scholarly journals Determining the content of vesicles captured by golgin tethers using LOPIT-DC

2019 ◽  
Author(s):  
John J.H. Shin ◽  
Oliver M. Crook ◽  
Alicia Borgeaud ◽  
Jérôme Cattin-Ortolá ◽  
Sew-Yeu Peak-Chew ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Transmembrane Protein ◽  
Coiled Coil ◽  
Retrograde Transport ◽  
Statistical Modelling ◽  
Lysosomal Hydrolases ◽  
Transport Pathways ◽  
Transient Nature ◽  
Transport Vesicles ◽  
Cargo Proteins

AbstractThe internal organisation of the cell depends on tethers at destination organelles to selectively capture incoming transport vesicles to facilitate SNARE-mediated fusion. The golgin long coiled-coil proteins function as tethers that contributes to this specificity at the Golgi (1). Golgin-97, golgin-245 and GCC88 golgins of the trans-Golgi capture vesicles derived from endosomes, which serve to recycle the critical Golgi machinery required to deliver lysosomal hydrolases and to maintain exocytosis. Retrograde trafficking from endosomes to the trans-Golgi network (TGN) is a complex process that involves the sorting of transmembrane cargo proteins into distinct transport vesicles by adaptors from multiple pathways. The content of these distinct vesicles, which golgin they target and the factors that mediate this targeting are not well understood. The major challenges that have limited advances in these areas is the transient nature of vesicle tethering, and the redundancies in their mechanisms that confound experimental dissection. To gain better insight into these problems, we performed organelle proteomics using the Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC) method on a system in which an ectopic golgin causes vesicles to accumulate in a tethered state (2). By incorporating Bayesian statistical modelling into our analysis (3), we determined that over 45 transmembrane proteins and 51 peripheral membrane proteins of the endosomal network are on vesicles captured by golgin-97, including known cargo and components of the clathrin/AP-1, retromer-dependent and -independent transport pathways. We also determined a distinct class of vesicles shared by golgin-97, golgin-245 and GCC88 that is enriched in TMEM87A, a multi-pass transmembrane protein of unknown function that has previously been implicated in endosome-to-Golgi retrograde transport (4). Finally, we categorically demonstrate that the vesicles that these golgins capture are retrograde transport vesicles based on the lack of enrichment of lysosomal hydrolases in our LOPIT-DC data, and from correlative light electron tomography images of spherical vesicles captured by golgin-97. Together, our study demonstrates the power of combining LOPIT-DC with Bayesian statistical analysis in interrogating the dynamic spatial movement of proteins in transport vesicles.

scholarly journals Vps51p Mediates the Association of the GARP (Vps52/53/54) Complex with the Late Golgi t-SNARE Tlg1p

2003 ◽  
Vol 14 (4) ◽  
pp. 1610-1623 ◽  
Author(s):  
Elizabeth Conibear ◽  
Jessica N. Cleck ◽  
Tom H. Stevens
Keyword(s):  
Coiled Coil ◽  
Retrograde Transport ◽  
The Other ◽  
Snare Protein ◽  
Transport Pathways ◽  
Distinct Phenotype ◽  
Cog Complex ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Target Membrane

Multisubunit tethering complexes may contribute to the specificity of membrane fusion events by linking transport vesicles to their target membrane in an initial recognition event that promotes SNARE assembly. However, the interactions that link tethering factors to the other components of the vesicle fusion machinery are still largely unknown. We have previously identified three subunits of a Golgi-localized complex (the Vps52/53/54 complex) that is required for retrograde transport to the late Golgi. This complex interacts with a Rab and a SNARE protein found at the late Golgi and is related to two other multisubunit tethering complexes: the COG complex and the exocyst. Here we show that the Vps52/53/54 complex has an additional subunit, Vps51p. All four members of this tetrameric GARP (Golgi-associated retrograde protein) complex are required for two distinct retrograde transport pathways, from both early and late endosomes, back to the TGN.vps51 mutants exhibit a distinct phenotype suggestive of a regulatory role. Indeed, we find that Vps51p mediates the interaction between Vps52/53/54 and the t-SNARE Tlg1p. The binding of this small, coiled-coil protein to the conserved N-terminal domain of the t-SNARE therefore provides a crucial link between components of the tethering and the fusion machinery.

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.

Assembly of COPI and COPII Vesicular Coat Proteins on Membranes

2018 ◽  
Vol 47 (1) ◽  
pp. 63-83 ◽  
Author(s):  
Julien Béthune ◽  
Felix T. Wieland
Keyword(s):  
Electron Tomography ◽  
Coat Proteins ◽  
Structural Elements ◽  
X Ray Crystallography ◽  
Transport Vesicles ◽  
Cryo Electron Tomography ◽  
Functional Implications ◽  
Cargo Proteins ◽  
Intracellular Compartments ◽  
Proteins And Peptides

In eukaryotes, distinct transport vesicles functionally connect various intracellular compartments. These carriers mediate transport of membranes for the biogenesis and maintenance of organelles, secretion of cargo proteins and peptides, and uptake of cargo into the cell. Transport vesicles have distinct protein coats that assemble on a donor membrane where they can select cargo and curve the membrane to form a bud. A multitude of structural elements of coat proteins have been solved by X-ray crystallography. More recently, the architectures of the COPI and COPII coats were elucidated in context with their membrane by cryo-electron tomography. Here, we describe insights gained from the structures of these two coat lattices and discuss the resulting functional implications.

