scholarly journals Golgi-resident Small GTPase Rab33B Interacts with Atg16L and Modulates Autophagosome Formation

2008 ◽  
Vol 19 (7) ◽  
pp. 2916-2925 ◽  
Author(s):  
Takashi Itoh ◽  
Naonobu Fujita ◽  
Eiko Kanno ◽  
Akitsugu Yamamoto ◽  
Tamotsu Yoshimori ◽  
...  
Keyword(s):  
De Novo ◽  
Membrane Dynamics ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Membrane Structures ◽  
Autophagosome Formation ◽  
Membrane Formation ◽  
Double Membrane ◽  
Sequestosome 1 ◽  
Mechanism Of Degradation

Macroautophagy is a mechanism of degradation of cytoplasmic components in all eukaryotic cells. In macroautophagy, cytoplasmic components are wrapped by double-membrane structures called autophagosomes, whose formation involves unique membrane dynamics, i.e., de novo formation of a double-membrane sac called the isolation membrane and its elongation. However, the precise regulatory mechanism of isolation membrane formation and elongation remains unknown. In this study, we showed that Golgi-resident small GTPase Rab33B (and Rab33A) specifically interacts with Atg16L, an essential factor in isolation membrane formation, in a guanosine triphosphate-dependent manner. Expression of a GTPase-deficient mutant Rab33B (Rab33B-Q92L) induced the lipidation of LC3, which is an essential process in autophagosome formation, even under nutrient-rich conditions, and attenuated macroautophagy, as judged by the degradation of p62/sequestosome 1. In addition, overexpression of the Rab33B binding domain of Atg16L suppressed autophagosome formation. Our findings suggest that Rab33 modulates autophagosome formation through interaction with Atg16L.

scholarly journals Curvature-sensitive trans-assembly of human Atg8-family proteins in autophagy-related membrane tethering

2019 ◽  
Author(s):  
Saki Taniguchi ◽  
Masayuki Toyoshima ◽  
Tomoyo Takamatsu ◽  
Joji Mima
Keyword(s):  
Lipid Membranes ◽  
De Novo ◽  
Membrane Curvature ◽  
Membrane Dynamics ◽  
Membrane Structures ◽  
Autophagosome Formation ◽  
Molar Ratios ◽  
Membrane Tethering ◽  
Membrane Attachment

In macroautophagy, de novo formation of the double membrane-bound organelles, termed autophagosomes, is essential for engulfing and sequestering the cytoplasmic contents to be degraded in the lytic compartments such as vacuoles and lysosomes. Atg8-family proteins have been known to be responsible for autophagosome formation via membrane tethering and fusion events of precursor membrane structures. Nevertheless, how Atg8 proteins act directly upon autophagosome formation still remains enigmatic. Here, to further gain molecular insights into Atg8-mediated autophagic membrane dynamics, we study the two representative human Atg8 orthologs, LC3B and GATE-16, by quantitatively evaluating their intrinsic potency to physically tether lipid membranes in a chemically defined reconstitution system using purified Atg8 proteins and synthetic liposomes. Both LC3B and GATE-16 retained the capacities to trigger efficient membrane tethering at the protein-to-lipid molar ratios ranging from 1:100 to 1:5,000. These human Atg8-mediated membrane tethering reactions require trans-assembly between the membrane-anchored forms of LC3B and GATE-16 and can be reversibly and strictly controlled by the membrane attachment and detachment cycles. Strikingly, we further uncovered distinct membrane curvature dependences of LC3B- and GATE-16-mediated membrane tethering reactions: LC3B can drive tethering more efficiently than GATE-16 for highly-curved small vesicles (e.g. 50 nm in diameter), although GATE-16 turns out to be a more potent tether than LC3B for flatter large vesicles (e.g. 200 and 400 nm in diameter). Our findings establish curvature-sensitive trans-assembly of human Atg8-family proteins in reconstituted membrane tethering, which recapitulates an essential subreaction of the biogenesis of autophagosomes in vivo.

