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 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 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 FIP200 organizes the autophagy machinery at p62-ubiquitin condensates beyond activation of the ULK1 kinase

2020 ◽  
Author(s):  
Eleonora Turco ◽  
Irmgard Fischer ◽  
Sascha Martens
Keyword(s):  
Kinase Activity ◽  
De Novo ◽  
Super Resolution ◽  
Membrane Vesicles ◽  
Degradation Pathway ◽  
Autophagosome Formation ◽  
Ubiquitinated Proteins ◽  
The Many ◽  
Super Resolution Microscopy ◽  
Insight Into

AbstractMacroautophagy is a conserved degradation pathway, which mediates cellular homeostasis by the delivery of harmful substances into lysosomes. This is achieved by the sequestration of these substances referred to as cargo within double membrane vesicles, the autophagosomes, which form de novo. Among the many cargoes that are targeted by autophagy are condensates containing p62 and ubiquitinated proteins. p62 recruits the FIP200 protein to initiate autophagosome formation at the condensates. How FIP200 in turn organizes the autophagy machinery is unclear. Here we show that FIP200 is dispensable for the recruitment of the upstream autophagy machinery to the condensates, but it is necessary for phosphatidylinositol 3-phosphate formation and WIPI2 recruitment. We further find that FIP200 is required for the activation of the ULK1 kinase. Surprisingly, ULK1 kinase activity is not strictly required for autophagosome formation at p62 condensates. Super-resolution microscopy of p62 condensates revealed that FIP200 surrounds the condensates where it spatially organizes ATG13 and ATG9A for productive autophagosome formation. Our data provide a mechanistic insight into how FIP200 orchestrates autophagosome initiation at the cargo.

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.

WIPI β-propellers at the crossroads of autophagosome and lipid droplet dynamics

2014 ◽  
Vol 42 (5) ◽  
pp. 1414-1417 ◽  
Author(s):  
Simon G. Pfisterer ◽  
Daniela Bakula ◽  
Alice Cezanne ◽  
Horst Robenek ◽  
Tassula Proikas-Cezanne
Keyword(s):  
De Novo ◽  
Membrane Vesicles ◽  
Autophagosome Formation ◽  
Droplet Dynamics ◽  
Atg Proteins ◽  
Cytoplasmic Material ◽  
Storage Reservoirs ◽  
Close Proximity ◽  
Cytoplasmic Content ◽  
Catabolic Process

Macroautophagy (autophagy hereafter) is an evolutionarily highly conserved catabolic process activated by eukaryotes in order to counteract cellular starvation. Autophagy leads to bulk degradation of cytoplasmic content in the lysosomal compartment, thereby clearing the cytoplasm and generating nutrients and energy. Upon autophagy initiation, cytoplasmic material becomes sequestered in newly formed double-membrane vesicles termed ‘autophagosomes’ that subsequently acquire acidic hydrolases for content destruction. The de novo biogenesis of autophagosomes often occurs at the endoplasmic reticulum (ER) and, in many cases, in close proximity to lipid droplets (LDs), intracellular neutral lipid storage reservoirs. LDs are targets of autophagic destruction, but have recently also been shown to contribute to autophagosome formation. In fact, some autophagy-related (Atg) proteins, such as microtubule-associated protein light chain 3 (LC3), Atg2 and Atg14L, functionally contribute to both LD and autophagosome biogenesis. In the present paper, we discuss Atg proteins, including members of the human WD-repeat protein interacting with phosphoinositides (WIPI) family that co-localize prominently with LC3, Atg2 and Atg14L to conceivably integrate LD and autophagosome dynamics.

scholarly journals Remodeling of ER-exit sites initiates a membrane supply pathway for autophagosome biogenesis

2017 ◽  
Author(s):  
Liang Ge ◽  
Min Zhang ◽  
Samuel J Kenny ◽  
Dawei Liu ◽  
Miharu Maeda ◽  
...  
Keyword(s):  
Super Resolution ◽  
Membrane Vesicles ◽  
Membrane Component ◽  
Autophagosome Formation ◽  
Er Exit Sites ◽  
Intracellular Compartments ◽  
A Subunit ◽  
Intermediate Compartment ◽  
Copii Vesicles ◽  
Super Resolution Microscopy

