Abstract
Copper is an essential yet toxic trace element. The Ctr1 family of proteins plays a critical role for copper uptake in eukaryotes. However, the mechanisms of action of Ctr1 are largely unknown. Our previous data demonstrated that copper transport induces conformational changes in the cytosolic C terminus of the yeast Saccharomyces cerevisiae Ctr1. To define the physiological significance of this molecular event and gain better insights into the mechanism of Ctr1-mediated copper uptake, we have characterized the functional roles of the Ctr1 C terminus. A Ctr1 mutant lacking the entire C-terminal cytosolic tail is functional in high affinity copper uptake; however, yeast cells expressing this mutant are extremely sensitive to excess copper. Toxic copper uptake is not attributed to elevated expression or distinct subcellular localization of this mutant as compared with wild type Ctr1. Further characterization of the function of Ctr1 containing deletions or site-directed mutations at the C terminus indicates a structural role for the C terminus in controlling Ctr1 activities. In response to excess copper, Ctr1-mediated copper transport is rapidly blocked in a C terminus-dependent mechanism associated with direct binding of copper. We propose that conformational changes in the cytosolic tail of yeast Ctr1 by copper sensing within this domain lead to the inhibition of Ctr1-mediated copper transport. These data suggest a new regulatory mechanism by which yeast cells maintain homeostatic copper acquisition.
Highlights
Identification and characterization of the molecular mechanisms involved in transport, distribution, and excretion of copper and the regulation of these processes have revealed how cells maintain homeostatic copper metabolism (3, 4)
C-terminal Cytosolic Tail of Ctr[1] Is Dispensable for Copper Transport—To determine the roles for the cytosolic C-terminal tail of Ctr[1] in copper transport, we examined functionality of Ctr[1] with deletion of the C terminus encompassing the last 106 amino acids (Ctr1(300)) (Fig. 1A). ⌬ctr[1] cells were not able to grow on the YPEG media containing solely non-fermentable carbon sources because of the defect in copper-requiring cytochrome c oxidase (Fig. 1B)
Cells were cultured to exponential growth phase in synthetic complete (SC) media supplemented with copper chelator bathocuproine disulfonate (BCS) (10 M) for 12 h, and 64Cu (0.1 M, 64CuCl2) uptake was measured for 5 min
Summary
N-terminal extracellular domain and C-terminal cytosolic tail are distinct in length and amino acid sequence. Several lines of evidence indicate that Ctr[1] forms a multimer complex (30 –32), and a projection structure of the human Ctr[1] confirmed that Ctr[1] forms a compact trimer with a novel channel-like architecture and no structural occlusion of the predicted pore for copper (33). It is largely unknown how Ctr[1] transports copper. Our data suggest a new regulatory mechanism of Ctr[1] by which yeast cells prevent toxic accumulation of copper
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