scholarly journals Vps10p Transport from the trans-Golgi Network to the Endosome Is Mediated by Clathrin-coated Vesicles

2001 ◽  
Vol 12 (2) ◽  
pp. 475-485 ◽  
Author(s):  
Olivier Deloche ◽  
Bonny G. Yeung ◽  
Gregory S. Payne ◽  
Randy Schekman
Keyword(s):  
Large Subunit ◽  
Adaptor Protein ◽  
Coated Vesicles ◽  
Carboxypeptidase Y ◽  
Yeast Saccharomyces Cerevisiae ◽  
Trans Golgi Network ◽  
Protein Receptor ◽  
Vacuolar Protein ◽  
Golgi Network

A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12Δ andvps34Δ, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10CtΔp mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.

scholarly journals Proteomic Characterization of Isolated Arabidopsis Clathrin-Coated Vesicles Reveals Evolutionarily Conserved and Plant Specific Components

2021 ◽  
Author(s):  
Dana A. Dahhan ◽  
Gregory D. Reynolds ◽  
Jessica J. Cárdenas ◽  
Dominique Eeckhout ◽  
Alexander Johnson ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Adaptor Protein ◽  
Coated Vesicles ◽  
Mass Spectrometry Analysis ◽  
Chemical Labeling ◽  
Spectrometry Analysis ◽  
Trans Golgi Network ◽  
Evolutionarily Conserved ◽  
Golgi Network

In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein AP-1 complex operates as part of the secretory pathway at the trans-Golgi network, while the AP-2 complex and the TPLATE complex (TPC) jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched trans-Golgi network/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

scholarly journals Acidic Di-leucine Motif Essential for AP-3–dependent Sorting and Restriction of the Functional Specificity of the Vam3p Vacuolar t-SNARE

1998 ◽  
Vol 142 (4) ◽  
pp. 913-922 ◽  
Author(s):  
Tamara Darsow ◽  
Christopher G. Burd ◽  
Scott D. Emr
Keyword(s):  
Protein Sorting ◽  
Adaptor Protein ◽  
Specific Protein ◽  
Carboxypeptidase Y ◽  
Yeast Saccharomyces Cerevisiae ◽  
Protein Protein Interaction ◽  
Sorting Signals ◽  
Interaction Domains ◽  
Dependent Transport ◽  
Vacuolar Protein

The transport of newly synthesized proteins through the vacuolar protein sorting pathway in the budding yeast Saccharomyces cerevisiae requires two distinct target SNAP receptor (t-SNARE) proteins, Pep12p and Vam3p. Pep12p is localized to the pre-vacuolar endosome and its activity is required for transport of proteins from the Golgi to the vacuole through a well defined route, the carboxypeptidase Y (CPY) pathway. Vam3p is localized to the vacuole where it mediates delivery of cargoes from both the CPY and the recently described alkaline phosphatase (ALP) pathways. Surprisingly, despite their organelle-specific functions in sorting of vacuolar proteins, overexpression of VAM3 can suppress the protein sorting defects of pep12Δ cells. Based on this observation, we developed a genetic screen to identify domains in Vam3p (e.g., localization and/or specific protein–protein interaction domains) that allow it to efficiently substitute for Pep12p. Using this screen, we identified mutations in a 7–amino acid sequence in Vam3p that lead to missorting of Vam3p from the ALP pathway into the CPY pathway where it can substitute for Pep12p at the pre-vacuolar endosome. This region contains an acidic di-leucine sequence that is closely related to sorting signals required for AP-3 adaptor–dependent transport in both yeast and mammalian systems. Furthermore, disruption of AP-3 function also results in the ability of wild-type Vam3p to compensate for pep12 mutants, suggesting that AP-3 mediates the sorting of Vam3p via the di-leucine signal. Together, these data provide the first identification of an adaptor protein–specific sorting signal in a t-SNARE protein, and suggest that AP-3–dependent sorting of Vam3p acts to restrict its interaction with compartment-specific accessory proteins, thereby regulating its function. Regulated transport of cargoes such as Vam3p through the AP-3–dependent pathway may play an important role in maintaining the unique composition, function, and morphology of the vacuole.

scholarly journals A Family of Proteins with γ-Adaptin and Vhs Domains That Facilitate Trafficking between the Trans-Golgi Network and the Vacuole/Lysosome

2000 ◽  
Vol 149 (1) ◽  
pp. 67-80 ◽  
Author(s):  
Jennifer Hirst ◽  
Winnie W.Y. Lui ◽  
Nicholas A. Bright ◽  
Nicholas Totty ◽  
Matthew N.J. Seaman ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Coated Vesicles ◽  
Carboxypeptidase Y ◽  
Golgi Stack ◽  
Trans Golgi Network ◽  
Terminal Domain ◽  
Vhs Domain ◽  
Golgi Network ◽  
Terminal Domains

We have cloned and characterized members of a novel family of proteins, the GGAs. These proteins contain an NH2-terminal VHS domain, one or two coiled-coil domains, and a COOH-terminal domain homologous to the COOH-terminal “ear” domain of γ-adaptin. However, unlike γ-adaptin, the GGAs are not associated with clathrin-coated vesicles or with any of the components of the AP-1 complex. GGA1 and GGA2 are also not associated with each other, although they colocalize on perinuclear membranes. Immunogold EM shows that these membranes correspond to trans elements of the Golgi stack and the TGN. GST pulldown experiments indicate that the GGA COOH-terminal domains bind to a subset of the proteins that bind to the γ-adaptin COOH-terminal domain. In yeast there are two GGA genes. Deleting both of these genes results in missorting of the vacuolar enzyme carboxypeptidase Y, and the cells also have a defective vacuolar morphology phenotype. These results indicate that the function of the GGAs is to facilitate the trafficking of proteins between the TGN and the vacuole, or its mammalian equivalent, the lysosome.

