Poliovirus Infection Transiently Increases COPII Vesicle Budding

Poliovirus (PV) requires membranes of the host cell's secretory pathway to generate replication complexes (RCs) for viral RNA synthesis. Recent work identified the intermediate compartment and the Golgi apparatus as the precursors of the replication “organelles” of PV (N. Y. Hsu et al., Cell 141:799–811, 2010). In this study, we examined the effect of PV on COPII vesicles, the secretory cargo carriers that bud from the endoplasmic reticulum and homotypically fuse to form the intermediate compartment that matures into the Golgi apparatus. We found that infection by PV results in a biphasic change in functional COPII vesicle biogenesis in cells, with an early enhancement and subsequent inhibition. Concomitant with the early increase in COPII vesicle formation, we found an increase in the membrane fraction of Sec16A, a key regulator of COPII vesicle formation. We suggest that the early burst in COPII vesicle formation detected benefits PV by increasing the precursor pool required for the formation of its RCs.

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MIA3/TANGO1 enables efficient secretion by constraining COPII vesicle budding

10.1101/2021.02.24.432632 ◽

2021 ◽

Author(s):

Janine McCaughey ◽

Judith M. Mantell ◽

Chris R. Neal ◽

Kate Heesom ◽

David J. Stephens

Keyword(s):

Endoplasmic Reticulum ◽

Light Microscopy ◽

Secretory Pathway ◽

Genome Engineering ◽

High Volume ◽

Vesicle Budding ◽

Early Secretory Pathway ◽

Copii Vesicle ◽

Intermediate Compartment ◽

Golgi Organization

AbstractComplex machinery is required to drive secretory cargo export from the endoplasmic reticulum. In vertebrates, this includes transport and Golgi organization protein 1 (TANGO1), encoded by the Mia3 gene. Here, using genome engineering of human cells light microscopy, secretion assays, and proteomics, we show loss of Mia3/TANGO1 results in formation of numerous vesicles and a loss of early secretory pathway integrity. This restricts secretion not only of large proteins like procollagens but of all types of secretory cargo. Our data shows that Mia3/TANGO1 constrains the propensity of COPII to form vesicles promoting instead the formation of the ER-Golgi intermediate compartment. Thus, Mia3/TANGO1 facilities the secretion of complex and high volume cargoes from vertebrate cells.

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Structural basis for the initiation of COPII vesicle biogenesis

10.1101/2020.10.08.331793 ◽

2020 ◽

Author(s):

Aaron M.N. Joiner ◽

J. Christopher Fromme

Keyword(s):

Secretory Pathway ◽

Membrane Surface ◽

Structural Basis ◽

Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Copii Vesicle ◽

Cargo Proteins ◽

Vesicle Biogenesis ◽

Copii Vesicles ◽

Gtpase Activation

AbstractThe first stage of the eukaryotic secretory pathway is the packaging of cargo proteins into COPII vesicles exiting the endoplasmic reticulum (ER). The cytoplasmic COPII vesicle coat machinery is recruited to the ER membrane by the activated, GTP-bound, form of the conserved Sar1 GTPase. Activation of Sar1 on the surface of the ER by Sec12, a membrane-anchored GEF (guanine nucleotide exchange factor), is therefore the initiating step of the secretory pathway. Here we report the structure of the complex between Sar1 and the cytoplasmic GEF domain of Sec12, both from Saccharomyces cerevisiae. This structure, representing the key nucleotide-free activation intermediate, reveals how the potassium ion-binding K-loop disrupts the nucleotide binding site of Sar1. We describe an unexpected orientation of the GEF domain relative to the membrane surface and propose a mechanism for how Sec12 facilitates membrane insertion of the amphipathic helix exposed by Sar1 upon GTP-binding.

