Figure 5.

RNF31 ubiquitinates ATG13 on multiple lysine residues. (A) Interaction of ATG13 or its truncation mutants with RNF31. ATG13 truncation constructs were generated based on the domain information. 1–191aa was the HORMA domain. 383–433aa was the MIM motif. HEK293 FT cells were transfected with Flag-RNF31 and MYC-ATG13 or the indicated truncation mutants. Flag pulldown was performed with anti-Flag beads. Samples were analyzed by immunoblotting with the indicated antibodies. (B) Ubiquitination of MYC-ATG13 and its mutants in RNF31-overexpressing cells. HEK293 FT cells were transfected with MYC-ATG13 or the indicated mutants and untagged ubiquitin, along with the control or Flag-RNF31. Ubiquitinated proteins were immunoprecipitated with anti-MYC beads under denaturing conditions and samples were analyzed by immunoblotting with the indicated antibodies