Branched Actin Maintains Acetylated Microtubule Network in the Early Secretory Pathway

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

eLife ◽

10.7554/elife.04132 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 29

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.

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

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.619022 ◽

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.

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The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells.

Biochemical Society Symposium ◽

10.1042/bss0720001 ◽

2005 ◽

Vol 72 ◽

pp. 1-13 ◽

Cited By ~ 24

Author(s):

Krysten J. Palmer ◽

Peter Watson ◽

David J. Stephens

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Secretory Pathway ◽

Motor Proteins ◽

Microtubule Cytoskeleton ◽

Early Secretory Pathway ◽

Golgi Transport ◽

Intracellular Compartments ◽

Retrograde Traffic

The organization of intracellular compartments and the transfer of components between them are central to the correct functioning of mammalian cells. Proteins and lipids are transferred between compartments by the formation, movement and subsequent specific fusion of transport intermediates. These vesicles and membrane clusters must be coupled to the cytoskeleton and to motor proteins that drive motility. Anterograde ER (endoplasmic reticulum)-to-Golgi transport, and the converse step of retrograde traffic from the Golgi to the ER, are now known to involve coupling of membranes to the microtubule cytoskeleton. Here we shall discuss our current understanding of the mechanisms that link membrane traffic in the early secretory pathway to the microtubule cytoskeleton in mammalian cells. Recent data have also provided molecular detail of functional co-ordination of motor proteins to specify directionality, as well as mechanisms for regulating motor activity by protein phosphorylation.

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Morphological and Functional Association of Sec22b/ERS-24 with the pre-Golgi Intermediate Compartment

Molecular Biology of the Cell ◽

10.1091/mbc.10.2.435 ◽

1999 ◽

Vol 10 (2) ◽

pp. 435-453 ◽

Cited By ~ 44

Author(s):

Tao Zhang ◽

Siew Heng Wong ◽

Bor Luen Tang ◽

Yue Xu ◽

Wanjin Hong

Keyword(s):

Golgi Apparatus ◽

Mammalian Cells ◽

Intact Cell ◽

Brefeldin A ◽

Cell System ◽

Fusion Event ◽

Homologous Proteins ◽

Golgi Transport ◽

Protein Receptor ◽

Intermediate Compartment

Yeast Sec22p participates in both anterograde and retrograde vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus by functioning as a v-SNARE (solubleN-ethylmaleimide-sensitive factor [NSF] attachment protein receptor) of transport vesicles. Three mammalian proteins homologous to Sec22p have been identified and are referred to as Sec22a, Sec22b/ERS-24, and Sec22c, respectively. The existence of three homologous proteins in mammalian cells calls for detailed cell biological and functional examinations of each individual protein. The epitope-tagged forms of all three proteins have been shown to be primarily associated with the ER, although functional examination has not been carefully performed for any one of them. In this study, using antibodies specific for Sec22b/ERS-24, it is revealed that endogenous Sec22b/ERS-24 is associated with vesicular structures in both the perinuclear Golgi and peripheral regions. Colabeling experiments for Sec22b/ERS-24 with Golgi mannosidase II, the KDEL receptor, and the envelope glycoprotein G (VSVG) of vesicular stomatitis virus (VSV) en route from the ER to the Golgi under normal, brefeldin A, or nocodazole-treated cells suggest that Sec22b/ERS-24 is enriched in the pre-Golgi intermediate compartment (IC). In a well-established semi-intact cell system that reconstitutes transport from the ER to the Golgi, transport of VSVG is inhibited by antibodies against Sec22b/ERS-24. EGTA is known to inhibit ER–Golgi transport at a stage after vesicle/transport intermediate docking but before the actual fusion event. Antibodies against Sec22b/ERS-24 inhibit ER–Golgi transport only when they are added before the EGTA-sensitive stage. Transport of VSVG accumulated in pre-Golgi IC by incubation at 15°C is also inhibited by Sec22b/ERS-24 antibodies. Morphologically, VSVG is transported from the ER to the Golgi apparatus via vesicular intermediates that scatter in the peripheral as well as the Golgi regions. In the presence of antibodies against Sec22b/ERS-24, VSVG is seen to accumulate in these intermediates, suggesting that Sec22b/ERS-24 functions at the level of the IC in ER–Golgi transport.

