ATG9 resides on a unique population of small vesicles in presynaptic nerve terminals

ABSTRACT In neurons, autophagosome biogenesis occurs mainly in distal axons, followed by maturation during retrograde transport. Autophagosomal growth depends on the supply of membrane lipids which requires small vesicles containing ATG9, a lipid scramblase essential for macroautophagy/autophagy. Here, we show that ATG9-containing vesicles are enriched in synapses and resemble synaptic vesicles in size and density. The proteome of ATG9-containing vesicles immuno-isolated from nerve terminals showed conspicuously low levels of trafficking proteins except of the AP2-complex and some enzymes involved in endosomal phosphatidylinositol metabolism. Super resolution microscopy of nerve terminals and isolated vesicles revealed that ATG9-containing vesicles represent a distinct vesicle population with limited overlap not only with synaptic vesicles but also other membranes of the secretory pathway, uncovering a surprising heterogeneity in their membrane composition. Our results are compatible with the view that ATG9-containing vesicles function as lipid shuttles that scavenge membrane lipids from various intracellular membranes to support autophagosome biogenesis. Abbreviations: AP: adaptor related protein complex: ATG2: autophagy related 2; ATG9: autophagy related 9; DNA PAINT: DNA-based point accumulation for imaging in nanoscale topography; DyMIN STED: dynamic minimum stimulated emission depletion; EL: endosome and lysosome; ER: endoplasmic reticulum; GA: Golgi apparatus; iBAQ: intensity based absolute quantification; LAMP: lysosomal-associated membrane protein; M6PR: mannose-6-phosphate receptor, cation dependent; Minflux: minimal photon fluxes; Mito: mitochondria; MS: mass spectrometry; PAS: phagophore assembly site; PM: plasma membrane; Px: peroxisome; RAB26: RAB26, member RAS oncogene family; RAB3A: RAB3A, member RAS oncogene family; RAB5A: RAB5A, member RAS oncogene family; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment receptor; SVs: synaptic vesicles; SYP: synaptophysin; TGN: trans-Golgi network; TRAPP: transport protein particle; VTI1: vesicle transport through interaction with t-SNAREs.

[1]  S. Jakobs,et al.  DNA-PAINT MINFLUX nanoscopy , 2022, Nature Methods.

[2]  P. De Camilli,et al.  ATG9 vesicles comprise the seed membrane of mammalian autophagosomes , 2022, bioRxiv.

[3]  David K. Sidibe,et al.  Retrograde Axonal Autophagy and Endocytic Pathways Are Parallel and Separate in Neurons , 2022, The Journal of Neuroscience.

[4]  P. De Camilli,et al.  Synaptic vesicle proteins and ATG9A self-organize in distinct vesicle phases within synapsin condensates , 2022, bioRxiv.

[5]  C. Garner,et al.  Organization of Presynaptic Autophagy-Related Processes , 2022, Frontiers in Synaptic Neuroscience.

[6]  S. Hell,et al.  Colocalization of different neurotransmitter transporters on synaptic vesicles is sparse except for VGLUT1 and ZnT3 , 2022, Neuron.

[7]  M. Aridor.  A tango for coats and membranes: New insights into ER-to-Golgi traffic. , 2022, Cell reports.

[8]  Marie-Françoise Bourvon,et al.  Formation , 2022, European Company Law.

[9]  A. Brazma,et al.  The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences , 2021, Nucleic Acids Res..

[10]  C. Kraft,et al.  Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context , 2021, Cell metabolism.

[11]  E. Soderblom,et al.  BioID reveals an ATG9A interaction with ATG13‐ATG101 in the degradation of p62/SQSTM1‐ubiquitin clusters , 2021, EMBO reports.

[12]  J. Paulo,et al.  Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy , 2021, bioRxiv.

[13]  C. Kraft,et al.  Small but mighty: Atg8s and Rabs in membrane dynamics during autophagy , 2021, Biochimica et biophysica acta. Molecular cell research.

[14]  S. Tooze,et al.  Membrane supply and remodeling during autophagosome biogenesis. , 2021, Current opinion in cell biology.

