Abstract
After Ctr1-mediated uptake into human cells, copper (Cu) ions are transported by the cytoplasmic Cu chaperone Atox1 to the Wilson disease protein (ATP7B) in the Golgi network. Cu transfer occurs via direct protein-protein interactions and leads to incorporation of Cu into Cu-dependent enzymes. ATP7B is a large multi-domain membrane-spanning protein which, in contrast to homologs, has six cytoplasmic metal-binding domains (MBDs). The reason for multiple MBDs is proposed to be indirect modulation of activity but mechanistic studies of full-length ATP7B are limited. We here developed a system that probes Cu flow through human Atox1 and ATP7B proteins when expressed in yeast. Using this assay, we assessed the roles of the different MBDs in ATP7B and found that the presence of the most N-terminal MBD increased, whereas the third MBD decreased, overall ATP7B-mediated Cu transport activity. Upon removal of all MBDs in ATP7B, the ability to transport Cu disappeared. The designed system can be expanded to include other yeast viability parameters and will be a useful tool for further mechanistic insights on human Cu transport as well as diseases involving Cu imbalance.
Highlights
ATP7A/B are large, multi-domain proteins with several domains protruding in the cytoplasm as well as membrane spanning parts that harbors a channel for Cu
To explore metal-binding domains (MBDs) domain–domain interactions in ATP7B, we have previously investigated several multi-MBD constructs and, through in vitro and in silico analyzes, identified correlated domain–domain structural dynamics in two-domain constructs,[26] linked domain– domain interactions in WD56 to thermal stability, Cu binding and Atox1-mediated Cu transfer,[27] and discovered that Cu binding triggers compaction of MBD1–4.23 the studies with truncated ATP7B constructs ca only indirectly address in vivo mechanisms
We expressed the human Atox[1] and ATP7B genes on high copy plasmids in order to test for complementation and thereby rescue of the phenotype
Summary
ATP7A/B are large (about 1400 residues), multi-domain proteins with several domains protruding in the cytoplasm as well as membrane spanning parts that harbors a channel for Cu. Metallomics as well as Atox[1], has a ferredoxin-like a/b fold and a surfaceexposed invariant CXXC motif (X = any residue) in which a single Cu can bind to the cysteine sulfurs. Because Atox[1] can deliver Cu to the MBDs,[14,15,16,17,18,19,20] through direct protein–protein interactions,[21,22] one may speculate that Cu-triggered conformational changes among these domains might initiate the catalytic cycle after Atox1mediated Cu delivery.[23] it remains unclear if Cu is being transferred via the MBDs or if Atox[1], like its bacterial homolog CopZ,[24] delivers Cu directly to a binding site at the membrane-spanning parts of ATP7A/B
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