Figure 5
ATPase-dependent helicase activity of ASCC3 is essential for RQC induction. (A) Top: Domain structure of ASCC3. Bottom: Amino acid sequence alignment of the conserved residues in the RecA1 motif I of indicated proteins. (B) FLAG-ASCC3-WT or K505R mutant was co-transfected into ASCC3 KD cells along with the V5-GFP-K(AAA)24-FLAG-HIS3 reporter, and protein was analyzed by western blotting with anti-V5 antibodies. (C) HA-ASCC2, and FLAG-ASCC3-WT or K505R mutant were co-transfected to HEK293T cells. Cell lysates were immunoprecipitated with anti-FLAG antibody beads. FLAG-tagged proteins were eluted with FLAG peptides, and the elution fraction was analyzed by western blotting with antibodies against FLAG and HA. (B,C) Cropped blots are displayed; full uncropped blots are available in Supplemental Fig. S6. Blots shown are representative of three independent experiments. (D) FLAG-ASCC3-WT or K505R mutant was transfected into ASCC3 KD cells, and polysome analysis was performed. Top: A254 of each fraction is plotted. Bottom: Protein distribution of ASCC3 was analyzed by western blotting with anti-FLAG antibodies. Cropped blots are displayed; full uncropped blots are available in Supplemental Fig. S7. Blots shown are representative of three independent experiments. (E) Two independent blots of indicated samples were quantified, and relative ASCC3 distribution is displayed. Error bars represent S.E. from N = 2.
