title: A region within the C-terminal domain of Ure2p is shown to interact with the molecular chaperone Ssa1p by the use of cross-linkers and mass spectrometry.
abstract: The propagation of yeast prion phenotypes is highly dependent on molecular chaperones. We previously demonstrated that the molecular chaperone Ssa1p sequesters Ure2p in high molecular weight, assembly incompetent oligomeric species. We also determined the affinity of Ssa1p for Ure2p, and its globular domain. To map the Ure2p-Ssa1p interface, we have used chemical cross-linkers and MS. We demonstrate that Ure2p and Ssa1p form a 1 : 1 complex. An analytical strategy combining in-gel digestion of cross-linked protein complexes, and both MS and MS/MS analysis of proteolytic peptides, allowed us to identify a number of peptides that were modified because they are exposed to the solvent. A difference in the exposure to the solvent of a single lysine residue, lysine 339 of Ure2p, was detected upon Ure2p-Ssa1p complex formation. These observations strongly suggest that lysine 339 and its flanking amino acid stretches are involved in the interaction between Ure2p and Ssa1p. They also reveal that the Ure2p amino-acid stretch spanning residues 327-339 plays a central role in the assembly into fibrils.
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France. virginie.redeker@lebs.cnrs-gif.fr
authors.forename: Virginie
authors.initials: V
authors.lastname: Redeker
authors.forename: Jonathan
authors.initials: J
authors.lastname: Bonnefoy
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Le Caer
authors.forename: Samantha
authors.initials: S
authors.lastname: Pemberton
authors.forename: Olivier
authors.initials: O
authors.lastname: Laprévote
authors.forename: Ronald
authors.initials: R
authors.lastname: Melki
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005979:Glutathione Peroxidase / Q000737:chemistry / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D011328:Prions / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D011328:Prions; D029701:Saccharomyces cerevisiae Proteins; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae; D008239:Lysine