Interaction of Nop17 with the other subunits of the R
TP complex. (A) Analysis of the interactions between Nop17 and Rvb1, and Rvb2 through the two-hybrid assay. BD-Nop17 interacts with both AD-Rvb1 and AD-Rvb2, as seen by the expression of the reporter genes HIS3 and lacZ. BD-Rvb2 + AD-Nop17 is stronger than BD-Rvb1-AD-Nop17. BD-Nip7/AD-Rrp43 and BD-Nip7/AD-Nop8 were used as positive controls for interaction [21,22] (B) Pull-down assay to confirm direct interaction between Nop17 and Rvb1/2. GST or GST-Nop17 were bound to glutathione-sepharose beads, followed by the incubation with His-Rvb1, or His-Rvb2, in the absence, or presence of 1 mM ATP or ADP at 4°C for 2 hours. Fractions from total extract (TE), flow through (FT), wash (W), or bound (B) were separated by SDS-PAGE and subjected to western blot with anti-His or anti-GST sera. Interaction Nop17-Rvb2 is independent of ATP. (C) Two-hybrid assay for the analysis of Nop17-Tah1 and Nop17-Hsp90 interaction. BD-Nop17 did not interact with AD-Hsp90, whereas BD-Nop17 interacted with AD-Tah1. Mutation of Nop17 in the position 306 disrupts interaction with Tah1.