scholarly journals Structural catalog of core Atg proteins opens new era of autophagy research

2021 ◽  
Author(s):  
Kazuaki Matoba ◽  
Nobuo N Noda
Keyword(s):  
De Novo ◽  
Structural Information ◽  
Membrane Dynamics ◽  
Autophagosome Formation ◽  
New Era ◽  
Intracellular Degradation ◽  
Atg Proteins ◽  
Biological Studies ◽  
Evolutionarily Conserved ◽  
Biological Methods

Summary Autophagy, which is an evolutionarily conserved intracellular degradation system, involves de novo generation of autophagosomes that sequester and deliver diverse cytoplasmic materials to the lysosome for degradation. Autophagosome formation is mediated by approximately 20 core autophagy-related (Atg) proteins, which collaborate to mediate complicated membrane dynamics during autophagy. To elucidate the molecular functions of these Atg proteins in autophagosome formation, many researchers have tried to determine the structures of Atg proteins by using various structural biological methods. Although not sufficient, the basic structural catalog of all core Atg proteins was established. In this review article, we summarize structural biological studies of core Atg proteins, with an emphasis on recently unveiled structures, and describe the mechanistic breakthroughs in autophagy research that have derived from new structural information.

scholarly journals Autophagosome formation in relation to the endoplasmic reticulum

2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Yo-hei Yamamoto ◽  
Takeshi Noda
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
De Novo ◽  
Transmembrane Protein ◽  
Lipid Transfer ◽  
Membrane Structures ◽  
Autophagosome Formation ◽  
Selective Autophagy ◽  
The Relationship ◽  
Er Membrane

Abstract Autophagy is a process in which a myriad membrane structures called autophagosomes are formed de novo in a single cell, which deliver the engulfed substrates into lysosomes for degradation. The size of the autophagosomes is relatively uniform in non-selective autophagy and variable in selective autophagy. It has been recently established that autophagosome formation occurs near the endoplasmic reticulum (ER). In this review, we have discussed recent advances in the relationship between autophagosome formation and endoplasmic reticulum. Autophagosome formation occurs near the ER subdomain enriched with phospholipid synthesizing enzymes like phosphatidylinositol synthase (PIS)/CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and choline/ethanolamine phosphotransferase 1 (CEPT1). Autophagy-related protein 2 (Atg2), which is involved in autophagosome formation has a lipid transfer capacity and is proposed to directly transfer the lipid molecules from the ER to form autophagosomes. Vacuole membrane protein 1 (VMP1) and transmembrane protein 41b (TMEM41b) are ER membrane proteins that are associated with the formation of the subdomain. Recently, we have reported that an uncharacterized ER membrane protein possessing the DNAJ domain, called ERdj8/DNAJC16, is associated with the regulation of the size of autophagosomes. The localization of ERdj8/DNAJC16 partially overlaps with the PIS-enriched ER subdomain, thereby implying its association with autophagosome size determination.

scholarly journals Autophagy in the context of the cellular membrane-trafficking system: the enigma of Atg9 vesicles

2017 ◽  
Vol 45 (6) ◽  
pp. 1323-1331 ◽  
Author(s):  
Takeshi Noda
Keyword(s):  
Membrane Protein ◽  
Membrane Trafficking ◽  
De Novo ◽  
Membrane Lipids ◽  
Cellular Membrane ◽  
Current Understanding ◽  
Membrane Structures ◽  
Autophagosome Formation ◽  
Intracellular Degradation

Macroautophagy is an intracellular degradation system that involves the de novo formation of membrane structures called autophagosomes, although the detailed process by which membrane lipids are supplied during autophagosome formation is yet to be elucidated. Macroautophagy is thought to be associated with canonical membrane trafficking, but several mechanistic details are still missing. In this review, the current understanding and potential mechanisms by which membrane trafficking participates in macroautophagy are described, with a focus on the enigma of the membrane protein Atg9, for which the proximal mechanisms determining its movement are disputable, despite its key role in autophagosome formation.