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Reconstitution of COPII vesicle fusion to generate a pre-Golgi intermediate compartment

The Journal of Cell Biology ◽

10.1083/jcb.200408135 ◽

2004 ◽

Vol 167 (6) ◽

pp. 997-1003 ◽

Cited By ~ 67

Author(s):

Dalu Xu ◽

Jesse C. Hay

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Fusion ◽

Secretory Pathway ◽

Golgi Stack ◽

Golgi Membranes ◽

Transport Vesicles ◽

Copii Vesicle ◽

Intermediate Compartment ◽

Copii Vesicles

What is the first membrane fusion step in the secretory pathway? In mammals, transport vesicles coated with coat complex (COP) II deliver secretory cargo to vesicular tubular clusters (VTCs) that ferry cargo from endoplasmic reticulum exit sites to the Golgi stack. However, the precise origin of VTCs and the membrane fusion step(s) involved have remained experimentally intractable. Here, we document in vitro direct tethering and SNARE-dependent fusion of endoplasmic reticulum–derived COPII transport vesicles to form larger cargo containers. The assembly did not require detectable Golgi membranes, preexisting VTCs, or COPI function. Therefore, COPII vesicles appear to contain all of the machinery to initiate VTC biogenesis via homotypic fusion. However, COPI function enhanced VTC assembly, and early VTCs acquired specific Golgi components by heterotypic fusion with Golgi-derived COPI vesicles.

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A role for Yip1p in COPII vesicle biogenesis

The Journal of Cell Biology ◽

10.1083/jcb.200306118 ◽

2003 ◽

Vol 163 (1) ◽

pp. 57-69 ◽

Cited By ~ 53

Author(s):

Matthew Heidtman ◽

Catherine Z. Chen ◽

Ruth N. Collins ◽

Charles Barlowe

Keyword(s):

Secretory Pathway ◽

Protein Transport ◽

Genetic Interaction ◽

Temperature Sensitive ◽

Rab Gtpases ◽

Direct Role ◽

Interacting Protein ◽

Copii Vesicle ◽

Vesicle Biogenesis

Yeast Ypt1p-interacting protein (Yip1p) belongs to a conserved family of transmembrane proteins that interact with Rab GTPases. We encountered Yip1p as a constituent of ER-derived transport vesicles, leading us to hypothesize a direct role for this protein in transport through the early secretory pathway. Using a cell-free assay that recapitulates protein transport from the ER to the Golgi complex, we find that affinity-purified antibodies directed against the hydrophilic amino terminus of Yip1p potently inhibit transport. Surprisingly, inhibition is specific to the COPII-dependent budding stage. In support of this in vitro observation, strains bearing the temperature-sensitive yip1-4 allele accumulate ER membranes at a nonpermissive temperature, with no apparent accumulation of vesicle intermediates. Genetic interaction analyses of the yip1-4 mutation corroborate a function in ER budding. Finally, ordering experiments show that preincubation of ER membranes with COPII proteins decreases sensitivity to anti-Yip1p antibodies, indicating an early requirement for Yip1p in vesicle formation. We propose that Yip1p has a previously unappreciated role in COPII vesicle biogenesis.

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Erv14p Directs a Transmembrane Secretory Protein into COPII-coated Transport Vesicles

Molecular Biology of the Cell ◽

10.1091/mbc.01-10-0499 ◽

2002 ◽

Vol 13 (3) ◽

pp. 880-891 ◽

Cited By ~ 81

Author(s):

Jacqueline Powers ◽

Charles Barlowe

Keyword(s):

Endoplasmic Reticulum ◽

Secretory Pathway ◽

Integral Membrane Protein ◽

Secretory Protein ◽

Conserved Residues ◽

Early Secretory Pathway ◽

Transport Vesicles ◽

Copii Vesicle ◽

Copii Vesicles ◽

Selection Of

Erv14p is a conserved integral membrane protein that traffics in COPII-coated vesicles and localizes to the early secretory pathway in yeast. Deletion of ERV14 causes a defect in polarized growth because Axl2p, a transmembrane secretory protein, accumulates in the endoplasmic reticulum and is not delivered to its site of function on the cell surface. Herein, we show that Erv14p is required for selection of Axl2p into COPII vesicles and for efficient formation of these vesicles. Erv14p binds to subunits of the COPII coat and binding depends on conserved residues in a cytoplasmically exposed loop domain of Erv14p. When mutations are introduced into this loop, an Erv14p-Axl2p complex accumulates in the endoplasmic reticulum, suggesting that Erv14p links Axl2p to the COPII coat. Based on these results and further genetic experiments, we propose Erv14p coordinates COPII vesicle formation with incorporation of specific secretory cargo.

