scholarly journals The ULK1-FBXW5-SEC23B nexus controls autophagy

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Yeon-Tae Jeong ◽  
Daniele Simoneschi ◽  
Sarah Keegan ◽  
David Melville ◽  
Natalia S Adler ◽  
...  
Keyword(s):  
Coat Protein ◽  
Protein Complex ◽  
Proteasomal Degradation ◽  
Nutrient Starvation ◽  
Nutrient Deprivation ◽  
Autophagic Flux ◽  
Complex Ii ◽  
Intermediate Compartment ◽  
F Box Protein ◽  
Efficient Execution

In response to nutrient deprivation, the cell mobilizes an extensive amount of membrane to form and grow the autophagosome, allowing the progression of autophagy. By providing membranes and stimulating LC3 lipidation, COPII (Coat Protein Complex II) promotes autophagosome biogenesis. Here, we show that the F-box protein FBXW5 targets SEC23B, a component of COPII, for proteasomal degradation and that this event limits the autophagic flux in the presence of nutrients. In response to starvation, ULK1 phosphorylates SEC23B on Serine 186, preventing the interaction of SEC23B with FBXW5 and, therefore, inhibiting SEC23B degradation. Phosphorylated and stabilized SEC23B associates with SEC24A and SEC24B, but not SEC24C and SEC24D, and they re-localize to the ER-Golgi intermediate compartment, promoting autophagic flux. We propose that, in the presence of nutrients, FBXW5 limits COPII-mediated autophagosome biogenesis. Inhibition of this event by ULK1 ensures efficient execution of the autophagic cascade in response to nutrient starvation.

scholarly journals The ULK1-FBXW5-SEC23B nexus controls autophagy

2018 ◽  
Author(s):  
Yeon-Tae Jeong ◽  
Daniele Simoneschi ◽  
Sarah Keegan ◽  
David Melville ◽  
Natalia S. Adler ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Protein Complex ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Proteasomal Degradation ◽  
Nutrient Deprivation ◽  
Autophagic Flux ◽  
Intermediate Compartment ◽  
F Box Protein

ABSTRACTIn response to nutrient deprivation, the cell needs to mobilize an extensive amount of membrane to form and grow the autophagosome, allowing the progression of autophagy. By providing membranes and a source for LC3 lipidation, COPII (Coat Protein Complex II) localizes to the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and promotes autophagosome biogenesis. However, the molecular mechanisms that, in response to starvation, divert COPII from the secretory pathway to the autophagic pathway are largely unknown. Here, we show that the F-box protein FBXW5 targets SEC23B, a component of COPII, for proteasomal degradation and that this event limits the autophagic flux in the presence of nutrients. In response to starvation, ULK1 phosphorylates SEC23B on Serine 186, preventing the interaction of SEC23B with FBXW5 and, therefore, inhibiting its degradation. Phosphorylated and stabilized SEC23B associates with SEC24A and SEC24B, but not SEC24C and SEC24D, and they re-localize to the ERGIC, promoting autophagic flux. Induction of autophagy and localization of both SEC23B and SEC24B to the ERGIC in response to nutrient deprivation are significantly reduced in SEC23B(S186A) knock-in cells. We propose that, in the presence of nutrients, FBXW5 limits COPII-mediated autophagosome biogenesis. Inhibition of this event by ULK1 ensures efficient execution of the autophagic cascade in response to nutrient starvation.

scholarly journals Expression of Sar1b Enhances Chylomicron Assembly and Key Components of the Coat Protein Complex II System Driving Vesicle Budding

2011 ◽  
Vol 31 (11) ◽  
pp. 2692-2699 ◽  
Author(s):  
Emile Levy ◽  
Elodie Harmel ◽  
Martine Laville ◽  
Rocio Sanchez ◽  
Léa Emonnot ◽  
...  
Keyword(s):  
Coat Protein ◽  
Protein Complex ◽  
Vesicle Budding ◽  
Complex Ii ◽  
Coat Protein Complex

scholarly journals Nm23H2 Facilitates Coat Protein Complex II Assembly and Endoplasmic Reticulum Export in Mammalian Cells

2005 ◽  
Vol 16 (2) ◽  
pp. 835-848 ◽  
Author(s):  
Lori Kapetanovich ◽  
Cassandra Baughman ◽  
Tina H. Lee
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Protein Complex ◽  
Mammalian Cells ◽  
Complex Ii ◽  
Permeabilized Cells ◽  
Er Export ◽  
Coat Protein Complex