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Poliovirus Infection Transiently Increases COPII Vesicle Budding

Journal of Virology ◽

10.1128/jvi.01159-12 ◽

2012 ◽

Vol 86 (18) ◽

pp. 9675-9682 ◽

Cited By ~ 19

Author(s):

Meg Trahey ◽

Hyung Suk Oh ◽

Craig E. Cameron ◽

Jesse C. Hay

Keyword(s):

Golgi Apparatus ◽

Secretory Pathway ◽

Vesicle Formation ◽

Vesicle Budding ◽

Precursor Pool ◽

Copii Vesicle ◽

Early Increase ◽

Vesicle Biogenesis ◽

Intermediate Compartment ◽

Copii Vesicles

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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The Mammalian Protein (rbet1) Homologous to Yeast Bet1p Is Primarily Associated with the Pre-Golgi Intermediate Compartment and Is Involved in Vesicular Transport from the Endoplasmic Reticulum to the Golgi Apparatus

The Journal of Cell Biology ◽

10.1083/jcb.139.5.1157 ◽

1997 ◽

Vol 139 (5) ◽

pp. 1157-1168 ◽

Cited By ~ 45

Author(s):

Tao Zhang ◽

Siew Heng Wong ◽

Bor Luen Tang ◽

Yue Xu ◽

Frank Peter ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

G Protein ◽

Vesicular Transport ◽

Dependent Manner ◽

Golgi Transport ◽

Golgi Region ◽

Intermediate Compartment ◽

Kdel Receptor ◽

Mammalian Protein

Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15°C. However, upon warming up from 15 to 37°C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER- derived vesicles with the cis-Golgi membrane.

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cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells

Molecular Biology of the Cell ◽

10.1091/mbc.e12-01-0034 ◽

2012 ◽

Vol 23 (16) ◽

pp. 3203-3214 ◽

Cited By ~ 49

Author(s):

Yoko Ito ◽

Tomohiro Uemura ◽

Keiko Shoda ◽

Masaru Fujimoto ◽

Takashi Ueda ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Fluorescent Proteins ◽

Plant Cells ◽

Brefeldin A ◽

Eukaryotic Cells ◽

Er Exit Sites ◽

Intermediate Compartment ◽

Golgi Protein ◽

And Function

The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.

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The COPII pathway and hematologic disease

Blood ◽

10.1182/blood-2012-01-292086 ◽

2012 ◽

Vol 120 (1) ◽

pp. 31-38 ◽

Cited By ~ 30

Author(s):

Rami Khoriaty ◽

Matthew P. Vasievich ◽

David Ginsburg

Keyword(s):

Secretory Pathway ◽

Coagulation Factor ◽

Coagulation Factors ◽

Early Secretory Pathway ◽

Golgi Transport ◽

Factor Deficiency ◽

Mannose Binding Protein ◽

Mannose Binding ◽

Coagulation Factor Deficiency ◽

Combined Deficiency

Abstract Multiple diseases, hematologic and nonhematologic, result from defects in the early secretory pathway. Congenital dyserythropoietic anemia type II (CDAII) and combined deficiency of coagulation factors V and VIII (F5F8D) are the 2 known hematologic diseases that result from defects in the endoplasmic reticulum (ER)–to–Golgi transport system. CDAII is caused by mutations in the SEC23B gene, which encodes a core component of the coat protein complex II (COPII). F5F8D results from mutations in either LMAN1 (lectin mannose-binding protein 1) or MCFD2 (multiple coagulation factor deficiency protein 2), which encode the ER cargo receptor complex LMAN1-MCFD2. These diseases and their molecular pathogenesis are the focus of this review.

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