scholarly journals COPII collar defines the boundary between ER and ER exit site and does not coat cargo containers

2021 ◽  
Vol 220 (6) ◽  
Author(s):  
Olga Shomron ◽  
Inbar Nevo-Yassaf ◽  
Tamar Aviad ◽  
Yakey Yaffe ◽  
Eitan Erez Zahavi ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Membrane Vesicles ◽  
Living Cells ◽  
Protein Export ◽  
Coat Proteins ◽  
Exit Site ◽  
Golgi Transport ◽  
Selection Processes ◽  
Er Exit Sites ◽  
Genetic Perturbations

COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER–ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins’ role in ER-to-Golgi transport.

scholarly journals Uncoating of COPII from ER exit site membranes precedes cargo accumulation and membrane fission

2019 ◽  
Author(s):  
Olga Shomron ◽  
Inbar Nevo-Yassaf ◽  
Tamar Aviad ◽  
Yakey Yaffe ◽  
Eitan Erez Zahavi ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Membrane Vesicles ◽  
Coat Proteins ◽  
Direct Transfer ◽  
Exit Site ◽  
Membrane Fission ◽  
Golgi Transport ◽  
Er Exit Sites ◽  
Genetic Perturbations

SummaryCOPII and COPI are considered to be analogous sets of vesicle coat protein heterocomplexes. Coupled to cargo selection, they mediate the formation of membrane vesicles translocating in opposite directions to differ rent destinations within the secretory pathway. Here, live cell and electron microscopy provided evidence for a different localization and mode of function of the COPII coat during protein export from the endoplasmic reticulum (ER). Pharmaceutical and genetic perturbations of ER-Golgi transport were used to demonstrate that COPII is recruited to membranes defining the boundary of ER-ER Exit Sites (ERES) where it facilitates selective cargo concentration. Uncoating of COPII membranes precedes cargo accumulation and fission of Golgi-bound carriers. Moreover, we report what may be direct transfer of cargo to the Golgi apparatus from Golgi-associated BFA sensitive ERESs. Finally, in ldlF cells the stably expressed functional ε-COPI-EYFP labeled both ERESs and anterograde carriers. These findings change our understanding of the role of coat proteins in ER to Golgi transport.

scholarly journals Vesicular and uncoated Rab1-dependent cargo carriers facilitate ER to Golgi transport

2020 ◽  
Vol 133 (14) ◽  
pp. jcs239814 ◽  
Author(s):  
Laura M. Westrate ◽  
Melissa J. Hoyer ◽  
Michael J. Nash ◽  
Gia K. Voeltz
Keyword(s):  
Endoplasmic Reticulum ◽  
De Novo ◽  
Coated Vesicle ◽  
First Person ◽  
Coat Proteins ◽  
Animal Cells ◽  
Golgi Transport ◽  
Er Exit Sites ◽  
Copii Vesicles ◽  
Export System

ABSTRACTSecretory cargo is recognized, concentrated and trafficked from endoplasmic reticulum (ER) exit sites (ERES) to the Golgi. Cargo export from the ER begins when a series of highly conserved COPII coat proteins accumulate at the ER and regulate the formation of cargo-loaded COPII vesicles. In animal cells, capturing live de novo cargo trafficking past this point is challenging; it has been difficult to discriminate whether cargo is trafficked to the Golgi in a COPII-coated vesicle. Here, we describe a recently developed live-cell cargo export system that can be synchronously released from ERES to illustrate de novo trafficking in animal cells. We found that components of the COPII coat remain associated with the ERES while cargo is extruded into COPII-uncoated, non-ER associated, Rab1 (herein referring to Rab1a or Rab1b)-dependent carriers. Our data suggest that, in animal cells, COPII coat components remain stably associated with the ER at exit sites to generate a specialized compartment, but once cargo is sorted and organized, Rab1 labels these export carriers and facilitates efficient forward trafficking.This article has an associated First Person interview with the first author of the paper.

scholarly journals Tales of the ER-Golgi Frontier: Drosophila-Centric Considerations on Tango1 Function