The cytosolic coat protein complex II (COPII) mediates vesicle formation from the endoplasmic reticulum (ER) and is essential for ER-to-Golgi trafficking. The minimal machinery for COPII assembly is well established. However, additional factors may regulate the process in mammalian cells. Here, a morphological COPII assembly assay using purified COPII proteins and digitonin-permeabilized cells has been applied to demonstrate a role for a novel component of the COPII assembly pathway. The factor was purified and identified by mass spectrometry as Nm23H2, one of eight isoforms of nucleoside diphosphate kinase in mammalian cells. Importantly, recombinant Nm23H2, as well as a catalytically inactive version, promoted COPII assembly in vitro, suggesting a noncatalytic role for Nm23H2. Consistent with a function for Nm23H2 in ER export, Nm23H2 localized to a reticular network that also stained for the ER marker calnexin. Finally, an in vivo role for Nm23H2 in COPII assembly was confirmed by isoform-specific knockdown of Nm23H2 by using short interfering RNA. Knockdown of Nm23H2, but not its most closely related isoform Nm23H1, resulted in diminished COPII assembly at steady state and reduced kinetics of ER export. These results strongly suggest a previously unappreciated role for Nm23H2 in mammalian ER export.

scholarly journals Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

2015 ◽  
Vol 89 (15) ◽  
pp. 8026-8041 ◽  
Author(s):  
Philippe Metz ◽  
Abhilash Chiramel ◽  
Laurent Chatel-Chaix ◽  
Gualtiero Alvisi ◽  
Peter Bankhead ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dengue Virus ◽  
Viral Replication ◽  
Normal Growth ◽  
Proteasomal Degradation ◽  
Denv Infection ◽  
Replication Cycle ◽  
Nutrient Deprivation ◽  
Autophagic Flux ◽  
Initial Activation

ABSTRACTAutophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV), several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. In addition, endolysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable overexpression of p62 significantly suppressed DENV replication, suggesting a novel role for p62 as a viral restriction factor. Overall, our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an antiviral role, which is countered by DENV.IMPORTANCEAutophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we used high-content, imaging-based flow cytometry to quantify autophagic flux and endolysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endolysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a proviral to an antiviral cellular process, which is counteracted by the virus.

scholarly journals Erv26p Directs Pro-Alkaline Phosphatase into Endoplasmic Reticulum–derived Coat Protein Complex II Transport Vesicles

2006 ◽  
Vol 17 (11) ◽  
pp. 4780-4789 ◽  
Author(s):  
Catherine A. Bue ◽  
Christine M. Bentivoglio ◽  
Charles Barlowe
Keyword(s):  
Alkaline Phosphatase ◽  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Membrane Protein ◽  
Protein Complex ◽  
Integral Membrane Protein ◽  
Secretory Proteins ◽  
Complex Ii ◽  
Transport Vesicles ◽  
Copii Vesicles

Secretory proteins are exported from the endoplasmic reticulum (ER) in transport vesicles formed by the coat protein complex II (COPII). We detected Erv26p as an integral membrane protein that was efficiently packaged into COPII vesicles and cycled between the ER and Golgi compartments. The erv26Δ mutant displayed a selective secretory defect in which the pro-form of vacuolar alkaline phosphatase (pro-ALP) accumulated in the ER, whereas other secretory proteins were transported at wild-type rates. In vitro budding experiments demonstrated that Erv26p was directly required for packaging of pro-ALP into COPII vesicles. Moreover, Erv26p was detected in a specific complex with pro-ALP when immunoprecipitated from detergent-solublized ER membranes. Based on these observations, we propose that Erv26p serves as a transmembrane adaptor to link specific secretory cargo to the COPII coat. Because ALP is a type II integral membrane protein in yeast, these findings imply that an additional class of secretory cargo relies on adaptor proteins for efficient export from the ER.

scholarly journals Small sequence variations between two mammalian paralogs of the small GTPase SAR1 underlie functional differences in coat protein complex II assembly

2020 ◽  
Vol 295 (25) ◽  
pp. 8401-8412 ◽  
Author(s):  
David B. Melville ◽  
Sean Studer ◽  
Randy Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Binding Site ◽  
Protein Complex ◽  
Small Gtpase ◽  
The Other ◽  
Complex Ii ◽  
Gtp Binding ◽  
Lipoprotein Secretion ◽  
Coat Protein Complex

Vesicles that are coated by coat protein complex II (COPII) are the primary mediators of vesicular traffic from the endoplasmic reticulum to the Golgi apparatus. Secretion-associated Ras-related GTPase 1 (SAR1) is a small GTPase that is part of COPII and, upon GTP binding, recruits the other COPII proteins to the endoplasmic reticulum membrane. Mammals have two SAR1 paralogs that genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion in the case of SAR1B. Here we identified two amino acid clusters that have conserved SAR1 paralog–specific sequences. We observed that one cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site for two COPII components, SEC31 homolog A COPII coat complex component (SEC31) and SEC23. We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A. Unlike SAR1B, SAR1A was prone to oligomerize on a membrane surface. SAR1B knockdown caused loss of lipoprotein secretion, overexpression of SAR1B but not of SAR1A could restore secretion, and a divergent cluster adjacent to the SEC31/SEC23-binding site was critical for this SAR1B function. These results highlight that small primary sequence differences between the two mammalian SAR1 paralogs lead to pronounced biochemical differences that significantly affect COPII assembly and identify a specific function for SAR1B in lipoprotein secretion, providing insights into the mechanisms of large cargo secretion that may be relevant for COPII-related diseases.

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