ATP7B that reside in the membranes of the trans-Golgi network, handle copper ion export (Lutsenko et al 2002; Voskoboinik and Camakaris 2002)
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