The environmental toxin cadmium is excreted from cells by the membrane P-type ATPase transporter, called Pca1 in Saccharomyces cerevisiae. In yeast, the abundance of Pca1 is controlled by the rate of the ubiquitinylation and proteasomal degradation of the protein, not through transcriptional or translational mechanisms. As a transmembrane protein, Pca1 is synthesized in the endoplasmic reticulum (ER). Adle et al. identified CUE1, which encodes a protein involved in ER-associated degradation (ERAD), in a screen of yeast genetic knockouts that exhibited increased abundance of green fluorescent protein (GFP)–tagged Pca1. Knockout of DOA10 or UBC7, other genes implicated in the ERAD-C pathway, which degrades ER-associated proteins with lesions in the cytosolic portions, also stabilized GFP-Pca1. Release from the ER was not required for Pca1 stabilization or degradation, as Pca1 was efficiently degraded in yeast defective in ER exit, and cadmium promoted its stabilization. Cadmium did not trigger the unfolded protein response, thus ERAD was not activated because of a nonspecific effect of cadmium on protein folding or damage. Transfer of the N-terminal degron sequence that is necessary for regulation by cadmium and Pca1 degradation, either to another transporter (synthesized in the ER) or to cytosolic GFP, allowed both of these proteins to be stabilized by cadmium and degraded through a DOA10-dependent ERAD process. The Pca1 degron mediated a direct interaction between Pca1 and DOA10 as seen with chemical cross-linking and coimmunoprecipitation experiments. The Pca1 degron bound copper, and this interaction was competed by cadmium. Trypsin sensitivity assays suggested that metal binding to the Pca1 degron alters the conformation of this region, which may prevent the interaction with DOA10 and allow rapid stabilization of Pca1. Thus, by selectively targeting proteins for ERAD-mediated degradation, cells may rapidly respond to changes in environmental conditions.
D. J. Adle, W. Wei, N. Smith, J. J. Bies, J. Lee, Cadmium-mediated rescue from ER-associated degradation induces expression of its exporter. Proc. Natl. Acad. Sci. U.S.A. 106, 10189–10194 (2009). [Abstract] [Full Text]