Abstract
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; Kd: 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
Highlights
Autophagy is an evolutionarily conserved catabolic process mainly to recycle or eliminate dysfunctional cellular orga nelles or proteins
The production of PtdIns3P and subsequent recruit ment of WD repeat domain phosphoinositide interacting 2B (WIPI2B) to membranes were independent of RAB33B. This is the first report of an endogenous protein that can induce conformational change of a small GTPase from the inactive form to the active form without nucleotide exchange, which is usually the driving force for such a process. It appears that the binding of ATG16L1 with switch I and switch II regions of RAB33B provides energy for such a conformational change in these regions (Figure S3C)
E186 and R193 in the other molecule of the dimeric ATG16L1 contributed to the stabilization of the active conformation of guanosine dipho sphate (GDP)-RAB33B via hydrogen bonding with R94 of the switch II region and the main chain of the switch I region, respectively (Figure 2B)
Summary
Autophagy is an evolutionarily conserved catabolic process mainly to recycle or eliminate dysfunctional cellular orga nelles or proteins. The process involves the formation of double-membrane vesicle structures, termed autophagosomes, which sequester and engulf cytoplasmic components consti tutively, or upon nutrient deprivation or stress. The subse quent autophagosome maturation is achieved by fusing with lysosomes to form autolysosomes, leading to the exposure of the cargo to lysosomal hydrolases for digestion. Autophagy plays an important role in cellular physiology including cell development and has been associated with diverse human diseases, including cancer, neurodegeneration and pathogen infection [1,2,3]. Autophagosomes initiate from phago phores throughout the cytosol. Phagophores expand, enfold cytosolic cargo and close, forming autophagosomes. Two ubiquitin-like conjugation systems are crucial for the Supplemental data for this article can be accessed here
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