scholarly journals Auxilin facilitates membrane traffic in the early secretory pathway

2016 ◽  
Vol 27 (1) ◽  
pp. 127-136 ◽  
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.

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

2002 ◽  
Vol 13 (3) ◽  
pp. 880-891 ◽  
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.

scholarly journals COPII-coated membranes function as transport carriers of intracellular procollagen-1

2017 ◽  
Author(s):  
Amita Gorur ◽  
Lin Yuan ◽  
Samuel J Kenny ◽  
Satoshi Baba ◽  
Ke Xu ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Imaging Techniques ◽  
Coated Vesicles ◽  
Intact Cells ◽  
Vesicle Budding ◽  
Coated Membranes ◽  
Copii Vesicles ◽  
Optical Reconstruction

AbstractThe coat protein complex II (COPII) is essential for the secretion of large cargo, such as the 300 nm precursor fibrils of procollagen I (PC1). Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles to over 300 nm in diameter and accelerates the secretion of PC1; however, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy (STORM), correlated light electron microscopy (CLEM), and live cell imaging we report the existence of mobile COPII-coated vesicles that completely encapsulate the cargo PC1 and are physically separated from ER. We have also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude from in vivo and in vitro evidence that large COPII vesicles are bona fide carriers of PC1.SummaryCOPII may play a direct or indirect role in the traffic of large protein complexes such as procollagen. Using high resolution imaging techniques in intact cells and in vitro reconstituted vesicles, Gorur et al. show that COPII coated vesicles carry procollagen1.

scholarly journals MIA3/TANGO1 enables efficient secretion by constraining COPII vesicle budding

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.

Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway v1

2022 ◽  
Author(s):  
Javier Manzano-Lopez†* ◽  
Sofia Rodriguez-Gallardo† ◽  
Susana Sabido-Bozo† ◽  
Alejandro Cortes-Gomez ◽  
Ana Maria Perez-Linero ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Protein Complexes ◽  
Secretory Protein ◽  
Extracellular Proteins ◽  
Transient Nature ◽  
Transport Vesicles ◽  
System P ◽  
Cargo Receptor ◽  
Cargo Proteins

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to stabilize the transient and/or weak protein interactions. Here, we describe a protocol of protein cross-linking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system.

Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway v1

2021 ◽  
Author(s):  
Javier Manzano-Lopez † ◽  
Sofia Rodriguez-Gallardo † ◽  
Susana Sabido-Bozo ◽  
Ana Maria Perez-Linero ◽  
Rafael Lucena ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Protein Complexes ◽  
Secretory Protein ◽  
Extracellular Proteins ◽  
Transient Nature ◽  
Transport Vesicles ◽  
System P ◽  
Cargo Receptor ◽  
Cargo Proteins

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to immobilize the transient and/or weak protein interactions. Here, we describe a protocol of protein cross-linking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system.

scholarly journals Molecular basis for KDEL-mediated retrieval of escaped ER-resident proteins – SWEET talking the COPs

2020 ◽  
Vol 133 (19) ◽  
pp. jcs250100
Author(s):  
Simon Newstead ◽  
Francis Barr
Keyword(s):  
Secretory Pathway ◽  
Signal Sequence ◽  
Structural Similarity ◽  
Retrograde Transport ◽  
Coated Vesicles ◽  
Early Secretory Pathway ◽  
Cargo Proteins ◽  
Protein Localisation ◽  
And Function ◽  
Trafficking Receptor

