title: ATP-dependent steps in the binding of ubiquitin conjugates to the 26S proteasome that commit to degradation.
abstract: Eukaryotic cells target proteins for degradation by the 26S proteasome by attaching a ubiquitin chain. Using a rapid assay, we analyzed the initial binding of ubiquitinated proteins to purified 26S particles as an isolated process at 4°C. Subunits Rpn10 and Rpn13 contribute equally to the high-affinity binding of ubiquitin chains, but in their absence, ubiquitin conjugates bind to another site with 4-fold lower affinity. Conjugate binding is stimulated 2- to 4-fold by binding of ATP or the nonhydrolyzable analog, ATPγS (but not ADP), to the 19S ATPases. Following this initial, reversible association, ubiquitin conjugates at 37°C become more tightly bound through a step that requires ATP hydrolysis and a loosely folded domain on the protein, but appears independent of ubiquitin. Unfolded or loosely folded polypeptides can inhibit this tighter binding. This commitment step precedes substrate deubiquitination and allows for selection of ubiquitinated proteins capable of being unfolded and efficiently degraded.
authors.affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Andreas
authors.initials: A
authors.lastname: Peth
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Uchiki
authors.forename: Alfred L
authors.initials: AL
authors.lastname: Goldberg
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001681:Biological Assay; D006868:Hydrolysis; D008239:Lysine / Q000378:metabolism; D025821:Polyubiquitin / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D058767:Protein Unfolding; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; C122019:RPN10 protein, S cerevisiae; C529711:RPN13 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D025821:Polyubiquitin; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D008239:Lysine