In vitro reconstitution of autophagic processes

2020 ◽  
Vol 48 (5) ◽  
pp. 2003-2014
Author(s):  
Jahangir Md. Alam ◽  
Nobuo N. Noda
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Membrane Structures ◽  
Autophagosome Formation ◽  
The Past ◽  
Atg Proteins ◽  
In Vitro Reconstitution ◽  
Complicated Process

Autophagy is a lysosomal degradation system that involves de novo autophagosome formation. A lot of factors are involved in autophagosome formation, including dozens of Atg proteins that form supramolecular complexes, membrane structures including vesicles and organelles, and even membraneless organelles. Because these diverse higher-order structural components cooperate to mediate de novo formation of autophagosomes, it is too complicated to be elaborated only by cell biological approaches. Recent trials to regenerate each step of this phenomenon in vitro have started to elaborate on the molecular mechanisms of such a complicated process by simplification. In this review article, we outline the in vitro reconstitution trials in autophagosome formation, mainly focusing on the reports in the past few years and discussing the molecular mechanisms of autophagosome formation by comparing in vitro and in vivo observations.

scholarly journals Tuba activates Cdc42 during neuronal polarization downstream of the small GTPase Rab8a

2019 ◽  
Author(s):  
Pamela J. Urrutia ◽  
Felipe Bodaleo ◽  
Daniel A. Bórquez ◽  
Victoria Rozes-Salvador ◽  
Cristopher Villablanca ◽  
...  
Keyword(s):  
Small Gtpases ◽  
Membrane Dynamics ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Neuronal Polarity ◽  
Dependent Manner ◽  
Gain Of Function ◽  
Molecular Determinants ◽  
Axon Specification ◽  
Neuronal Polarization

ABSTRACTThe acquisition of neuronal polarity is a complex molecular process that involves several different cellular mechanisms that need to be finely coordinated to define the somatodendritic and axonal compartments. Amongst such mechanisms, cytoskeleton and membrane dynamics control both the morphological transitions that define neuronal polarity acquisition as well as provide molecular determinants to specific sites in neurons at a defined time point. Small GTPases from the Rab and Rho families are well known molecular determinants of neuronal differentiation. However, during axon specification, a molecular link that couples proteins from these two families has yet to be identified. In this paper, we describe the role of Tuba, a Cdc42-specific guanine nucleotide-exchange factor (GEF), in neuronal polarity through a Rab8a-dependent mechanism. Rab8a or Tuba gain-of-function generates neurons with supernumerary axons whereas Rab8a or Tuba loss-of-function abrogated axon specification, phenocopying the well-established effect of Cdc42 on neuronal polarity. Neuronal polarization associated to Rab8a is also evidenced in vivo, since a dominant negative version of Rab8a severely impaired neuronal migration.Remarkably, Rab8a activates Cdc42 in a Tuba-dependent manner, and dominant negative mutants of both GTPases reciprocally prevent the effect over polarity acquisition in the gain-of-function scenarios. Our results strongly suggest that a positive feedback loop linking Rab8a and Cdc42 activities via Tuba, is a primary event in neuronal polarization. In addition, we identified the GEF responsible for Cdc42 activation that is essential to specify axons in cultured neurons.

scholarly journals COPII vesicles contribute to autophagosomal membranes

2019 ◽  
Vol 218 (5) ◽  
pp. 1503-1510 ◽  
Author(s):  
Takayuki Shima ◽  
Hiromi Kirisako ◽  
Hitoshi Nakatogawa
Keyword(s):  
De Novo ◽  
Membrane Vesicles ◽  
Membrane Dynamics ◽  
Fundamental Question ◽  
Autophagosome Formation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Definitive Answer ◽  
Copii Vesicle ◽  
Copii Vesicles ◽  
Labeling System

