Evolutionary diversification of the autophagy initiation complex: reduced Atg101 dependency and changes in Atg9 binding to Atg13

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

巨自噬（Macroautophagy）/自噬（autophagy）是一类进化保守的过程，细胞通过自噬体（autophagosome）降解胞质物质。在自噬体形成的起始阶段，UNC-51样自噬激活激酶/自噬相关基因1（ULK/Atg1）复合物作为支架，招募并调控下游自噬相关（ATG/Atg）蛋白以及含ATG9/Atg9的囊泡。尽管ULK/Atg1复合物发挥着不可或缺的作用，但其组分在进化过程中发生了改变：哺乳动物中的ULK复合物由ULK1（或ULK2）、RB1诱导卷曲螺旋1（RB1 inducible coiled-coil 1, RB1CC1）、ATG13和ATG101组成，而酿酒酵母（Saccharomyces cerevisiae）中的Atg1复合物缺少Atg101，取而代之的是与Atg17一同存在的Atg29和Atg31。本研究探讨了这类组分变化的演化历程。针对主要真核生物支系的基本局部比对搜索工具（BLAST, basic local alignment search tool）分析显示，在哺乳动物中对自噬至关重要的ATG101，在部分全真菌（Holomycota）谱系中，于其共同祖先获得ATG29和ATG31之后发生了丢失。此外，Atg13的帽结构域的出现早于ATG101的丢失。不过，部分全真菌物种同时保留了ATG101与ATG29-ATG31，包括米曲霉（Aspergillus oryzae）和巴斯德毕赤酵母（Komagataella phaffii）。酵母双杂交实验结果表明，ATG101在哺乳动物中是介导ATG13与ATG9相互作用的必需因子，但在米曲霉中并非必需，这一现象可能源于米曲霉Atg13的AoAtg9结合位点发生了偏移。我们在巴斯德毕赤酵母的饥饿诱导自噬实验中发现，atg101与atg31的缺失存在叠加效应。进一步研究证实，巴斯德毕赤酵母中的KpAtg101和KpAtg31均参与了Atg1复合物的组装。上述研究结果表明，Atg101在Atg13-Atg9相互作用以及Atg1复合物组装过程中的重要性降低，使得包括酿酒酵母在内的部分全真菌物种最终丢失了ATG101。
Ao: 米曲霉（Aspergillus oryzae）；3-AT: 3-氨基-1,2,4-三唑（3-amino-1,2,4-triazole）；ATG: 自噬相关基因（autophagy related）；ATG9C: ATG9的C端区域（C-terminal region of ATG9）；ATG9N: ATG9的N端区域（N-terminal region of ATG9）；ATG13H: ATG13的HORMA结构域（HORMA domain of ATG13）；BLASTP: 蛋白质基本局部比对搜索工具（protein basic local alignment search tool）；BUSCO: 基准通用单拷贝同源基因（benchmarking universal single-copy orthologs）；CFP: 青色荧光蛋白（cyan fluorescent protein）；Co: 卡氏孢子虫（Capsaspora owczarzaki）；DDM: n-十二烷基-β-D-麦芽糖苷（n-dodecyl-β-d-maltoside）；DELTA-BLAST: 域增强搜索加速基本局部比对搜索工具（domain enhanced lookup time accelerated basic local alignment search tool）；DMEM: 杜尔贝科改良伊格尔培养基（Dulbecco’s Modified Eagle Medium）；GFP: 绿色荧光蛋白（green fluorescent protein）；HORMA: Hop1、Rev7和MAD2结构域（Hop1, Rev7, and MAD2）；Hs: 智人（Homo sapiens）；KO: 敲除（knockout）；Kp: 巴斯德毕赤酵母（Komagataella phaffii）；LECA: 最后真核共同祖先（last eukaryotic common ancestor）；MEF: 小鼠胚胎成纤维细胞（mouse embryonic fibroblast）；Nc: 粗糙脉孢菌（Neurospora crassa）；Op: 多形汉逊酵母（Ogataea polymorpha）；PAS: 自噬体组装位点（phagophore assembly site）；PBS: 磷酸盐缓冲液（phosphate-buffered saline）；PCR: 聚合酶链式反应（polymerase chain reaction）；Pgk1: 磷酸甘油酸激酶1（phosphoglycerate kinase 1）；PtdIns3K: 磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase）；PSI-BLAST: 位置特异性迭代基本局部比对搜索工具（position-specific iterative basic local alignment search tool）；RB1CC1: RB1诱导卷曲螺旋1（RB1 inducible coiled-coil 1）；Sc: 酿酒酵母（Saccharomyces cerevisiae）；SD+DO: 含缺失混合物的合成限定培养基（synthetic defined medium with dropout mix）；SD-N: 无氮源合成限定培养基（synthetic defined medium without nitrogen sources）；SLiCE: 无缝连接克隆提取（seamless ligation cloning extract）；Sp: 裂殖酵母（Schizosaccharomyces pombe）；TMR: 四甲基罗丹明（tetramethylrhodamine）；ULK: UNC-51样自噬激活激酶（unc-51 like autophagy activating kinase）；WT: 野生型（wild type）；YPD: 酵母提取物-蛋白胨-葡萄糖培养基（yeast extract, peptone, dextrose）