TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes

Autophagy is a bulk degradation process characterized by the formation of double membrane vesicles called autophagosomes. The exact molecular mechanism of autophagosome formation and the origin of the autophagosomal membrane remain unclear. We screened 38 human Tre-2/Bub2/Cdc16 domain–containing Rab guanosine triphosphatase–activating proteins (GAPs) and identified 11 negative regulators of starvation-induced autophagy. One of these putative RabGAPs, TBC1D14, colocalizes and interacts with the autophagy kinase ULK1. Overexpressed TBC1D14 tubulates ULK1-positive recycling endosomes (REs), impairing their function and inhibiting autophagosome formation. TBC1D14 binds activated Rab11 but is not a GAP for Rab11, and loss of Rab11 prevents TBC1D14-induced tubulation of REs. Furthermore, Rab11 is required for autophagosome formation. ULK1 and Atg9 are found on Rab11- and transferrin (Tfn) receptor (TfnR)–positive recycling endosomes. Amino acid starvation causes TBC1D14 to relocalize from REs to the Golgi complex, whereas TfnR and Tfn localize to forming autophagosomes, which are ULK1 and LC3 positive. Thus, TBC1D14- and Rab11-dependent vesicular transport from REs contributes to and regulates starvation-induced autophagy.

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Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum

The Journal of Cell Biology ◽

10.1083/jcb.200803137 ◽

2008 ◽

Vol 182 (4) ◽

pp. 685-701 ◽

Cited By ~ 1081

Author(s):

Elizabeth L. Axe ◽

Simon A. Walker ◽

Maria Manifava ◽

Priya Chandra ◽

H. Llewelyn Roderick ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Dynamic Equilibrium ◽

Membrane Vesicles ◽

Amino Acid Starvation ◽

Autophagosome Formation ◽

Double Membrane ◽

Dynamic Relationship ◽

Golgi Membranes ◽

Membrane Compartments ◽

Phosphatidylinositol 3

Autophagy is the engulfment of cytosol and organelles by double-membrane vesicles termed autophagosomes. Autophagosome formation is known to require phosphatidylinositol 3-phosphate (PI(3)P) and occurs near the endoplasmic reticulum (ER), but the exact mechanisms are unknown. We show that double FYVE domain–containing protein 1, a PI(3)P-binding protein with unusual localization on ER and Golgi membranes, translocates in response to amino acid starvation to a punctate compartment partially colocalized with autophagosomal proteins. Translocation is dependent on Vps34 and beclin function. Other PI(3)P-binding probes targeted to the ER show the same starvation-induced translocation that is dependent on PI(3)P formation and recognition. Live imaging experiments show that this punctate compartment forms near Vps34-containing vesicles, is in dynamic equilibrium with the ER, and provides a membrane platform for accumulation of autophagosomal proteins, expansion of autophagosomal membranes, and emergence of fully formed autophagosomes. This PI(3)P-enriched compartment may be involved in autophagosome biogenesis. Its dynamic relationship with the ER is consistent with the idea that the ER may provide important components for autophagosome formation.

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ATG9A shapes the forming autophagosome through Arfaptin 2 and phosphatidylinositol 4-kinase IIIβ

The Journal of Cell Biology ◽

10.1083/jcb.201901115 ◽

2019 ◽

Vol 218 (5) ◽

pp. 1634-1652 ◽

Cited By ~ 48

Author(s):

Delphine Judith ◽

Harold B.J. Jefferies ◽

Stefan Boeing ◽

David Frith ◽

Ambrosius P. Snijders ◽

...

Keyword(s):

Quantitative Analysis ◽

Amino Acid ◽

Membrane Protein ◽

Initiation Site ◽

Amino Acid Starvation ◽

Autophagosome Formation ◽

Metabolizing Enzymes ◽

Bar Domain ◽

Golgi Membranes ◽

Phosphatidylinositol 4

ATG9A is a multispanning membrane protein essential for autophagy. Normally resident in Golgi membranes and endosomes, during amino acid starvation, ATG9A traffics to sites of autophagosome formation. ATG9A is not incorporated into autophagosomes but is proposed to supply so-far-unidentified proteins and lipids to the autophagosome. To address this function of ATG9A, a quantitative analysis of ATG9A-positive compartments immunoisolated from amino acid–starved cells was performed. These ATG9A vesicles are depleted of Golgi proteins and enriched in BAR-domain containing proteins, Arfaptins, and phosphoinositide-metabolizing enzymes. Arfaptin2 regulates the starvation-dependent distribution of ATG9A vesicles, and these ATG9A vesicles deliver the PI4-kinase, PI4KIIIβ, to the autophagosome initiation site. PI4KIIIβ interacts with ATG9A and ATG13 to control PI4P production at the initiation membrane site and the autophagic response. PI4KIIIβ and PI4P likely function by recruiting the ULK1/2 initiation kinase complex subunit ATG13 to nascent autophagosomes.