ABSTRACTProtein localisation in the cell is controlled through the function of trafficking receptors, which recognise specific signal sequences and direct cargo proteins to different locations. The KDEL receptor (KDELR) was one of the first intracellular trafficking receptors identified and plays an essential role in maintaining the integrity of the early secretory pathway. The receptor recognises variants of a canonical C-terminal Lys-Asp-Glu-Leu (KDEL) signal sequence on ER-resident proteins when these escape to the Golgi, and targets these proteins to COPI- coated vesicles for retrograde transport back to the ER. The empty receptor is then recycled from the ER back to the Golgi by COPII-coated vesicles. Crystal structures of the KDELR show that it is structurally related to the PQ-loop family of transporters that are found in both pro- and eukaryotes, and shuttle sugars, amino acids and vitamins across cellular membranes. Furthermore, analogous to PQ-loop transporters, the KDELR undergoes a pH-dependent and ligand-regulated conformational cycle. Here, we propose that the striking structural similarity between the KDELR and PQ-loop transporters reveals a connection between transport and trafficking in the cell, with important implications for understanding trafficking receptor evolution and function.

scholarly journals ADP-ribosylation factor and coatomer couple fusion to vesicle budding

1994 ◽  
Vol 124 (4) ◽  
pp. 415-424 ◽  
Author(s):  
Z Elazar ◽  
L Orci ◽  
J Ostermann ◽  
M Amherdt ◽  
G Tanigawa ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Retrograde Transport ◽  
Coated Vesicles ◽  
Coat Proteins ◽  
Vesicle Budding ◽  
Adp Ribosylation ◽  
Golgi Membranes ◽  
Transport Vesicles ◽  
Donor And Acceptor ◽  
Adp Ribosylation Factor

The coat proteins required for budding COP-coated vesicles from Golgi membranes, coatomer and ADP-ribosylation factor (ARF) protein, are shown to be required to reconstitute the orderly process of transport between Golgi cisternae in which fusion of transport vesicles begins only after budding ends. When either coat protein is omitted, fusion is uncoupled from budding-donor and acceptor compartments pair directly without an intervening vesicle. Coupling may therefore results from the sequestration of fusogenic membrane proteins into assembling coated vesicles that are only exposed when the coat is removed after budding is complete. This mechanism of coupling explains the phenomenon of "retrograde transport" triggered by uncouplers such as the drug brefeldin A.

scholarly journals Poliovirus Infection Transiently Increases COPII Vesicle Budding

2012 ◽  
Vol 86 (18) ◽  
pp. 9675-9682 ◽  
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.

scholarly journals Distinct stages in the recognition, sorting, and packaging of proTGFα into COPII-coated transport vesicles

2016 ◽  
Vol 27 (12) ◽  
pp. 1938-1947 ◽  
Author(s):  
Pengcheng Zhang ◽  
Randy Schekman
Keyword(s):  
Secretory Pathway ◽  
Transforming Growth Factor ◽  
Transmembrane Protein ◽  
Transforming Growth Factor Α ◽  
Transport Vesicles ◽  
Cargo Protein ◽  
Copii Vesicles ◽  
Factor Α ◽  
Cytosolic Factor

In addition to its role in forming vesicles from the endoplasmic reticulum (ER), the coat protein complex II (COPII) is also responsible for selecting specific cargo proteins to be packaged into COPII transport vesicles. Comparison of COPII vesicle formation in mammalian systems and in yeast suggested that the former uses more elaborate mechanisms for cargo recognition, presumably to cope with a significantly expanded repertoire of cargo that transits the secretory pathway. Using proTGFα, the transmembrane precursor of transforming growth factor α (TGFα), as a model cargo protein, we demonstrate in cell-free assays that at least one auxiliary cytosolic factor is specifically required for the efficient packaging of proTGFα into COPII vesicles. Using a knockout HeLa cell line generated by CRISPR/Cas9, we provide functional evidence showing that a transmembrane protein, Cornichon-1 (CNIH), acts as a cargo receptor of proTGFα. We show that both CNIH and the auxiliary cytosolic factor(s) are required for efficient recruitment of proTGFα to the COPII coat in vitro. Moreover, we provide evidence that the recruitment of cargo protein by the COPII coat precedes and may be distinct from subsequent cargo packaging into COPII vesicles.

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