ABSTRACT

Autophagosome formation is a fundamental process in macroautophagy/autophagy, a conserved self-eating mechanism in all eukaryotes, which requires the conjugating ATG (autophagy related) protein complex, ATG12–ATG5-ATG16L1 and lipidated MAP1LC3/LC3 (microtubule associated protein 1 light chain 3). How the ATG12–ATG5-ATG16L1 complex is recruited to membranes is not fully understood. Here, we demonstrated that RAB33B plays a key role in recruiting the ATG16L1 complex to phagophores during starvation-induced autophagy. Crystal structures of RAB33B bound to the coiled-coil domain (CCD) of ATG16L1 revealed the recognition mechanism between RAB33B and ATG16L1. ATG16L1 is a novel RAB-binding protein (RBP) that can induce RAB proteins to adopt active conformation without nucleotide exchange. RAB33B and ATG16L1 mutually determined the localization of each other on phagophores. RAB33B-ATG16L1 interaction was required for LC3 lipidation and autophagosome formation. Upon starvation, a fraction of RAB33B translocated from the Golgi to phagophores and recruited the ATG16L1 complex. In this work, we reported a new mechanism for the recruitment of the ATG12–ATG5-ATG16L1 complex to phagophores by RAB33B, which is required for autophagosome formation.

Abbreviations

: ATG: autophagy-related; Cα: alpha carbon; CCD: coiled-coil domain; CLEM: correlative light and electron microscopy; DTE: dithioerythritol; EBSS: Earle’s balanced salt solution; EDTA: ethylenediaminetetraacetic acid; EGFP: enhanced green fluorescent protein; FBS: fetal bovine serum; FLIM: fluorescence lifetime imaging microscopy; FRET: Förster resonance energy transfer; GDP: guanosine diphosphate; GOLGA2/GM130: golgin A2; GppNHp: guanosine 5ʹ-[β,γ-imido]triphosphate; GST: glutathione S-transferase; GTP: guanosine triphosphate; GTPγS: guanosine 5ʹ-O-[gamma-thio]triphosphate; HA (tag): hemagglutinin (tag); HEK: human embryonic kidney; HeLa: Henrietta Lacks; HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); IgG: immunoglobulin G; K_d: dissociation constant; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCF7: Michigan cancer foundation-7; MEF: mouse embryonic fibroblast; MEM: minimum essential medium Eagle; MST: microscale thermophoresis; NEAA: non-essential amino acids; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RAB: RAS-associated binding; RB1CC1/FIP200: RB1 inducible coiled-coil protein 1; RBP: RAB-binding protein; SD: standard deviation; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TBS-T: tris-buffered saline-tween 20; WD (repeat): tryptophan-aspartic acid (repeat); WIPI2B: WD repeat domain phosphoinositide interacting 2B; WT: wild type

摘要

自噬体形成是巨自噬/自噬的基本过程，这是所有真核生物中保守的自食机制，需要结合 ATG（自噬相关）蛋白复合物、ATG12-ATG5-ATG16L1 和脂化 MAP1LC3/LC3（微管相关蛋白 1 轻链） 3）。ATG12-ATG5-ATG16L1 复合物如何被招募到膜上尚不完全清楚。在这里，我们证明了在饥饿诱导的自噬过程中，RAB33B 在将 ATG16L1 复合物募集到吞噬细胞中起关键作用。RAB33B 的晶体结构与 ATG16L1 的卷曲螺旋结构域 (CCD) 结合，揭示了 RAB33B 和 ATG16L1 之间的识别机制。ATG16L1 是一种新型 RAB 结合蛋白 (RBP)，可诱导 RAB 蛋白在不发生核苷酸交换的情况下采用活性构象。RAB33B 和 ATG16L1 相互确定了彼此在吞噬细胞上的定位。LC3 脂化和自噬体形成需要 RAB33B-ATG16L1 相互作用。饥饿时，一小部分 RAB33B 从高尔基体转移到吞噬细胞并募集 ATG16L1 复合物。在这项工作中，我们报道了一种新机制，用于通过 RAB33B 将 ATG12-ATG5-ATG16L1 复合物募集到吞噬细胞中，这是自噬体形成所必需的。

缩写

：ATG：自噬相关；Cα：α碳；CCD：盘绕线圈域；CLEM：相关光学和电子显微镜；DTE：二硫赤藓糖醇；EBSS：厄尔平衡盐溶液；EDTA：乙二胺四乙酸；EGFP：增强型绿色荧光蛋白；FBS：胎牛血清；FLIM：荧光寿命成像显微镜；FRET：福斯特共振能量转移；GDP：鸟苷二磷酸；GOLGA2/GM130：高尔金A2；GppNHp：鸟苷 5ʹ-[β,γ-亚氨基]三磷酸；GST：谷胱甘肽 S-转移酶；GTP：鸟苷三磷酸；GTPγS：鸟苷 5ʹ-O-[γ-硫代]三磷酸；HA（标签）：血凝素（标签）；HEK：人胚肾；海拉：亨丽埃塔拉克斯；HEPES：（4-（2-羟乙基）-1-哌嗪乙磺酸）；IgG：免疫球蛋白G；ķ _{_}: 解离常数; MAP1LC3/LC3：微管相关蛋白1轻链3；MCF7：密歇根癌症基金会-7；MEF：小鼠胚胎成纤维细胞；MEM：最低必需培养基 Eagle；MST：微尺度热泳；NEAA：非必需氨基酸；PBS：磷酸盐缓冲液；PE：磷脂酰乙醇胺；PtdIns3P：3-磷酸磷脂酰肌醇；RAB：RAS相关结合；RB1CC1/FIP200：RB1 诱导型卷曲螺旋蛋白 1；RBP：RAB结合蛋白；SD：标准差；SDS：十二烷基硫酸钠；SQSTM1/p62：隔离体 1；TBS-T：tris-缓冲盐水-吐温 20；WD（重复）：色氨酸-天冬氨酸（重复）；WIPI2B：WD 重复结构域磷酸肌醇相互作用 2B；WT：野生型