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Title: In vitro analysis of the cytosolic copper metallochaperones in saccharomyces cerevisiae
Authors: Brusby, Kerrie
Issue Date: 2020
Publisher: Newcastle University
Abstract: In yeast, Cu,Zn-superoxide dismutase (Sod1) is activated by the copper metallochaperone Ccs1, mainly in the cytosol. This involves the delivery of Cu1+ and the formation of a structurally important disulfide bond. Ccs1 consists of three domains, with domain 1 (D1) and domain 3 (D3) containing copper-binding CXXC and CXC motifs respectively. In human CCS1, D1 is the site of copper transfer and D3 is responsible for disulfide bond formation in SOD1. In yeast cells, D3 of Ccs1 is essential for Sod1 activation, whereas D1 is required only under copper-limiting conditions. The Cu1+-binding ability of Ccs1 has been investigated using mutants in which the Cys residues in either the D1 (C17S/C20S-Ccs1) or D3 (C229S/C231S-Ccs1) copper-binding motif have been mutated to Ser residues, as well as a mutant lacking D3 (D1/2-Ccs1). Both D1 and D3 of Ccs1 are able to bind a single equivalent of Cu1+, and dimerisation occurs upon Cu1+-binding both via a copper mediated process as well as by disulfide bond formation. The Cu1+ affinity of D1 is (2.6 ± 0.5) x 1018 M-1 at pH 7.5, while that of D3 is six times weaker. D1 of human CCS1 has a Cu1+ affinity which is over an order of magnitude greater than that of D3. Aerobic incubation of reduced E,Zn-Sod1 with all Cu1+-loaded Ccs1 proteins produces catalytically active Cu,Zn-Sod1 containing the catalytically active disulfide bond. Activation with C17S/C20S-Ccs1 is slightly lower than with the wild type protein, however removal of the copper-binding site in D3 (C229S/C231S-Ccs1 and D1/2-Ccs1) has a much more marked effect. A second cytosolic metallochaperone, Atx1, delivers Cu1+ to the copper-transporting P-type ATPase Ccc2 for incorporation into the membrane protein Fet3p. The Cu1+ affinity of Atx1 is greater than those of the copper-binding motifs of Ccs1. Exchange of Cu1+ between D1 and D3 of Ccs1 and Atx1 occurs as expected on the basis of their Cu1+ affinities. Ccs1 and Atx1 may collect Cu1+ from the same source, and cross-talk between them and their copper-trafficking pathways could occur.
Description: PhD Thesis
Appears in Collections:Institute for Cell and Molecular Biosciences

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