A hallmark of autophagy is the de novo formation of double-membrane vesicles called autophagosomes, which sequester various cellular constituents for degradation in lysosomes or vacuoles. The membrane dynamics underlying the biogenesis of autophagosomes, including the origin of the autophagosomal membrane, are still elusive. Although previous studies suggested that COPII vesicles are closely associated with autophagosome biogenesis, it remains unclear whether these vesicles serve as a source of the autophagosomal membrane. Using a recently developed COPII vesicle–labeling system in fluorescence and immunoelectron microscopy in the budding yeast Saccharomyces cerevisiae, we show that the transmembrane cargo Axl2 is loaded into COPII vesicles in the ER. Axl2 is then transferred to autophagosome intermediates, ultimately becoming part of autophagosomal membranes. This study provides a definitive answer to a long-standing, fundamental question regarding the mechanisms of autophagosome formation by implicating COPII vesicles as a membrane source for autophagosomes.

scholarly journals Atg9 vesicles are an important membrane source during early steps of autophagosome formation

2012 ◽  
Vol 198 (2) ◽  
pp. 219-233 ◽  
Author(s):  
Hayashi Yamamoto ◽  
Soichiro Kakuta ◽  
Tomonobu M. Watanabe ◽  
Akira Kitamura ◽  
Takayuki Sekito ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Membrane Structures ◽  
Autophagosome Formation ◽  
Early Step ◽  
Double Membrane ◽  
Lytic Compartment ◽  
Insight Into

During the process of autophagy, cytoplasmic materials are sequestered by double-membrane structures, the autophagosomes, and then transported to a lytic compartment to be degraded. One of the most fundamental questions about autophagy involves the origin of the autophagosomal membranes. In this study, we focus on the intracellular dynamics of Atg9, a multispanning membrane protein essential for autophagosome formation in yeast. We found that the vast majority of Atg9 existed on cytoplasmic mobile vesicles (designated Atg9 vesicles) that were derived from the Golgi apparatus in a process involving Atg23 and Atg27. We also found that only a few Atg9 vesicles were required for a single round of autophagosome formation. During starvation, several Atg9 vesicles assembled individually into the preautophagosomal structure, and eventually, they are incorporated into the autophagosomal outer membrane. Our findings provide conclusive linkage between the cytoplasmic Atg9 vesicles and autophagosomal membranes and offer new insight into the requirement for Atg9 vesicles at the early step of autophagosome formation.

scholarly journals Rab32 uses its effector reticulon 3L to trigger autophagic degradation of mitochondria-associated membrane (MAM) proteins

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Maria Sol Herrera-Cruz ◽  
Megan C. Yap ◽  
Nasser Tahbaz ◽  
Keelie Phillips ◽  
Laurel Thomas ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Transmembrane Protein ◽  
Membrane Dynamics ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Related Protein ◽  
Intracellular Membrane ◽  
Selective Autophagy ◽  
Large Panel ◽  
Autophagic Degradation

Abstract Background Rab32 is a small GTPase associated with multiple organelles but is particularly enriched at the endoplasmic reticulum (ER). Here, it controls targeting to mitochondria-ER contacts (MERCs), thus influencing composition of the mitochondria-associated membrane (MAM). Moreover, Rab32 regulates mitochondrial membrane dynamics via its effector dynamin-related protein 1 (Drp1). Rab32 has also been reported to induce autophagy, an essential pathway targeting intracellular components for their degradation. However, no autophagy-specific effectors have been identified for Rab32. Similarly, the identity of the intracellular membrane targeted by this small GTPase and the type of autophagy it induces are not known yet. Results To investigate the target of autophagic degradation mediated by Rab32, we tested a large panel of organellar proteins. We found that a subset of MERC proteins, including the thioredoxin-related transmembrane protein TMX1, are specifically targeted for degradation in a Rab32-dependent manner. We also identified the long isoform of reticulon-3 (RTN3L), a known ER-phagy receptor, as a Rab32 effector. Conclusions Rab32 promotes degradation of mitochondrial-proximal ER membranes through autophagy with the help of RTN3L. We propose to call this type of selective autophagy “MAM-phagy”.

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