The Rrp4 G58V and Rrp4 G226D variants do not associate with the RNA exosome complex in the presence of wild-type Rrp4, but the Rrp4 G226D variant can associate with the RNA exosome complex. (A) The Rrp4 G226D and Rrp4 G58V variants do not associate with the RNA exosome core subunit Rrp43 in the presence of a wild-type copy of Rrp4. TAP-tagged Rrp43 was immunoprecipitated from RRP43-TAP cells expressing Myc-tagged Rrp4, Rrp4 G58V, or Rrp4 G226D in the presence of wild-type Rrp4 grown at 30°C using IgG Sepharose beads and analyzed by immunoblotting. As a control, immunoprecipitations were also performed from untagged RRP43 cells (No TAP Control) expressing Myc-tagged Rrp4 proteins. The bound/input level of Rrp4-Myc was detected with an anti-Myc antibody and bound/input level of Rrp43-TAP was detected with a peroxidase antiperoxidase (PAP) antibody. Bound Rrp43-TAP was also detected by the anti-Myc antibody as the Protein A moiety of the TAP tag binds to antibody. The input level of 3-phosphoglycerate kinase (Pgk1) was detected with an anti-Pgk1 antibody as a loading control. The stain-free signal from input protein is also included as a loading control. (B) The Rrp4 G226D variant associates with the RNA exosome core subunit Rrp43 at a level similar to wild-type Rrp4 when the variant is the sole form of Rrp4. TAP-tagged Rrp43 was immunoprecipitated from RRP43-TAP rrp4Δ cells expressing either Myc-tagged wild-type Rrp4 or Rrp4 G226D, which were grown at 30°C using IgG Sepharose beads and analyzed by immunoblotting. As a control, immunoprecipitations were also performed from untagged RRP43 rrp4Δ cells (No TAP Control) expressing Myc-tagged Rrp4 proteins. The bound/input level of Rrp4-Myc was assessed with an anti-Myc antibody and bound/input level of Rrp43-TAP was detected with a peroxidase anti-peroxidase (PAP) antibody. Bound Rrp43-TAP was also detected by the anti-Myc antibody as the Protein A moiety of the TAP tag binds to antibody. The input level of 3-phosphoglycerate kinase (Pgk1) was detected with an anti-Pgk1 antibody as a loading control. The immunoblots are representative of triplicate experiments; coimmunoprecipitations were performed as described in Materials and Methods.
