Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and autophagy.

UFMylation involves the covalent modification of substrate proteins with UFM1 (Ubiquitin‐fold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ER‐bound ribosomes and activates C53‐mediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8‐interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIM‐mediated ATG8 interaction remains unknown. Here, we show that C53 and sAIMs are conserved across eukaryotes but secondarily lost in fungi and various algal lineages. Biochemical assays showed that the unicellular alga Chlamydomonas reinhardtii has a functional UFMylation pathway, refuting the assumption that UFMylation is linked to multicellularity. Comparative structural analyses revealed that both UFM1 and ATG8 bind sAIMs in C53, but in a distinct way. Conversion of sAIMs into canonical AIMs impaired binding of C53 to UFM1, while strengthening ATG8 binding. Increased ATG8 binding led to the autoactivation of the C53 pathway and sensitization of Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral role of sAIMs in UFMylation‐dependent fine‐tuning of C53‐mediated autophagy activation. Synopsis: Phylogenomic profiling has shown that UFMylation coevolves with the shuffled ATG8‐interacting motifs (sAIMs) in the ER‐phagy receptor C53. Further mechanistic analyses demonstrated that sAIMs regulate C53‐mediated autophagy by competitively binding UFM1 and ATG8.UFMylation and C53 autophagy pathways are conserved in eukaryotes, but secondarily lost in fungi and various parasitic and algal species.UFMylation is crucial for ER stress tolerance in the unicellular alga Chlamydomonas reinhardtii.Both UFM1 and ATG8 bind the shuffled AIM sequences within the intrinsically disordered domain of C53, yet their binding modes are different.Competitive binding of either UFM1 or ATG8 to C53 shuffled AIM sequences regulates C53‐mediated autophagy and ER stress tolerance. [ABSTRACT FROM AUTHOR]