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Detoxification and homeostatic acquisition of metal ions are vital for all living organisms. Non-physiological heavy metals are toxic at low concentrations and represent major environmental hazards to human health. In particular, cadmium is a toxic environmental pollutant linked to a number of ailments including cancer, kidney and bone disease and reproductive disorders. The biological effects of cadmium toxicity which lead to human disease and the cellular mechanisms for cadmium defense are ill defined. Thus, the study of heavy metal detoxification systems represents an important research avenue to help combat cadmium related disorders.
The goal of this research project was to identify novel factors involved in metal ion defense in the model eukaryote, Saccharomyces cerevisiae. A genetic screen led to the discovery of Pca1, a P-type ATPase which functions as a cadmium-specific efflux pump. Unexpectedly, the PCA1 allele in common laboratory yeast strains possesses an inactivating mis-sense mutation which has complicated previous attempts to characterize its function.
A unique feature of Pca1 is a cysteine-rich cytosolic amino terminal extension that can participate in metal coordination and regulate expression levels. Under normal conditions, Pca1 is an unstable protein that is ubiquitinated and rapidly degraded by the proteasome. However, in the presence of cadmium, ubiquitination is inhibited resulting in rapid up-regulation and trafficking of Pca1 to the cell surface for cadmium efflux. An autonomous degradation signal within the cysteine-rich domain is necessary and sufficient for metal responsive regulation.
Finally, a second genetic screen was devised to uncover factors involved in Pca1 degradation. Unexpectedly, components of the ER-associated degradation (ERAD) pathway were found to be required for Pca1 turnover in the absence of cadmium. Conformational changes associated with cadmium binding to the metal sensing degradation signal lead to the escape of Pca1 from ERAD.
In summary, our data have revealed a novel metal detoxification system in a eukaryotic organism mediated by a P-type ATPase that is unique in structure and substrate specificity. Furthermore, we have described an unprecedented mode of ligand regulated degradation of a cell surface transporter at the ER and have provided the mechanistic basis of this regulation.