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.

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 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 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.

Membrane re-modelling by BAR domain superfamily proteins via molecular and non-molecular factors

2018 ◽  
Vol 46 (2) ◽  
pp. 379-389 ◽  
Author(s):  
Tamako Nishimura ◽  
Nobuhiro Morone ◽  
Shiro Suetsugu
Keyword(s):  
Lipid Membranes ◽  
Intracellular Transport ◽  
Lipid Membrane ◽  
Membrane Curvature ◽  
Membrane Dynamics ◽  
Cytoskeletal Proteins ◽  
Cellular Functions ◽  
High Concentration ◽  
Bar Domain ◽  
Intracellular Organelles

Lipid membranes are structural components of cell surfaces and intracellular organelles. Alterations in lipid membrane shape are accompanied by numerous cellular functions, including endocytosis, intracellular transport, and cell migration. Proteins containing Bin–Amphiphysin–Rvs (BAR) domains (BAR proteins) are unique, because their structures correspond to the membrane curvature, that is, the shape of the lipid membrane. BAR proteins present at high concentration determine the shape of the membrane, because BAR domain oligomers function as scaffolds that mould the membrane. BAR proteins co-operate with various molecular and non-molecular factors. The molecular factors include cytoskeletal proteins such as the regulators of actin filaments and the membrane scission protein dynamin. Lipid composition, including saturated or unsaturated fatty acid tails of phospholipids, also affects the ability of BAR proteins to mould the membrane. Non-molecular factors include the external physical forces applied to the membrane, such as tension and friction. In this mini-review, we will discuss how the BAR proteins orchestrate membrane dynamics together with various molecular and non-molecular factors.

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.

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 Recent advances in the understanding of autophagosome biogenesis

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 212 ◽  
Author(s):  
Martin Graef
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Phospholipid Biosynthesis ◽  
Key Factors ◽  
Lysosomal Degradation ◽  
Autophagosome Formation ◽  
Recent Advances ◽  
Membrane Tethering ◽  
Catabolic Process

Autophagy is a conserved catabolic process critical for cell homeostasis with broad implications for aging and age-associated diseases. A defining feature of autophagy is the de novo formation of a specialized transient organelle, the double-membrane autophagosome. Autophagosomes originate from small vesicular precursors after rapid membrane expansion resulting in the engulfment of a broad spectrum of cytoplasmic cargoes within a few minutes for vacuolar or lysosomal degradation. Recent advances have provided exciting new insights into the molecular mechanisms underlying the assembly of autophagic membranes during autophagosome biogenesis. Specifically, the phospholipid biosynthesis activity of the endoplasmic reticulum and a dedicated membrane-tethering complex between nascent autophagosomes and the endoplasmic reticulum have emerged as key factors in autophagosome formation.

scholarly journals Mammalian phospholipid homeostasis: evidence that membrane curvature elastic stress drives homeoviscous adaptation in vivo

2016 ◽  
Vol 13 (121) ◽  
pp. 20160228 ◽  
Author(s):  
Marcus K. Dymond
Keyword(s):  
Mammalian Cells ◽  
Elastic Stress ◽  
De Novo ◽  
Acyl Chain ◽  
Membrane Curvature ◽  
Homeoviscous Adaptation ◽  
Membrane Order ◽  
A Value ◽  
Modelling Studies

Several theories of phospholipid homeostasis have postulated that cells regulate the molecular composition of their bilayer membranes, such that a common biophysical membrane parameter is under homeostatic control. Two commonly cited theories are the intrinsic curvature hypothesis, which states that cells control membrane curvature elastic stress, and the theory of homeoviscous adaptation, which postulates cells control acyl chain packing order (membrane order). In this paper, we present evidence from data-driven modelling studies that these two theories correlate in vivo. We estimate the curvature elastic stress of mammalian cells to be 4–7 × 10 −12 N, a value high enough to suggest that in mammalian cells the preservation of membrane order arises through a mechanism where membrane curvature elastic stress is controlled. These results emerge from analysing the molecular contribution of individual phospholipids to both membrane order and curvature elastic stress in nearly 500 cellular compositionally diverse lipidomes. Our model suggests that the de novo synthesis of lipids is the dominant mechanism by which cells control curvature elastic stress and hence membrane order in vivo . These results also suggest that cells can increase membrane curvature elastic stress disproportionately to membrane order by incorporating polyunsaturated fatty acids into lipids.

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