[15]  Sung Rae Kim,et al.  Neuronal Autophagy: Characteristic Features and Roles in Neuronal Pathophysiology , 2021, Biomolecules & therapeutics.

[16]  V. Haucke,et al.  Mechanism of synaptic protein turnover and its regulation by neuronal activity , 2021, Current Opinion in Neurobiology.

[17]  Fabian A. Mikulasch,et al.  Presynaptic activity and protein turnover are correlated at the single-synapse level. , 2021, Cell reports.

[18]  S. Hell,et al.  MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope , 2021, Nature Communications.

[19]  Daniel A. Colón-Ramos,et al.  Presynaptic autophagy is coupled to the synaptic vesicle cycle via ATG-9 , 2020, Neuron.

[20]  S. Takamori,et al.  Hidden proteome of synaptic vesicles in the mammalian brain , 2020, Proceedings of the National Academy of Sciences.

[21]  Oliver M. Crook,et al.  Spatial proteomics defines the content of trafficking vesicles captured by golgin tethers , 2020, Nature Communications.

[22]  Y. Sugita,et al.  Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion , 2020, Nature Structural & Molecular Biology.

[23]  N. Grishin,et al.  Structure, lipid scrambling activity and role in autophagosome formation of ATG9A , 2020, Nature Structural & Molecular Biology.

[24]  S. Hell,et al.  Multicolor 3D MINFLUX nanoscopy of mitochondrial MICOS proteins , 2020, Proceedings of the National Academy of Sciences.

[25]  J. Bonifacino,et al.  Structure of Human ATG9A, the Only Transmembrane Protein of the Core Autophagy Machinery. , 2020, Cell reports.

[26]  Taki Nishimura,et al.  Emerging roles of ATG proteins and membrane lipids in autophagosome formation , 2020, Cell Discovery.

[27]  Z. Mao,et al.  The emerging roles of vacuolar-type ATPase-dependent Lysosomal acidification in neurodegenerative diseases , 2020, Translational Neurodegeneration.

[28]  S. Tooze,et al.  The Golgi as an Assembly Line to the Autophagosome. , 2020, Trends in biochemical sciences.

[29]  Daniel A. Colón-Ramos,et al.  The Journey of the Synaptic Autophagosome: A Cell Biological Perspective , 2020, Neuron.

[30]  J. Ellenberg,et al.  MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells , 2020, Nature Methods.

[31]  A. Ballabio,et al.  Lysosomes as dynamic regulators of cell and organismal homeostasis , 2019, Nature Reviews Molecular Cell Biology.

[32]  H. Stenmark,et al.  The many functions of ESCRTs , 2019, Nature Reviews Molecular Cell Biology.

[33]  E. Holzbaur,et al.  Autophagy in Neurons. , 2019, Annual review of cell and developmental biology.

[34]  Hong-Gang Wang,et al.  TOM40 Targets Atg2 to Mitochondria-Associated ER Membranes for Phagophore Expansion , 2019, Cell reports.

[35]  A. Snijders,et al.  ATG9A shapes the forming autophagosome through Arfaptin 2 and phosphatidylinositol 4-kinase IIIβ , 2019, The Journal of cell biology.

[36]  T. Osawa,et al.  Atg2 mediates direct lipid transfer between membranes for autophagosome formation , 2019, Nature Structural & Molecular Biology.

[37]  A. Brunger,et al.  The pre-synaptic fusion machinery , 2019, Current opinion in structural biology.

[38]  Oliver M. Crook,et al.  Combining LOPIT with differential ultracentrifugation for high-resolution spatial proteomics , 2019, Nature Communications.

[39]  M. Vaccaro,et al.  Initial Steps in Mammalian Autophagosome Biogenesis , 2018, Front. Cell Dev. Biol..

[40]  T. L. Archuleta,et al.  AP-4 vesicles contribute to spatial control of autophagy via RUSC-dependent peripheral delivery of ATG9A , 2018, Nature Communications.

[41]  Miroslav P. Milev,et al.  TRAPPopathies: An emerging set of disorders linked to variations in the genes encoding transport protein particle (TRAPP)‐associated proteins , 2018, Traffic.

