title: Cadmium and Secondary Structure-dependent Function of a Degron in the Pca1p Cadmium Exporter.
abstract: Protein turnover is a critical cellular process regulating biochemical pathways and destroying terminally misfolded or damaged proteins. Pca1p, a cadmium exporter in the yeast Saccharomyces cerevisiae, is rapidly degraded by the endoplasmic reticulum-associated degradation (ERAD) system via a cis-acting degron that exists at the 250-350 amino acid region of Pca1p and is transferable to other proteins to serve as a degradation signal. Cadmium stabilizes Pca1p in a manner dependent on the degron. This suggested that cadmium-mediated masking of the degron impedes its interaction with the molecular factors involved in the ERAD. The characteristics and mechanisms of action of the degron in Pca1p and most of those in other proteins however remain to be determined. The results presented here indicate that specific cysteine residues in a degron of Pca1p sense cadmium. An unbiased approach selecting non-functional degrons indicated a critical role of hydrophobic amino acids in the degron for its function. A secondary structure modeling predicted the formation of an amphipathic helix. Site-directed mutagenesis confirmed the functional significance of the hydrophobic patch. Last, hydrophobic amino acids in the degron- and cadmium-binding region affected the interaction of Pca1p with the Ssa1p molecular chaperone, which is involved in ERAD. These results reveal the mechanism of action of the degron, which might be useful for the identification and characterization of other degrons.
authors.affiliation: From the Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska, 68588-0664 and.
authors.forename: Nathan
authors.initials: N
authors.lastname: Smith
authors.affiliation: From the Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska, 68588-0664 and.
authors.forename: Wenzhong
authors.initials: W
authors.lastname: Wei
authors.affiliation: From the Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska, 68588-0664 and.
authors.forename: Miaoyun
authors.initials: M
authors.lastname: Zhao
authors.affiliation: From the Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska, 68588-0664 and the College of Life and Environmental Sciences, Shanghai Normal University, Shanghai 200234, China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Qin
authors.affiliation: From the Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska, 68588-0664 and.
authors.forename: Javier
authors.initials: J
authors.lastname: Seravalli
authors.affiliation: From the Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska, 68588-0664 and.
authors.forename: Heejeong
authors.initials: H
authors.lastname: Kim
authors.affiliation: From the Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska, 68588-0664 and jlee7@unl.edu.
authors.forename: Jaekwon
authors.initials: J
authors.lastname: Lee
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002104:Cadmium / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D060746:Endoplasmic Reticulum-Associated Degradation / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D027682:Cation Transport Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; C093603:PCA1 protein, S cerevisiae; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D002104:Cadmium; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae; D003545:Cysteine
keywords: ATPase; cadmium; degron; endoplasmic reticulum-associated protein degradation (ERAD); metal homeostasis; metal ion-protein interaction; molecular chaperone; transporter; yeast