AbstractAutophagosomes are double-membrane vesicles generated during autophagy. Biogenesis of the autophagosome requires membrane acquisition from intracellular compartments, the mechanisms of which are unclear. We previously found that a relocation of COPII machinery to the ER-Golgi intermediate compartment (ERGIC) generates ERGIC-derived COPII vesicles which serve as a membrane precursor for the lipidation of LC3, a key membrane component of the autophagosome. Here we employed super-resolution microscopy to show that starvation induces the enlargement of ER-exit sites (ERES) positive for the COPII activator, SEC12, and the remodeled ERES patches along the ERGIC. A SEC12 binding protein, CTAGE5, is required for the enlargement of ERES, SEC12 relocation to the ERGIC, and modulates autophagosome biogenesis. Moreover, FIP200, a subunit of the ULK protein kinase complex, facilitates the starvation-induced enlargement of ERES independent of the other subunits of this complex and associates via its C-terminal domain with SEC12. Our data indicate a pathway wherein FIP200 and CTAGE5 facilitate starvation-induced remodeling of the ERES, a prerequisite for the production of COPII vesicles budded from the ERGIC that contribute to autophagosome formation.

scholarly journals mBet3p is required for homotypic COPII vesicle tethering in mammalian cells

2006 ◽  
Vol 174 (3) ◽  
pp. 359-368 ◽  
Author(s):  
Sidney Yu ◽  
Ayano Satoh ◽  
Marc Pypaert ◽  
Karl Mullen ◽  
Jesse C. Hay ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vesicle Tethering ◽  
Transitional Endoplasmic Reticulum ◽  
Large Complex ◽  
Copii Vesicle ◽  
Copii Vesicles

TRAPPI is a large complex that mediates the tethering of COPII vesicles to the Golgi (heterotypic tethering) in the yeast Saccharomyces cerevisiae. In mammalian cells, COPII vesicles derived from the transitional endoplasmic reticulum (tER) do not tether directly to the Golgi, instead, they appear to tether to each other (homotypic tethering) to form vesicular tubular clusters (VTCs). We show that mammalian Bet3p (mBet3p), which is the most highly conserved TRAPP subunit, resides on the tER and adjacent VTCs. The inactivation of mBet3p results in the accumulation of cargo in membranes that colocalize with the COPII coat. Furthermore, using an assay that reconstitutes VTC biogenesis in vitro, we demonstrate that mBet3p is required for the tethering and fusion of COPII vesicles to each other. Consistent with the proposal that mBet3p is required for VTC biogenesis, we find that ERGIC-53 (VTC marker) and Golgi architecture are disrupted in siRNA-treated mBet3p-depleted cells. These findings imply that the TRAPPI complex is essential for VTC biogenesis.

Cellular factors important for the de novo formation of yeast prions

2008 ◽  
Vol 36 (5) ◽  
pp. 1083-1087 ◽  
Author(s):  
Mick Tuite ◽  
Klement Stojanovski ◽  
Frederique Ness ◽  
Gloria Merritt ◽  
Nadejda Koloteva-Levine
Keyword(s):  
De Novo ◽  
Prion Diseases ◽  
Underlying Mechanism ◽  
Structural Form ◽  
Yeast Saccharomyces Cerevisiae ◽  
Yeast Prions ◽  
Cellular Factors ◽  
Jakob Disease ◽  
Protein Rich ◽  
Cis And Trans

Prions represent an unusual structural form of a protein that is ‘infectious’. In mammals, prions are associated with fatal neurodegenerative diseases such as CJD (Creutzfeldt–Jakob disease), while in fungi they act as novel epigenetic regulators of phenotype. Even though most of the human prion diseases arise spontaneously, we still know remarkably little about how infectious prions form de novo. The [PSI+] prion of the yeast Saccharomyces cerevisiae provides a highly tractable model in which to explore the underlying mechanism of de novo prion formation, in particular identifying key cis- and trans-acting factors. Most significantly, the de novo formation of [PSI+] requires the presence of a second prion called [PIN+], which is typically the prion form of Rnq1p, a protein rich in glutamine and aspartic acid residues. The molecular mechanism by which the [PIN+] prion facilitates de novo [PSI+] formation is not fully established, but most probably involves some form of cross-seeding. A number of other cellular factors, in particular chaperones of the Hsp70 (heat-shock protein 70) family, are known to modify the frequency of de novo prion formation in yeast.

