A novel region within a conserved domain in ATG7 emerged in vertebrates.

The E1-like enzyme ATG7 belongs to a group of ATG proteins that mediate the autophagy process. Autophagy is a highly conserved degradation pathway important for maintaining homeostasis in eukaryotic cells. Here, we study the evolution of E1 enzymes and specifically describe a region of ATG7 that emerged early in vertebrates. This vertebrate-specific region (VSR) is situated within the adenylation domain of the protein, which is the most conserved domain of E1 enzymes and is of prokaryotic origin. A comparative analysis revealed that ATG7 is unique in this respect, as in other E1 enzyme family members this domain is highly conserved from yeast to humans and has not experienced insertions of extra amino acids. The function of the VSR domain is unknown, but two residues within the region, D522 and S531 have been linked with cancer in humans. Analysis of natural selection indicates positive selection on S531 only on the mammalian clade. Notably, this was the only residue in ATG7 showing this signal. Interestingly, structural analysis of ATG7 predicted that the VSR may be intrinsically disordered and could harbor a macro-molecular binding site. Analysis of cells expressing ATG7 lacking the VSR indicated that these cells are unable to facilitate the lipidation of LC3, suggesting an important role of this region in autophagy. Abbreviations: aBSREL - an adaptive branch-site random effects likelihood; AD - adenylation domain; ATGs - autophagy-related genes; Baf-A1 - Bafilomycin-A1; EV - empty-vector; CTD - C-terminal domain; ECTD - extreme C-terminal domain; EMT - epithelial-mesenchymal transition; FEL - fixed effects likelihood; GABARAP - gamma-aminobutyric acid receptor-associated protein; LC3 - microtubule-associated protein 1A/1B-light chain 3; MEFs - mouse embryonic fibroblasts; MOCS3 - molybdenum cofactor synthesis 3; NTD - N-terminal domain; UBL ubiquitin like protein; VSR - vertebrate specific region.