.(1980s)Hershko and Ciechanover purified the components of the first part of the reaction (APF-1-protein amide synthetase): E1, E2 and enzymes, by covalent'affinity chromatography over immobilized Ub s The protease step was further clarified with the characterization purification of the 26 proteasome complex, by martin Rechsteiner and colleagues(1986) .o It was first thought that multiple ubiquitin attached to different Lys sites in the substrate protein but Hershko( 1985)and then A xander Varshavsky and colleagues(1989)demonstrated that a single poly- ubiquitin chain could be form through attachment to a single internal Lys residue Attachment of subsequent Ub to the first one occurs through linkages of the Gly c-t of the next Ub to specific lysines present in the precedent Ub Poly-ubiquitin chains might have different roles dependin on the lys utilized K48> recognition by proteasome K63> signal for traffic and degradation in the lysosome, vacuole; subunit selectivity K29. K6. etc First evidence showing that ubiquitin constitutes a signal for protein degradation in vivo Hershko lab: 1982): immunochemical analysis of Ub adducts in cells> by using anti-Ub abs they saw that after incubation of cells in the presence of aa analogs the resulting abnormal proteins were short-lived and their rapid degradation was accompanied y a transient increase in Ub adducts But the strongest confirmation came with alexander varshavsky' s work™ (1980’s) Hershko and Ciechanover purified the components of the first part of the reaction (APF-1-protein amide synthetase): E1, E2 and E3 enzymes, by ‘covalent’ affinity chromatography over immobilized Ub. ™ The ‘protease’ step was further clarified with the characterization and purification of the 26 proteasome complex, by Martin Rechsteiner and colleagues (1986). ™ It was first thought that multiple ubiquitin attached to different Lys sites in the substrate protein but Hershko (1985) and then Alexander Varshavsky and colleagues (1989) demonstrated that a single poly-- ubiquitin chain could be form through attachment to a single internal Lys residue. Attachment of subsequent Ub to the first one occurs through linkages of the Gly C-t of the next Ub to specific lysines present in the precedent Ub. Poly-ubiquitin chains might have different roles depending on the Lys utilized: K48 Æ recognition by proteasome K63 Æ signal for traffic and degradation in the lysosome, vacuole; subunit selectivity K29, K6, etc.. ™ First evidence showing that ubiquitin constitutes a signal for protein degradation in vivo (Hershko lab; 1982): immunochemical analysis of Ub adducts in cells Æ by using anti-Ub Abs they saw that, after incubation of cells in the presence of aa analogs the resulting abnormal proteins were short-lived and their rapid degradation was accompanied by a transient increase in Ub adducts. But the strongest confirmation came with Alexander Varshavsky’s work