scholarly journals GCC185 plays independent roles in Golgi structure maintenance and AP-1–mediated vesicle tethering

2011 ◽  
Vol 194 (5) ◽  
pp. 779-787 ◽  
Author(s):  
Frank C. Brown ◽  
Carmel H. Schindelhaim ◽  
Suzanne R. Pfeffer
Keyword(s):  
Coiled Coil ◽  
Retrograde Transport ◽  
Vesicle Tethering ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Transport Vesicle ◽  
Golgi Structure ◽  
Clathrin Adaptor ◽  
Golgi Network

GCC185 is a long coiled-coil protein localized to the trans-Golgi network (TGN) that functions in maintaining Golgi structure and tethering mannose 6-phosphate receptor (MPR)–containing transport vesicles en route to the Golgi. We report the identification of two distinct domains of GCC185 needed either for Golgi structure maintenance or transport vesicle tethering, demonstrating the independence of these two functions. The domain needed for vesicle tethering binds to the clathrin adaptor AP-1, and cells depleted of GCC185 accumulate MPRs in transport vesicles that are AP-1 decorated. This study supports a previously proposed role of AP-1 in retrograde transport of MPRs from late endosomes to the Golgi and indicates that docking may involve the interaction of vesicle-associated AP-1 protein with the TGN-associated tethering protein GCC185.

scholarly journals The yeast p24 complex is required for the formation of COPI retrograde transport vesicles from the Golgi apparatus

2008 ◽  
Vol 180 (4) ◽  
pp. 713-720 ◽  
Author(s):  
Auxiliadora Aguilera-Romero ◽  
Joanna Kaminska ◽  
Anne Spang ◽  
Howard Riezman ◽  
Manuel Muñiz
Keyword(s):  
Golgi Apparatus ◽  
Direct Evidence ◽  
Family Members ◽  
Transmembrane Proteins ◽  
Retrograde Transport ◽  
Active Role ◽  
Small Gtpase ◽  
Vesicle Formation ◽  
Transport Vesicles ◽  
Cargo Proteins

The p24 family members are transmembrane proteins assembled into heteromeric complexes that continuously cycle between the ER and the Golgi apparatus. These cargo proteins were assumed to play a structural role in COPI budding because of their major presence in mammalian COPI vesicles. However, this putative function has not been proved conclusively so far. Furthermore, deletion of all eight yeast p24 family members does not produce severe transport phenotypes, suggesting that the p24 complex is not essential for COPI function. In this paper we provide direct evidence that the yeast p24 complex plays an active role in retrograde transport from Golgi to ER by facilitating the formation of COPI-coated vesicles. Therefore, our results demonstrate that p24 proteins are important for vesicle formation instead of simply being a passive traveler, supporting the model in which cargo together with a small GTPase of the ARF superfamily and coat subunits act as primer for vesicle formation.

Illuminating the secretory pathway: when do we need vesicles?

2001 ◽  
Vol 114 (6) ◽  
pp. 1053-1059 ◽  
Author(s):  
D.J. Stephens ◽  
R. Pepperkok
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Retrograde Transport ◽  
Time Lapse ◽  
Membrane Structures ◽  
Long Distance ◽  
Transport Pathways ◽  
Transport Vesicles ◽  
Range Transport

Recent studies using GFP-tagged markers and time-lapse microscopy have allowed direct visualisation of membrane traffic in the secretory pathway in living mammalian cells. This work shows that larger membrane structures, 300–500 nm in size, are the vehicles responsible for long distance, microtubule-dependent ER-to-Golgi and trans-Golgi to plasma membrane transport of secretory markers. At least two retrograde transport pathways from the Golgi to the ER exist, both of which are proposed to involve a further class of long, tubular membrane carrier that forms from the Golgi and fuses with the ER. Together, this has challenged established transport models, raising the question of whether larger pleiomorphic structures, rather than small 60–80 nm transport vesicles, mediate long-range transport between the ER and Golgi and between the Golgi and plasma membrane. http://www.biologists.com/JCS/movies/jcs2220.html

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 FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network

2010 ◽  
Vol 21 (17) ◽  
pp. 3041-3053 ◽  
Author(s):  
Jian Jing ◽  
Jagath R. Junutula ◽  
Christine Wu ◽  
Jemima Burden ◽  
Hugo Matern ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Binding Protein ◽  
Retrograde Transport ◽  
Recycling Endosome ◽  
Transport Pathways ◽  
Recycling Endosomes ◽  
Trans Golgi Network ◽  
C Terminus ◽  
Transport Vesicles ◽  
Golgi Network

Many proteins are retrieved to the trans-Golgi Network (TGN) from the endosomal system through several retrograde transport pathways to maintain the composition and function of the TGN. However, the molecular mechanisms involved in these distinct retrograde pathways remain to be fully understood. Here we have used fluorescence and electron microscopy as well as various functional transport assays to show that Rab11a/b and its binding protein FIP1/RCP are both required for the retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN, but not for the retrieval of mannose-6-phosphate receptor from late endosomes. Furthermore, by proteomic analysis we identified Golgin-97 as a FIP1/RCP-binding protein. The FIP1/RCP-binding domain maps to the C-terminus of Golgin-97, adjacent to its GRIP domain. Binding of FIP1/RCP to Golgin-97 does not affect Golgin-97 recruitment to the TGN, but appears to regulate the targeting of retrograde transport vesicles to the TGN. Thus, we propose that FIP1/RCP binding to Golgin-97 is required for tethering and fusion of recycling endosome-derived retrograde transport vesicles to the TGN.

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

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