[42]  Guangyu Wu,et al.  RAB26-dependent autophagy protects adherens junctional integrity in acute lung injury , 2018, Autophagy.

[43]  E. Fornasiero,et al.  Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission , 2018, The EMBO journal.

[44]  J. Bonifacino,et al.  Altered distribution of ATG9A and accumulation of axonal aggregates in neurons from a mouse model of AP-4 deficiency syndrome , 2018, PLoS genetics.

[45]  Ira Milosevic.  Revisiting the Role of Clathrin-Mediated Endoytosis in Synaptic Vesicle Recycling , 2018, Front. Cell. Neurosci..

[46]  S. Tooze,et al.  A molecular perspective of mammalian autophagosome biogenesis , 2018, The Journal of Biological Chemistry.

[47]  J. Bonifacino,et al.  AP-4 mediates export of ATG9A from the trans-Golgi network to promote autophagosome formation , 2017, Proceedings of the National Academy of Sciences.

[48]  S. Petri,et al.  Plekhg5-regulated autophagy of synaptic vesicles reveals a pathogenic mechanism in motoneuron disease , 2017, Nature Communications.

[49]  P. Hiesinger,et al.  The where, what, and when of membrane protein degradation in neurons , 2017, Developmental neurobiology.

[50]  S. Hell,et al.  Adaptive-illumination STED nanoscopy , 2017, Proceedings of the National Academy of Sciences.

[51]  Jüergen Cox,et al.  The MaxQuant computational platform for mass spectrometry-based shotgun proteomics , 2016, Nature Protocols.

[52]  D. Klionsky,et al.  Autophagy core machinery: overcoming spatial barriers in neurons , 2016, Journal of Molecular Medicine.

[53]  L. Collinson,et al.  Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles , 2016, Nature Communications.

[54]  Daniel A. Colón-Ramos,et al.  KIF1A/UNC-104 Transports ATG-9 to Regulate Neurodevelopment and Autophagy at Synapses. , 2016, Developmental cell.

[55]  Marco Y. Hein,et al.  The Perseus computational platform for comprehensive analysis of (prote)omics data , 2016, Nature Methods.

[56]  E. Holzbaur,et al.  Compartment-Specific Regulation of Autophagy in Primary Neurons , 2016, The Journal of Neuroscience.

[57]  H. Schmitt,et al.  Coat/Tether Interactions—Exception or Rule? , 2016, Front. Cell Dev. Biol..

[58]  Zhanna Lipatova,et al.  TRAPP Complexes in Secretion and Autophagy , 2016, Front. Cell Dev. Biol..

[59]  O. Bakke,et al.  Spatiotemporal Resolution of Rab9 and CI‐MPR Dynamics in the Endocytic Pathway , 2016, Traffic.

[60]  E. Schuman,et al.  The Regulation of Synaptic Protein Turnover* , 2015, The Journal of Biological Chemistry.

[61]  R. Jahn,et al.  The GTPase Rab26 links synaptic vesicles to the autophagy pathway , 2015, eLife.

[62]  E. Holzbaur,et al.  Autophagosome biogenesis in primary neurons follows an ordered and spatially regulated pathway. , 2014, Developmental cell.

[63]  H. Urlaub,et al.  Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins , 2014, Science.

[64]  Guy M. Hagen,et al.  ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging , 2014, Bioinform..

[65]  S. Rizzoli.  Synaptic vesicle recycling: steps and principles , 2014, The EMBO journal.

[66]  J. Mills,et al.  RAB26 coordinates lysosome traffic and mitochondrial localization , 2014, Journal of Cell Science.

[67]  Johannes B. Woehrstein,et al.  Multiplexed 3D Cellular Super-Resolution Imaging with DNA-PAINT and Exchange-PAINT , 2014, Nature Methods.

[68]  Christian Rosenmund,et al.  Ultrafast endocytosis at mouse hippocampal synapses , 2013, Nature.

[69]  I. Deyev,et al.  Clathrin-Mediated Endocytosis and Adaptor Proteins , 2013, Acta naturae.

[70]  J. Solinger,et al.  Tethering complexes in the endocytic pathway: CORVET and HOPS , 2013, The FEBS journal.

