scholarly journals Protein sorting at the ER–Golgi interface

2016 ◽  
Vol 215 (6) ◽  
pp. 769-778 ◽  
Author(s):  
Natalia Gomez-Navarro ◽  
Elizabeth Miller
Keyword(s):  
Secretory Pathway ◽  
Protein Sorting ◽  
Vesicle Traffic ◽  
Protein Traffic ◽  
Cellular Physiology ◽  
Transport Vesicles ◽  
Cargo Proteins ◽  
Vesicle Biogenesis ◽  
Membrane Bound ◽  
Insight Into

Protein traffic is of critical importance for normal cellular physiology. In eukaryotes, spherical transport vesicles move proteins and lipids from one internal membrane-bound compartment to another within the secretory pathway. The process of directing each individual protein to a specific destination (known as protein sorting) is a crucial event that is intrinsically linked to vesicle biogenesis. In this review, we summarize the principles of cargo sorting by the vesicle traffic machinery and consider the diverse mechanisms by which cargo proteins are selected and captured into different transport vesicles. We focus on the first two compartments of the secretory pathway: the endoplasmic reticulum and Golgi. We provide an overview of the complexity and diversity of cargo adaptor function and regulation, focusing on recent mechanistic discoveries that have revealed insight into protein sorting in 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 Biogenesis of γ-secretase early in the secretory pathway

2007 ◽  
Vol 179 (5) ◽  
pp. 951-963 ◽  
Author(s):  
Jinoh Kim ◽  
Bertrand Kleizen ◽  
Regina Choy ◽  
Gopal Thinakaran ◽  
Sangram S. Sisodia ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Direct Evidence ◽  
Surface Proteins ◽  
Cell Surface Proteins ◽  
Transport Vesicles ◽  
Cargo Proteins ◽  
Transport Vesicle ◽  
A Cell ◽  
Transient Intermediate ◽  
Important Enzyme

γ-Secretase is responsible for proteolytic maturation of signaling and cell surface proteins, including amyloid precursor protein (APP). Abnormal processing of APP by γ-secretase produces a fragment, Aβ42, that may be responsible for Alzheimer's disease (AD). The biogenesis and trafficking of this important enzyme in relation to aberrant Aβ processing is not well defined. Using a cell-free reaction to monitor the exit of cargo proteins from the endoplasmic reticulum (ER), we have isolated a transient intermediate of γ-secretase. Here, we provide direct evidence that the γ-secretase complex is formed in an inactive complex at or before the assembly of an ER transport vesicle dependent on the COPII sorting subunit, Sec24A. Maturation of the holoenzyme is achieved in a subsequent compartment. Two familial AD (FAD)–linked PS1 variants are inefficiently packaged into transport vesicles generated from the ER. Our results suggest that aberrant trafficking of PS1 may contribute to disease pathology.

scholarly journals Structural basis for the initiation of COPII vesicle biogenesis

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.

scholarly journals Charting the Secretory Pathway in a Simple Eukaryote

2010 ◽  
Vol 21 (22) ◽  
pp. 3781-3784 ◽  
Author(s):  
Randy Schekman
Keyword(s):  
Graduate Students ◽  
Cell Biology ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Protein Transport ◽  
Yeast Cells ◽  
Founding Father ◽  
Transport Vesicles ◽  
Cargo Proteins ◽  
American Society

George Palade, a founding father of cell biology and of the American Society for Cell Biology (ASCB), established the ultrastructural framework for an analysis of how proteins are secreted and membranes are assembled in eukaryotic cells. His vision inspired a generation of investigators to probe the molecular mechanisms of protein transport. My laboratory has dissected these pathways with complementary genetic and biochemical approaches. Peter Novick, one of my first graduate students, isolated secretion mutants of Saccharomyces cerevisiae, and through cytological analysis of single and double mutants and molecular cloning of the corresponding SEC genes, we established that yeast cells use a secretory pathway fundamentally conserved in all eukaryotes. A biochemical reaction that recapitulates the first half of the secretory pathway was used to characterize Sec proteins that comprise the polypeptide translocation channel in the endoplasmic reticulum (ER) membrane (Sec61) and the cytoplasmic coat protein complex (COPII) that captures cargo proteins into transport vesicles that bud from the ER.

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

2016 ◽  
Author(s):  
Pengcheng Zhang ◽  
Randy Schekman
Keyword(s):  
Secretory Pathway ◽  
Transforming Growth Factor ◽  
Transmembrane Protein ◽  
Transport Vesicles ◽  
Cargo Protein ◽  
Cargo Proteins ◽  
Factor Alpha ◽  
Copii Vesicles ◽  
Cytosolic Factor

AbstractIn 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 employs 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 alpha (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.AbbreviationsCNIHCornichon

scholarly journals Vps52p, Vps53p, and Vps54p Form a Novel Multisubunit Complex Required for Protein Sorting at the Yeast Late Golgi

2000 ◽  
Vol 11 (1) ◽  
pp. 305-323 ◽  
Author(s):  
Elizabeth Conibear ◽  
Tom H. Stevens
Keyword(s):  
Membrane Proteins ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Protein Sorting ◽  
Retrograde Transport ◽  
Carboxypeptidase Y ◽  
Golgi Membrane ◽  
Triple Mutant ◽  
Protein Traffic ◽  
Golgi Compartment

The late Golgi of the yeast Saccharomyces cerevisiaereceives membrane traffic from the secretory pathway as well as retrograde traffic from post-Golgi compartments, but the machinery that regulates these vesicle-docking and fusion events has not been characterized. We have identified three components of a novel protein complex that is required for protein sorting at the yeast late Golgi compartment. Mutation of VPS52, VPS53, orVPS54 results in the missorting of 70% of the vacuolar hydrolase carboxypeptidase Y as well as the mislocalization of late Golgi membrane proteins to the vacuole, whereas protein traffic through the early part of the Golgi complex is unaffected. Avps52/53/54 triple mutant strain is phenotypically indistinguishable from each of the single mutants, consistent with the model that all three are required for a common step in membrane transport. Native coimmunoprecipitation experiments indicate that Vps52p, Vps53p, and Vps54p are associated in a 1:1:1 complex that sediments as a single peak on sucrose velocity gradients. This complex, which exists both in a soluble pool and as a peripheral component of a membrane fraction, colocalizes with markers of the yeast late Golgi by immunofluorescence microscopy. Together, the phenotypic and biochemical data suggest that VPS52, VPS53, andVPS54 are required for the retrograde transport of Golgi membrane proteins from an endosomal/prevacuolar compartment. The Vps52/53/54 complex joins a growing list of distinct multisubunit complexes that regulate membrane-trafficking events.

Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway v2

2021 ◽  
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 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.

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 ◽  
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.

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