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Atg27 Is Required for Autophagy-dependent Cycling of Atg9

Molecular Biology of the Cell ◽

10.1091/mbc.e06-07-0612 ◽

2007 ◽

Vol 18 (2) ◽

pp. 581-593 ◽

Cited By ~ 112

Author(s):

Wei-Lien Yen ◽

Julie E. Legakis ◽

Usha Nair ◽

Daniel J. Klionsky

Keyword(s):

Saccharomyces Cerevisiae ◽

Golgi Complex ◽

Transmembrane Protein ◽

Eukaryotic Cells ◽

Vesicle Formation ◽

Catabolic Pathway ◽

Autophagosome Formation ◽

Yeast Saccharomyces Cerevisiae ◽

Double Membrane ◽

Selective Autophagy

Autophagy is a catabolic pathway for the degradation of cytosolic proteins or organelles and is conserved among all eukaryotic cells. The hallmark of autophagy is the formation of double-membrane cytosolic vesicles, termed autophagosomes, which sequester cytoplasm; however, the mechanism of vesicle formation and the membrane source remain unclear. In the yeast Saccharomyces cerevisiae, selective autophagy mediates the delivery of specific cargos to the vacuole, the analog of the mammalian lysosome. The transmembrane protein Atg9 cycles between the mitochondria and the pre-autophagosomal structure, which is the site of autophagosome biogenesis. Atg9 is thought to mediate the delivery of membrane to the forming autophagosome. Here, we characterize a second transmembrane protein Atg27 that is required for specific autophagy in yeast. Atg27 is required for Atg9 cycling and shuttles between the pre-autophagosomal structure, mitochondria, and the Golgi complex. These data support a hypothesis that multiple membrane sources supply the lipids needed for autophagosome formation.

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Pex3 confines pexophagy receptor activity of Atg36 to peroxisomes by regulating Hrr25-mediated phosphorylation and proteasomal degradation

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013565 ◽

2020 ◽

Vol 295 (48) ◽

pp. 16292-16298

Author(s):

Sota Meguro ◽

Xizhen Zhuang ◽

Hiromi Kirisako ◽

Hitoshi Nakatogawa

Keyword(s):

Saccharomyces Cerevisiae ◽

Recombinant Proteins ◽

Posttranslational Modifications ◽

Membrane Vesicles ◽

Proteasomal Degradation ◽

Regulatory Mechanisms ◽

Autophagosome Formation ◽

Peroxisomal Membrane ◽

Double Membrane ◽

Selective Autophagy

In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast Saccharomyces cerevisiae, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by posttranslational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36–Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36–Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.

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Rubicon in Metabolic Diseases and Ageing

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.816829 ◽

2022 ◽

Vol 9 ◽

Author(s):

Satoshi Minami ◽

Shuhei Nakamura ◽

Tamotsu Yoshimori

Keyword(s):

Potential Role ◽

Metabolic Diseases ◽

Close Association ◽

Membrane Vesicles ◽

Beclin 1 ◽

Negative Regulators ◽

Double Membrane ◽

Promising Therapeutic Target ◽

Independent Functions ◽

Positive Regulators

Autophagy is a conserved cellular degradation system that maintains intracellular homeostasis. Cytoplasmic components are engulfed into double-membrane vesicles called autophagosomes, which fuse with lysosomes, and resulting in the degradation of sequestered materials. Recently, a close association between autophagy and the pathogenesis of metabolic diseases and ageing has become apparent: autophagy is dysregulated during metabolic diseases and ageing; dysregulation of autophagy is intimately associated with the pathophysiology. Rubicon (Run domain Beclin-1 interacting and cysteine-rich containing protein) has been identified as a Beclin-1 associated protein. Notably, Rubicon is one of few negative regulators of autophagy whereas many autophagy-related genes are positive regulators of autophagy. Rubicon also has autophagy-independent functions including phagocytosis and endocytosis. In this mini-review, we focus on the various roles of Rubicon in different organs in the settings of metabolic diseases and ageing, and discuss its potential role as a promising therapeutic target.

