tailieunhanh - Báo cáo khoa học: 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

The propagation of yeast prion phenotypes is highly dependent on molecu-lar 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. | IFEBS Journal 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 Virginie Redeker1 Jonathan Bonnefoy1 Jean-Pierre Le Caer2 Samantha Pemberton1 Olivier Laprevote2 and Ronald Melki1 1 Laboratoire d Enzymologie et Biochimie Structurales CNRS Gif-sur-Yvette France 2 Institut de Chimie des Substances Naturelles CNRS Gif-sur-Yvette France Keywords cross-linker mass spectrometry molecular chaperone oligomerization Ure2p prion Correspondence Virginie Redeker or Ronald Melki Laboratoire d Enzymologie et Biochimie Structurales CNRS Avenue de la terrasse 91198 Gif-sur-Yvette Cedex France Fax 33 1 69 82 31 29 Tel 33 1 69 82 34 60 or 33 1 69 82 35 03 E-mail Received 28 July 2010 revised 5 October 2010 accepted 12 October 2010 doi The propagation of yeast prion phenotypes is highly dependent on molecular chaperones. We previously demonstrated that the molecular chaperone Ssalp 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 Ssalp 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 .

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