Figure 5: Ubiquitylation releases Rpn10 from the proteasome.

(a) Superimposition of the Ub-Rpn10 onto the cryo-EM structure²³. Colour code: grey, cryo-EM model; purple current model. The Ub moiety from the Ub-Rpn10 molecule (orange) clearly clashes into the Rpn9 subunit (pink). (b) Pull-down experiments of Rpn10 and Ub-Rpn10 by the indicated proteins. His₆-tagged proteins were immobilized on Ni²⁺ beads and used to pull-down apo-Rpn10 or Ub-Rpn10. Bound fractions were analysed by SDS–PAGE followed by western blot with an α-Rpn10 antibody. Apo-Rpn10 and Ub-Rpn10 inputs are indicated on the left side of the blot. SDS–PAGE analysis shows the immobilized protein inputs (right). (c) PCA analysis shows that Rpn10 ubiquitylation at K84 destabilizes the interaction with Rpn9 in vivo. Ten-fold serial dilutions of strains containing the N terminus fusions of RPN9 (F[1,2]::RPN9), combined with either [F3]::RPN10 or [F3]::rpn10-K84R (RPN10 mutated at lysine 84), were plated on a rich medium lacking (control) or supplemented with 200 μg ml⁻¹ methotrexate. CDC19::F[1,2]/MCK1::F[3] and MET18::F[1,2]/MCK1::F[3] fusion proteins were used as positive and negative controls, respectively. (d) Rpn10 ubiquitylation on purified proteasomes. rpt6Δ yeast strains expressing wild type or Rpn10-K84 mutant or Rpn10Δ from the native chromosomal locus were transformed with vector expressing Tap-proteinA-Rpt6. Proteasomes from the indicated strains were affinity-purified and immobilized on calmodulin resin. The immobilized proteasomes were incubated with Rpn10 ubiquitylation buffer that containing bacterial purified Ub, E1, Ubc4 and Rsp5. Bound and unbound fractions were separated on columns, and samples were resolved by SDS–PAGE, followed by western blot with the indicated antibodies. Ubiquitylated-Rpn10 was observed only in the unbound fraction.