Two ubiquitin-like conjugation systems that mediate membrane formation during autophagy

2013 ◽  
Vol 55 ◽  
pp. 39-50 ◽  
Author(s):  
Hitoshi Nakatogawa
Keyword(s):  
Light Chain ◽  
Membrane Dynamics ◽  
The Other ◽  
Aminobutyric Acid ◽  
Amino Group ◽  
Autophagosome Formation ◽  
Acid Receptor ◽  
Receptor Associated Protein ◽  
Atg Proteins ◽  
C Terminus

In autophagy, the autophagosome, a transient organelle specialized for the sequestration and lysosomal or vacuolar transport of cellular constituents, is formed via unique membrane dynamics. This process requires concerted actions of a distinctive set of proteins named Atg (autophagy-related). Atg proteins include two ubiquitin-like proteins, Atg12 and Atg8 [LC3 (light-chain 3) and GABARAP (γ-aminobutyric acid receptor-associated protein) in mammals]. Sequential reactions by the E1 enzyme Atg7 and the E2 enzyme Atg10 conjugate Atg12 to the lysine residue in Atg5, and the resulting Atg12–Atg5 conjugate forms a complex with Atg16. On the other hand, Atg8 is first processed at the C-terminus by Atg4, which is related to ubiquitin-processing/deconjugating enzymes. Atg8 is then activated by Atg7 (shared with Atg12) and, via the E2 enzyme Atg3, finally conjugated to the amino group of the lipid PE (phosphatidylethanolamine). The Atg12–Atg5–Atg16 complex acts as an E3 enzyme for the conjugation reaction of Atg8; it enhances the E2 activity of Atg3 and specifies the site of Atg8–PE production to be autophagy-related membranes. Atg8–PE is suggested to be involved in autophagosome formation at multiple steps, including membrane expansion and closure. Moreover, Atg4 cleaves Atg8–PE to liberate Atg8 from membranes for reuse, and this reaction can also regulate autophagosome formation. Thus these two ubiquitin-like systems are intimately involved in driving the biogenesis of the autophagosomal membrane.

Growth-dependent regulation of system A in SV40-transformed fetal rat hepatocytes

1988 ◽  
Vol 255 (3) ◽  
pp. C261-C270 ◽  
Author(s):  
M. E. Handlogten ◽  
M. S. Kilberg
Keyword(s):  
Cell Density ◽  
De Novo ◽  
Membrane Vesicles ◽  
Rat Hepatocytes ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
System A

Fetal RLA209-15 hepatocytes, transformed with a temperature-sensitive SV40 mutant, behave like fully differentiated cells at the growth-restrictive temperature of 40 degrees C. Conversely, incubation at the growth-permissive temperature of 33 degrees C results in a transformed phenotype characterized by rapid cell division and decreased production of liver-specific proteins. The results presented here demonstrate that the cells at 33 degrees C exhibited high rates of system A transport, but transfer to 40 degrees C reduced the activity greater than 50% within 24 h. This decline in transport was independent of cell density, although the basal rate of uptake was inversely proportional to cell density in rapidly dividing cells. Transfer of cells from 40 to 33 degrees C resulted in an enhancement of system A activity that was blocked by tunicamycin. Plasma membrane vesicles from cells maintained at either 33 or 40 degrees C retained uptake rates proportional to those in the intact cells; this difference in transport activity could also be demonstrated after detergent solubilization and reconstitution. Collectively, these data indicate that de novo synthesis of the system A carrier is regulated in conjunction with temperature-dependent cell growth in RLA209-15 hepatocytes.

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