[71]  H. Urlaub,et al.  Molecular Profiling of Synaptic Vesicle Docking Sites Reveals Novel Proteins but Few Differences between Glutamatergic and GABAergic Synapses , 2013, Neuron.

[72]  H. Balderhaar,et al.  CORVET and HOPS tethering complexes – coordinators of endosome and lysosome fusion , 2013, Journal of Cell Science.

[73]  R. Jahn,et al.  Molecular machines governing exocytosis of synaptic vesicles , 2012, Nature.

[74]  Pietro De Camilli,et al.  Synaptic vesicle endocytosis. , 2012, Cold Spring Harbor perspectives in biology.

[75]  N. Ziv,et al.  Formation of Golgi-Derived Active Zone Precursor Vesicles , 2012, The Journal of Neuroscience.

[76]  L. Collinson,et al.  Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy , 2012, Molecular biology of the cell.

[77]  E. Holzbaur,et al.  Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons , 2012, The Journal of cell biology.

[78]  P. Verveer,et al.  Coordinate-based colocalization analysis of single-molecule localization microscopy data , 2011, Histochemistry and Cell Biology.

[79]  Craig C. Garner,et al.  v-SNARE Composition Distinguishes Synaptic Vesicle Pools , 2011, Neuron.

[80]  M. Selbach,et al.  Global quantification of mammalian gene expression control , 2011, Nature.

[81]  C. Bartheld,et al.  Multivesicular bodies in neurons: Distribution, protein content, and trafficking functions , 2011, Progress in Neurobiology.

[82]  M. Mann,et al.  Andromeda: a peptide search engine integrated into the MaxQuant environment. , 2011, Journal of proteome research.

[83]  H. Urlaub,et al.  Quantitative Analysis of Synaptic Vesicle Rabs Uncovers Distinct Yet Overlapping Roles for Rab3a and Rab27b in Ca2+-Triggered Exocytosis , 2010, The Journal of Neuroscience.

[84]  Jemma L. Webber,et al.  New insights into the function of Atg9 , 2010, FEBS letters.

[85]  P. Pimpl,et al.  Coats of endosomal protein sorting: retromer and ESCRT. , 2009, Current opinion in plant biology.

[86]  H. Stenmark.  Rab GTPases as coordinators of vesicle traffic , 2009, Nature Reviews Molecular Cell Biology.

[87]  M. Mann,et al.  MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification , 2008, Nature Biotechnology.

[88]  Helmut Grubmüller,et al.  Molecular Anatomy of a Trafficking Organelle , 2006, Cell.

[89]  Leon Lagnado,et al.  Clathrin-Mediated Endocytosis Is the Dominant Mechanism of Vesicle Retrieval at Hippocampal Synapses , 2006, Neuron.

[90]  T. Sūdhof.  The synaptic vesicle cycle. , 2004, Annual review of neuroscience.

[91]  R. Parton,et al.  Rab23, a Negative Regulator of Hedgehog Signaling, Localizes to the Plasma Membrane and the Endocytic Pathway , 2003, Traffic.

[92]  D. Klionsky,et al.  Apg9p/Cvt7p Is an Integral Membrane Protein Required for Transport Vesicle Formation in the Cvt and Autophagy Pathways , 2000, The Journal of cell biology.

[93]  P. De Camilli,et al.  Localization of Rab5 to synaptic vesicles identifies endosomal intermediate in synaptic vesicle recycling pathway. , 1994, European journal of cell biology.

[94]  S. Pfeffer,et al.  Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network , 1994, The Journal of cell biology.

[95]  P. Greengard,et al.  Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation , 1983, The Journal of cell biology.

[96]  J. Elf,et al.  BIOPHYSICS: Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes , 2017 .

[97]  B. Gasnier,et al.  Vesicular neurotransmitter transporters: mechanistic aspects. , 2014, Current topics in membranes.

[98]  Cedric E. Ginestet.  ggplot2: Elegant Graphics for Data Analysis , 2011 .

[99]  W. Betz,et al.  Synaptic vesicle pools , 2005, Nature Reviews Neuroscience.