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Pumping lipids with P4-ATPases

Biological Chemistry ◽

10.1515/bc.2011.015 ◽

2011 ◽

Vol 392 (1-2) ◽

Cited By ~ 17

Author(s):

Rosa L. López-Marqués ◽

Joost C.M. Holthuis ◽

Thomas G. Pomorski

Keyword(s):

Vesicle Formation ◽

Potential Mechanism ◽

Vesicle Budding ◽

Vesicle Biogenesis ◽

Phospholipid Transport ◽

Endocytic Compartments

Abstract While accumulating evidence indicates that P4-ATPases catalyze phospholipid transport across cellular bilayers, their kinship to cation-pumping ATPases has raised fundamental questions concerning the underlying flippase mechanism. Loss of P4-ATPase function perturbs vesicle formation in late secretory and endocytic compartments. An intriguing concept is that P4-ATPases help drive vesicle budding by generating imbalances in transbilayer lipid numbers. Moreover, activation of P4-ATPases by phosphoinositides and other effectors of coat recruitment provide a potential mechanism to confine flippase activity to sites of vesicle biogenesis. These developments have raised considerable interest in understanding the mechanism, regulation and biological implications of P4-ATPase-catalyzed phospholipid transport.

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Auxilin facilitates membrane traffic in the early secretory pathway

Molecular Biology of the Cell ◽

10.1091/mbc.e15-09-0631 ◽

2016 ◽

Vol 27 (1) ◽

pp. 127-136 ◽

Cited By ~ 6

Author(s):

Jingzhen Ding ◽

Verónica A. Segarra ◽

Shuliang Chen ◽

Huaqing Cai ◽

Sandra K. Lemmon ◽

...

Keyword(s):

Secretory Pathway ◽

Protein Complexes ◽

Coated Vesicles ◽

Genetic Approach ◽

Vesicle Budding ◽

Early Secretory Pathway ◽

Transport Vesicles ◽

Copii Vesicles ◽

Mutant Cells ◽

Analogous Function

Coat protein complexes contain an inner shell that sorts cargo and an outer shell that helps deform the membrane to give the vesicle its shape. There are three major types of coated vesicles in the cell: COPII, COPI, and clathrin. The COPII coat complex facilitates vesicle budding from the endoplasmic reticulum (ER), while the COPI coat complex performs an analogous function in the Golgi. Clathrin-coated vesicles mediate traffic from the cell surface and between the trans-Golgi and endosome. While the assembly and structure of these coat complexes has been extensively studied, the disassembly of COPII and COPI coats from membranes is less well understood. We describe a proteomic and genetic approach that connects the J-domain chaperone auxilin, which uncoats clathrin-coated vesicles, to COPII and COPI coat complexes. Consistent with a functional role for auxilin in the early secretory pathway, auxilin binds to COPII and COPI coat subunits. Furthermore, ER–Golgi and intra-Golgi traffic is delayed at 15°C in swa2Δ mutant cells, which lack auxilin. In the case of COPII vesicles, we link this delay to a defect in vesicle fusion. We propose that auxilin acts as a chaperone and/or uncoating factor for transport vesicles that act in the early secretory pathway.

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Stacking the odds for Golgi cisternal maturation

eLife ◽

10.7554/elife.16231 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 12

Author(s):

Somya Mani ◽

Mukund Thattai

Keyword(s):

Golgi Apparatus ◽

Molecular Interactions ◽

Secretory Pathway ◽

Traffic Networks ◽

Minimal Set ◽

Vesicle Budding ◽

Vesicle Traffic

What is the minimal set of cell-biological ingredients needed to generate a Golgi apparatus? The compositions of eukaryotic organelles arise through a process of molecular exchange via vesicle traffic. Here we statistically sample tens of thousands of homeostatic vesicle traffic networks generated by realistic molecular rules governing vesicle budding and fusion. Remarkably, the plurality of these networks contain chains of compartments that undergo creation, compositional maturation, and dissipation, coupled by molecular recycling along retrograde vesicles. This motif precisely matches the cisternal maturation model of the Golgi, which was developed to explain many observed aspects of the eukaryotic secretory pathway. In our analysis cisternal maturation is a robust consequence of vesicle traffic homeostasis, independent of the underlying details of molecular interactions or spatial stacking. This architecture may have been exapted rather than selected for its role in the secretion of large cargo.