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AMPK Inhibits ULK1-Dependent Autophagosome Formation and Lysosomal Acidification via Distinct Mechanisms

Molecular and Cellular Biology ◽

10.1128/mcb.00023-18 ◽

2018 ◽

Vol 38 (10) ◽

Cited By ~ 26

Author(s):

Chinwendu Nwadike ◽

Leon E. Williamson ◽

Laura E. Gallagher ◽

Jun-Lin Guan ◽

Edmond Y. W. Chan

Keyword(s):

Amino Acid ◽

Amino Acid Starvation ◽

Glucose Starvation ◽

Autophagosome Formation ◽

Ampk Signaling ◽

Sequestosome 1 ◽

Acid Deficiency ◽

Ampk Activity ◽

Amp Activated Protein Kinase ◽

Lysosomal Acidification

ABSTRACT Autophagy maintains metabolism in response to starvation, but each nutrient is sensed distinctly. Amino acid deficiency suppresses mechanistic target of rapamycin complex 1 (MTORC1), while glucose deficiency promotes AMP-activated protein kinase (AMPK). The MTORC1 and AMPK signaling pathways converge onto the ULK1/2 autophagy initiation complex. Here, we show that amino acid starvation promoted formation of ULK1- and sequestosome 1/p62-positive early autophagosomes. Autophagosome initiation was controlled by MTORC1 sensing glutamine, leucine, and arginine levels together. In contrast, glucose starvation promoted AMPK activity, phosphorylation of ULK1 Ser555, and LC3-II accumulation, but with dynamics consistent with a block in autophagy flux. We studied the flux pathway and found that starvation of amino acid but not of glucose activated lysosomal acidification, which occurred independently of autophagy and ULK1. In addition to lack of activation, glucose starvation inhibited the ability of amino acid starvation to activate both autophagosome formation and the lysosome. Activation of AMPK and phosphorylation of ULK1 were determined to specifically inhibit autophagosome formation. AMPK activation also was sufficient to prevent lysosome acidification. These results indicate concerted but distinct AMPK-dependent mechanisms to suppress early and late phases of autophagy.

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Atg8 Controls Phagophore Expansion during Autophagosome Formation

Molecular Biology of the Cell ◽

10.1091/mbc.e07-12-1292 ◽

2008 ◽

Vol 19 (8) ◽

pp. 3290-3298 ◽

Cited By ~ 444

Author(s):

Zhiping Xie ◽

Usha Nair ◽

Daniel J. Klionsky

Keyword(s):

Membrane Vesicles ◽

Degradation Process ◽

Autophagosome Formation ◽

Multistage Model ◽

Intracellular Degradation ◽

Cytoplasmic Material ◽

New Genes ◽

Health And Disease ◽

Time Observation ◽

Related Proteins

Autophagy is a potent intracellular degradation process with pivotal roles in health and disease. Atg8, a lipid-conjugated ubiquitin-like protein, is required for the formation of autophagosomes, double-membrane vesicles responsible for the delivery of cytoplasmic material to lysosomes. How and when Atg8 functions in this process, however, is not clear. Here we show that Atg8 controls the expansion of the autophagosome precursor, the phagophore, and give the first real-time, observation-based temporal dissection of the autophagosome formation process. We demonstrate that the amount of Atg8 determines the size of autophagosomes. During autophagosome biogenesis, Atg8 forms an expanding structure and later dissociates from the site of vesicle formation. On the basis of the dynamics of Atg8, we present a multistage model of autophagosome formation. This model provides a foundation for future analyses of the functions and dynamics of known autophagy-related proteins and for screening new genes.

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A CRISPR screening approach for identifying novel autophagy-related factors and cytoplasm-to-lysosome trafficking routes

10.1101/229732 ◽

2017 ◽

Cited By ~ 2

Author(s):

Christopher J Shoemaker ◽

Tina Q Huang ◽

Nicholas R Weir ◽

Nicole Polyakov ◽

Vladimir Denic

Keyword(s):

Membrane Vesicles ◽

Genetic Interactions ◽

Endoplasmic Reticulum Membrane ◽

Autophagosome Formation ◽

Related Factors ◽

Double Membrane ◽

Receptor Proteins ◽

Selective Autophagy ◽

Genetic Fingerprint ◽

Genome Wide

SummarySelective autophagy comprises cytoplasm-to-lysosome trafficking routes that transport cargos using double-membrane vesicles (autophagosomes). Cargos are detected by receptor proteins, which typically also bind to lipid-conjugated LC3 proteins on autophagosome membranes. We dissected lysosomal delivery of four SQSTM1-like receptors by genome-wide CRISPR screening looking for novel autophagy-related (ATG) factors and trafficking routes. We uncovered new mammalian ATG factors including TMEM41B, an endoplasmic reticulum membrane protein required for autophagosome membrane expansion and/or closure. Furthermore, we found that certain receptors remain robustly targeted to the lysosome even in the absence of ATG7 or other LC3 conjugation factors. Lastly, we identified a unique genetic fingerprint behind receptor flux in ATG7KO cells, which includes factors implicated in nucleating autophagosome formation and vesicle trafficking factors. Our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms.

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