title: Transfer of Ho endonuclease and Ufo1 to the proteasome by the UbL-UbA shuttle protein, Ddi1, analysed by complex formation in vitro.
abstract: The F-box protein, Ufo1, recruits Ho endonuclease to the SCF(Ufo1) complex for ubiquitylation. Both ubiquitylated Ho and Ufo1 are transferred by the UbL-UbA protein, Ddi1, to the 19S Regulatory Particle (RP) of the proteasome for degradation. The Ddi1-UbL domain binds Rpn1 of the 19S RP, the Ddi1-UbA domain binds ubiquitin chains on the degradation substrate. Here we used complex reconstitution in vitro to identify stages in the transfer of Ho and Ufo1 from the SCF(Ufo1) complex to the proteasome. We report SCF(Ufo1) complex at the proteasome formed in the presence of Ho. Subsequently Ddi1 is recruited to this complex by interaction between the Ddi1-UbL domain and Ufo1. The core of Ddi1 binds both Ufo1 and Rpn1; this interaction confers specificity of SCF(Ufo1) for Ddi1. The substrate-shield model predicts that Ho would protect Ufo1 from degradation and we find that Ddi1 binds Ho, Ufo1, and Rpn1 simultaneously forming a complex for transfer of Ho to the 19S RP. In contrast, in the absence of Ho, Rpn1 displaces Ufo1 from Ddi1 indicating a higher affinity of the Ddi1-UbL for the 19S RP. However, at high Rpn1 levels there is synergistic binding of Ufo1 to Ddi1 that is dependent on the Ddi1-UbA domain. Our interpretation is that in the absence of substrate, the Ddi1-UbL binds Rpn1 while the Ddi1-UbA binds ubiquitin chains on Ufo1. This would promote degradation of Ufo1 and disassembly of SCF(Ufo1) complexes.
authors: Department of Life Sciences, Ben Gurion University of the Negev, Beersheba, Israel.
authors: Olga
authors: O
authors: Voloshin
authors: Anya
authors: A
authors: Bakhrat
authors: Sharon
authors: S
authors: Herrmann
authors: Dina
authors: D
authors: Raveh
mesh_terms: D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D044783:F-Box Proteins / Q000737:chemistry / Q000378:metabolism*; D055767:Immobilized Proteins / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: C108125:DDI1 protein, S cerevisiae; D044783:F-Box Proteins; D055767:Immobilized Proteins; D029701:Saccharomyces cerevisiae Proteins; C509156:UFO1 protein, S cerevisiae; C530845:HO protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D046988:Proteasome Endopeptidase Complex