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Branched Actin Maintains Acetylated Microtubule Network in the Early Secretory Pathway

Cells ◽

10.3390/cells11010015 ◽

2021 ◽

Vol 11 (1) ◽

pp. 15

Author(s):

Azumi Yoshimura ◽

Stéphanie Miserey-Lenkei ◽

Evelyne Coudrier ◽

Bruno Goud

Keyword(s):

Golgi Apparatus ◽

Transport Process ◽

Actin Polymerization ◽

Secretory Pathway ◽

Microtubule Network ◽

Early Secretory Pathway ◽

Golgi Transport ◽

Er Exit Sites ◽

Intermediate Compartment ◽

Stable Network

In the early secretory pathway, the delivery of anterograde cargoes from the endoplasmic reticulum (ER) exit sites (ERES) to the Golgi apparatus is a multi-step transport process occurring via the ER-Golgi intermediate compartment (IC, also called ERGIC). While the role microtubules in ER-to-Golgi transport has been well established, how the actin cytoskeleton contributes to this process remains poorly understood. Here, we report that Arp2/3 inhibition affects the network of acetylated microtubules around the Golgi and induces the accumulation of unusually long RAB1/GM130-positive carriers around the centrosome. These long carriers are less prone to reach the Golgi apparatus, and arrival of anterograde cargoes to the Golgi is decreased upon Arp2/3 inhibition. Our data suggest that Arp2/3-dependent actin polymerization maintains a stable network of acetylated microtubules, which ensures efficient cargo trafficking at the late stage of ER to Golgi transport.

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The recycling pathway of protein ERGIC-53 and dynamics of the ER-Golgi intermediate compartment

Journal of Cell Science ◽

10.1242/jcs.111.22.3411 ◽

1998 ◽

Vol 111 (22) ◽

pp. 3411-3425 ◽

Cited By ~ 16

Author(s):

J. Klumperman ◽

A. Schweizer ◽

H. Clausen ◽

B.L. Tang ◽

W. Hong ◽

...

Keyword(s):

Golgi Apparatus ◽

Secretory Pathway ◽

Immunofluorescence Microscopy ◽

Gradient Centrifugation ◽

Membrane System ◽

Cell Periphery ◽

Dynamic Membrane ◽

Early Secretory Pathway ◽

Intermediate Compartment ◽

Recycling Pathway

To establish recycling routes in the early secretory pathway we have studied the recycling of the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53 in HepG2 cells. Immunofluorescence microscopy showed progressive concentration of ERGIC-53 in the Golgi area at 15 degreesC. Upon rewarming to 37 degreesC ERGIC-53 redistributed into the cell periphery often via tubular processes that largely excluded anterograde transported albumin. Immunogold labeling of cells cultured at 37 degreesC revealed ERGIC-53 predominantly in characteristic beta-COP-positive tubulo-vesicular clusters both near the Golgi apparatus and in the cell periphery. Concentration of ERGIC-53 at 15 degreesC resulted from both accumulation of ERGIC-53 in the ERGIC and movement of ERGIC membranes closer to the Golgi apparatus. Upon rewarming to 37 degreesC the labeling of ERGIC-53 in the ERGIC rapidly returned to normal levels whereas ERGIC-53's labeling in the cis-Golgi was unchanged. Temperature manipulations had no effect on the average number of ERGIC-53 clusters. Density gradient centrifugation indicated that the surplus ERGIC-53 accumulating in the ERGIC at 15 degreesC was rapidly transported to the ER upon rewarming. These results suggest that the ERGIC is a dynamic membrane system composed of a constant average number of clusters and that the major recycling pathway of ERGIC-53 bypasses the Golgi apparatus.

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