Figure 6. Release of polyubiquitinated substrate from the Cdc48 ATPase complex.
(A) Photoconverted Ub(n)-Dendra, Cdc48, and Ufd1 were incubated with or without FLAG-tagged Npl4 in the presence of ADP or ATP. The Cdc48 complex was isolated with FLAG-antibody beads, and bead-bound material analyzed by SDS-PAGE, followed by immunoblotting (IB) with ubiquitin antibodies (anti-Ub; upper panel) and Coomassie-blue staining (lower panel).
(B) Scheme showing two conceivable scenarios for the post-translocation state.
(C) Dye-labeled Ub(n)-Dendra was incubated in the presence of ADP or ATP with Cdc48, Npl4-FLAG, and Ufd1 containing a TEV cleavage site following the UT3 domain (Ufd1TEV) (1st incubation). The Cdc48 complex was retrieved with FLAG-antibody beads, and bound material was treated with TEV protease in the presence of ADP, ATP, or ATPγS, a non-hydrolyzable ATP analog (2nd incubation). Bead-bound material was then analyzed by SDS-PAGE, followed by fluorescence scanning (upper panel) and Coomassie-blue staining (lower panel).
(D) As in (C), but with Ub(n)-sfGFP-Dendra. Note that sfGFP refolds after translocation, which would prevent backsliding of the polypeptide through the Cdc48 pore in scenario (a) (upper panel).
(E) Photoconverted Ub(n)-Dendra labeled with the fluorophore DyLight800 (Ub(n)-Dy800Dendra) was incubated with Cdc48-FLAG, SBP-Ufd1, and Npl4 in the presence of ADP or ATP (1st incubation). The Cdc48 complex was retrieved with streptavidin beads and eluted with biotin. The samples were then incubated with DyLight680-labeled, photoconverted Ub(n)-Dendra (Ub(n)-Dy680-Dendra) in the presence of ADP or ATP. Anti-FLAG antibody beads were added, and bound material analyzed by SDS-PAGE, followed by fluorescence scanning at 800nm and 680nm wavelength (upper two panels) and Coomassie-blue staining (lower panel). (F) Samples in (E), incubated in ADP or ATP during the first incubation, were tested for unfolding of photoconverted Ub(n)-Dy680-Dendra in ATP during the second incubation (panel E; lanes 9 versus 12).