2021 ◽  
Vol 8 ◽  
Author(s):  
Zhi Feng ◽  
Ke Yang ◽  
José C. Pastor-Pareja
Keyword(s):  
Secretory Pathway ◽  
Transmembrane Protein ◽  
Vesicular Transport ◽  
Fruit Fly ◽  
Evolutionary Perspective ◽  
Animal Evolution ◽  
Golgi Transport ◽  
Er Exit Sites ◽  
Related Proteins

In the secretory pathway, the transfer of cargo from the ER to the Golgi involves dozens of proteins that localize at specific regions of the ER called ER exit sites (ERES), where cargos are concentrated preceding vesicular transport to the Golgi. Despite many years of research, we are missing crucial details of how this highly dynamic ER-Golgi interface is defined, maintained and functions. Mechanisms allowing secretion of large cargos such as the very abundant collagens are also poorly understood. In this context, Tango1, discovered in the fruit fly Drosophila and widely conserved in animal evolution, has received a lot of attention in recent years. Tango1, an ERES-localized transmembrane protein, is the single fly member of the MIA/cTAGE family, consisting in humans of TANGO1 and at least 14 different related proteins. After its discovery in flies, a specific role of human TANGO1 in mediating secretion of collagens was reported. However, multiple studies in Drosophila have demonstrated that Tango1 is required for secretion of all cargos. At all ERES, through self-interaction and interactions with other proteins, Tango1 aids ERES maintenance and tethering of post-ER membranes. In this review, we discuss discoveries on Drosophila Tango1 and put them in relation with research on human MIA/cTAGE proteins. In doing so, we aim to offer an integrated view of Tango1 function and the nature of ER-Golgi transport from an evolutionary perspective.

scholarly journals Branched Actin Maintains Acetylated Microtubule Network in the Early Secretory Pathway

Cells ◽  
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.

SEC16 in COPII coat dynamics at ER exit sites

2015 ◽  
Vol 43 (1) ◽  
pp. 97-103 ◽  
Author(s):  
Joep Sprangers ◽  
Catherine Rabouille
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Functional Organization ◽  
Protein Export ◽  
Coated Vesicles ◽  
Loss Of Function ◽  
Exact Function ◽  
Er Exit Sites ◽  
Secretory Capacity

Protein export from the endoplasmic reticulum (ER), the first step in protein transport through the secretory pathway, is mediated by coatomer protein II (COPII)-coated vesicles at ER exit sites. COPII coat assembly on the ER is well understood and the conserved large hydrophilic protein Sec16 clearly has a role to play in COPII coat dynamics. Sec16 localizes to ER exit sites, its loss of function impairs their functional organization in all species where it has been studied, and it interacts with COPII coat subunits. However, its exact function in COPII dynamics is debated, as Sec16 is proposed to act as a scaffold to recruit COPII components and as a device to regulate the Sar1 activity in uncoating, in such a way that the coat is released only when the vesicle is fully formed and loaded with cargo. Furthermore, Sec16 has been shown to respond to nutrient signalling, thus coupling environmental stimuli to secretory capacity.

The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport

1991 ◽  
Vol 11 (6) ◽  
pp. 2980-2993
Author(s):  
R Ossig ◽  
C Dascher ◽  
H H Trepte ◽  
H D Schmitt ◽  
D Gallwitz
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Single Copy ◽  
Integral Membrane Protein ◽  
Carboxypeptidase Y ◽  
Null Mutant ◽  
Yeast Saccharomyces Cerevisiae ◽  
Golgi Transport ◽  
Gtp Binding

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.

scholarly journals Forward and retrograde trafficking in mitotic animal cells. ER-Golgi transport arrest restricts protein export from the ER into COPII-coated structures

1999 ◽  
Vol 112 (5) ◽  
pp. 589-600 ◽  
Author(s):  
T. Farmaki ◽  
S. Ponnambalam ◽  
A.R. Prescott ◽  
H. Clausen ◽  
B.L. Tang ◽  
...  
Keyword(s):  
Protein Transport ◽  
Protein Export ◽  
Sensitive Assay ◽  
Plasma Membrane Protein ◽  
Animal Cells ◽  
Golgi Stack ◽  
Golgi Transport ◽  
Intermediate Compartment ◽  
First Time ◽  
Mitotic Cells

