Fig. 3: Rqc1 interacts with Ltn1 and ubiquitin.

a Domain organization of Rqc1 delineating its NTD and CTD. Alpha-helical residue ranges involved with its interactions with Ltn1 and ubiquitin are indicated. b Close up view of Rqc1^CTD in complex with ubiquitin and Ltn1^RING. Gaussian-filtered map of the Rqc1-Ltn1-ubiquitin portion shown for clarity. c, d Focused views of Rqc1 interactions with Ltn1^RING and ubiquitin, respectively. Key interaction residues are labeled. Residues mutated to generated Rqc1^Ltn1 or Rqc1^Ub are indicated with a red outline. e Immunoblot of RQC co-IPs from wild-type or Rqc1 mutant (Rqc1^Ltn1 or Rqc1^Ub) backgrounds probed against total ubiquitin. Anti-FLAG (Rqc1) blot shown as loading control. f Same as in (d), but probed with anti-K48-linked poly-ubiquitin antibody. g Silver stained SDS-PAGE of RQC co-IPs reveal a loss of Cdc48 recovery by Rqc1^Ltn1 and Rqc1^Ub mutants. h Anti-Cdc48 immunoblot from Rqc1 co-IPs. Anti-FLAG (Rqc1) blot shown as loading control. i Immunoblot of whole cell lysate against nonstop substrate (anti-Protein A) in Rqc1∆ cells expressing Rqc1^Ltn1 or Rqc1^Ub mutants compared to wild-type control. Anti-Pgk1 blot shown as loading control. In (e–i), each experiment was repeated at least three times as independent biological replicates.