DOI: 10.6084/m9.figshare.30103091.v1
title: Evolutionary diversification of the autophagy initiation complex: reduced Atg101 dependency and changes in Atg9 binding to Atg13

description: Macroautophagy/autophagy is an evolutionarily conserved process through which cells degrade cytoplasmic substances via autophagosomes. During the initiation of autophagosome formation, the ULK/Atg1 complex serves as a scaffold that recruits and regulates downstream ATG/Atg proteins and ATG9/Atg9-containing vesicles. Despite the essential role of the ULK/Atg1 complex, its components have changed during evolution; the ULK complex in mammals consists of ULK1 (or ULK2), RB1CC1, ATG13, and ATG101, whereas the Atg1 complex in the yeast <i>Saccharomyces cerevisiae</i> lacks Atg101 but instead has Atg29 and Atg31 along with Atg17. In this study, we investigated how such changes have evolved. A BLAST analysis across the major eukaryotic clades revealed that <i>ATG101</i>, which is essential for autophagy in mammals, was lost in some Holomycota lineages after acquisition of <i>ATG29</i> and <i>ATG31</i> by their common ancestor. Additionally, the acquisition of a cap structure in Atg13 preceded the loss of <i>ATG101</i>. However, some Holomycota species have both <i>ATG101</i> and <i>ATG29-ATG31</i>, including <i>Aspergillus oryzae</i> and <i>Komagataella phaffii</i>. Yeast two-hybrid assays showed that ATG101 is required for ATG13-ATG9 interaction in mammals but dispensable in <i>A. oryzae</i>, probably because of a shift in the <i>Ao</i>Atg9-binding site in <i>Ao</i>Atg13. We found an additive effect between <i>atg101</i> and <i>atg31</i> deletions in starvation-induced autophagy in <i>K. phaffii</i>. Furthermore, both <i>Kp</i>Atg101 and <i>Kp</i>Atg31 are involved in Atg1 complex assembly in <i>K. phaffii</i>. These findings suggest that the reduced importance of Atg101 in the Atg13-Atg9 interaction and Atg1 complex assembly enabled the eventual loss of <i>ATG101</i> in some Holomycota species, including <i>S. cerevisiae</i>. <i>Ao</i>: <i>Aspergillus oryzae</i>; 3-AT: 3-amino-1,2,4-triazole; ATG: autophagy related; ATG9C: the C-terminal region of ATG9; ATG9N: the N-terminal region of ATG9; ATG13H: the HORMA domain of ATG13; BLASTP: protein basic local alignment search tool; BUSCO: benchmarking universal single-copy orthologs; CFP: cyan fluorescent protein; <i>Co</i>: <i>Capsaspora owczarzaki</i>; DDM: <i>n</i>-dodecyl-β-d-maltoside; DELTA-BLAST: domain enhanced lookup time accelerated basic local alignment search tool; DMEM: Dulbecco’s Modified Eagle Medium; GFP: green fluorescent protein; HORMA: Hop1, Rev7, and MAD2; <i>Hs</i>: <i>Homo sapiens</i>; KO: knockout; <i>Kp</i>: <i>Komagataella phaffii</i>; LECA: last eukaryotic common ancestor; MEF: mouse embryonic fibroblast; <i>Nc</i>: <i>Neurospora crassa</i>; <i>Op</i>: <i>Ogataea polymorpha</i>; PAS: phagophore assembly site; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; Pgk1: phosphoglycerate kinase 1; PtdIns3K: phosphatidylinositol 3-kinase; PSI-BLAST: position-specific iterative basic local alignment search tool; RB1CC1: RB1 inducible coiled-coil 1; <i>Sc</i>: <i>Saccharomyces cerevisiae</i>; SD+DO: synthetic defined medium with dropout mix; SD-N: synthetic defined medium without nitrogen sources; SLiCE, seamless ligation cloning extract; <i>Sp</i>: <i>Schizosaccharomyces pombe</i>; TMR: tetramethylrhodamine; ULK: unc-51 like autophagy activating kinase; WT: wild type; YPD: yeast extract, peptone, dextrose