Protein transport arrest occurs between the ER and Golgi stack of mitotic animal cells, but the location of this block is unknown. In this report we use the recycling intermediate compartment protein ERGIC 53/p58 and the plasma membrane protein CD8 to establish the site of transport arrest. Recycled ERGIC 53/p58 and newly synthesised CD8 accumulate in ER cisternae but not in COPII-coated export structures or more distal sites. During mitosis the tubulovesicular ER-related export sites were depleted of the COPII component Sec13p, as shown by immunoelectron microscopy, indicating that COPII budding structures are the target for mitotic inhibition. The extent of recycling of Golgi stack residents was also investigated. In this study we used oligosaccharide modifications on CD8 trapped in the ER of mitotic cells as a sensitive assay for recycling of Golgi stack enzymes. We find that modifications conferred by the Golgi stack-resident GalNac transferase do occur on newly synthesised CD8, but these modifications are entirely due to newly synthesised transferase rather than to enzyme recycled from the Golgi stack. Taken together our findings establish for the first time that the site of ER-Golgi transport arrest of mitotic cells is COPII budding structures, and they clearly speak against a role for recycling in partitioning of Golgi stack proteins via translocation to the ER.

scholarly journals A stress assembly that confers cell viability by preserving ERES components during amino-acid starvation

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Margarita Zacharogianni ◽  
Angelica Aguilera-Gomez ◽  
Tineke Veenendaal ◽  
Jan Smout ◽  
Catherine Rabouille
Keyword(s):  
Amino Acid ◽  
Cell Viability ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Liquid Droplet ◽  
Protein Translation ◽  
Amino Acid Starvation ◽  
Early Secretory Pathway ◽  
Er Exit Sites ◽  
Nutritional Restriction

Nutritional restriction leads to protein translation attenuation that results in the storage and degradation of free mRNAs in cytoplasmic assemblies. In this study, we show in Drosophila S2 cells that amino-acid starvation also leads to the inhibition of another major anabolic pathway, the protein transport through the secretory pathway, and to the formation of a novel reversible non-membrane bound stress assembly, the Sec body that incorporates components of the ER exit sites. Sec body formation does not depend on membrane traffic in the early secretory pathway, yet requires both Sec23 and Sec24AB. Sec bodies have liquid droplet-like properties, and they act as a protective reservoir for ERES components to rebuild a functional secretory pathway after re-addition of amino-acids acting as a part of a survival mechanism. Taken together, we propose that the formation of these structures is a novel stress response mechanism to provide cell viability during and after nutrient stress.

Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells

2000 ◽  
Author(s):  
Jens Rietdorf ◽  
David J. Stephens ◽  
Anthony Squire ◽  
Jeremy Simpson ◽  
David T. Shima ◽  
...  
Keyword(s):  
Cellular Structure ◽  
3D Imaging ◽  
Secretory Pathway ◽  
Living Cells ◽  
Structure And Function ◽  
Early Secretory Pathway ◽  
And Function

Transport Through the Secretory Pathway of VSVG Tagged With Green Fluorescent Protein: Role of Tubulovesicular Carriers and Microtubules

1997 ◽  
Vol 3 (S2) ◽  
pp. 139-140
Author(s):  
John Presley ◽  
Koret Hirschberg ◽  
Nelson Cole
Keyword(s):  
Green Fluorescent Protein ◽  
Membrane Transport ◽  
G Protein ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Dynamic Properties ◽  
Living Cells ◽  
Morphological Properties ◽  
Green Fluorescent

The ts045 mutant of VSV G protein has been used in numerous studies to identify biochemical and morphological properties of membrane transport, due to its reversible misfolding and retention in the ER at 40°C and ability to traffic out of the ER and into the Golgi complex upon temperature reduction to 32oC. The dynamic properties of membrane transport intermediates of the secretory pathway, including their lifetime and fate within cells, have not until now been explored due to the inability to follow transport in single living cells. Here, we attached green fluorescent protein to the cytoplasmic tail of VSV G protein in order to visualize ER-to-Golgi and Golgi-to-plasma membrane transport in living cells. VSVG-GFP expressed in Cos cells accumulated in the ER at 40°C and translocated to the Golgi complex when shifted to 32oC. Translocation of the protein was followed using time-lapse imaging of live cells on a confocal microscope. VSVG-GFP accumulated in tubulovesicular structures scattered throughout the cell upon shift from 40°C to 15°C for three hours.

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