scholarly journals Synthetic Genetic Interactions With Temperature-Sensitive Clathrin in Saccharomyces cerevisiae: Roles for Synaptojanin-Like Inp53p and Dynamin-Related Vps1p in Clathrin-Dependent Protein Sorting at the trans-Golgi Network

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 83-97
Author(s):  
Eric S Bensen ◽  
Giancarlo Costaguta ◽  
Gregory S Payne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mechanisms ◽  
Protein Transport ◽  
Protein Sorting ◽  
Genetic Interactions ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Trans Golgi Network ◽  
Vacuolar Protein ◽  
Golgi Network

Abstract Clathrin is involved in selective protein transport at the Golgi apparatus and the plasma membrane. To further understand the molecular mechanisms underlying clathrin-mediated protein transport pathways, we initiated a genetic screen for mutations that display synthetic growth defects when combined with a temperature-sensitive allele of the clathrin heavy chain gene (chc1-521) in Saccharomyces cerevisiae. Mutations, when present in cells with wild-type clathrin, were analyzed for effects on mating pheromone α-factor precursor maturation and sorting of the vacuolar protein carboxypeptidase Y as measures of protein sorting at the yeast trans-Golgi network (TGN) compartment. By these criteria, two classes of mutants were obtained, those with and those without defects in protein sorting at the TGN. One mutant with unaltered protein sorting at the TGN contains a mutation in PTC1, a type 2c serine/threonine phosphatase with widespread influences. The collection of mutants displaying TGN sorting defects includes members with mutations in previously identified vacuolar protein sorting genes (VPS), including the dynamin family member VPS1. Striking genetic interactions were observed by combining temperature-sensitive alleles of CHC1 and VPS1, supporting the model that Vps1p is involved in clathrin-mediated vesicle formation at the TGN. Also in the spectrum of mutants with TGN sorting defects are isolates with mutations in the following: RIC1, encoding a product originally proposed to participate in ribosome biogenesis; LUV1, encoding a product potentially involved in vacuole and microtubule organization; and INP53, encoding a synaptojanin-like inositol polyphosphate 5-phosphatase. Disruption of INP53, but not the related INP51 and INP52 genes, resulted in α-factor maturation defects and exacerbated α-factor maturation defects when combined with chc1-521. Our findings implicate a wide variety of proteins in clathrin-dependent processes and provide evidence for the selective involvement of Inp53p in clathrin-mediated protein sorting at the TGN.

scholarly journals Golgi-localized, γ-Ear-containing, ADP-Ribosylation Factor-binding Proteins: Roles of the Different Domains and Comparison with AP-1 and Clathrin

2001 ◽  
Vol 12 (11) ◽  
pp. 3573-3588 ◽  
Author(s):  
Jennifer Hirst ◽  
Margaret R. Lindsay ◽  
Margaret S. Robinson
Keyword(s):  
Mammalian Cells ◽  
Binding Proteins ◽  
Coated Vesicles ◽  
Factor Binding ◽  
Adp Ribosylation ◽  
Trans Golgi Network ◽  
Vhs Domain ◽  
Vacuolar Protein ◽  
Golgi Network ◽  
Adp Ribosylation Factor

We have previously identified a novel family of proteins called the GGAs (Golgi-localized, γ-ear-containing, ADP-ribosylation factor-binding proteins). These proteins consist of an NH2-terminal VHS domain, followed by a GAT domain, a variable domain, and a γ-adaptin ear homology domain. Studies from our own laboratory and others, making use of both yeast and mammals cells, indicate that the GGAs facilitate trafficking from the trans-Golgi network to endosomes. Here we have further investigated the function of the GGAs. We find that GGA-deficient yeast are not only defective in vacuolar protein sorting but they are also impaired in their ability to process α-factor. Using deletion mutants and chimeras, we show that the VHS domain is required for GGA function and that the VHS domain from Vps27p will not substitute for the GGA VHS domain. In contrast, the γ-adaptin ear homology domain contributes to GGA function but is not absolutely required, and full function can be restored by replacing the GGA ear domain with the γ-adaptin ear domain. Deleting the γ-adaptin gene together with the twoGGA genes exacerbates the phenotype in yeast, suggesting that they function on parallel pathways. In mammalian cells, the association of GGAs with the membrane is extremely unstable, which may account for their absence from purified clathrin-coated vesicles. Double- and triple-labeling immunofluorescence experiments indicate that the GGAs and AP-1 are associated with distinct populations of clathrin-coated vesicles budding from the trans-Golgi network. Together with results from other studies, our findings suggest that the GGAs act as monomeric adaptors, with the four domains involved in cargo selection, membrane localization, clathrin binding, and accessory protein recruitment.

scholarly journals A High Copy Suppressor Screen Reveals Genetic Interactions Between BET3 and a New Gene: Evidence for a Novel Complex in ER-to-Golgi Transport

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 833-841
Author(s):  
Yu Jiang ◽  
Al Scarpa ◽  
Li Zhang ◽  
Shelly Stone ◽  
Ed Feliciano ◽  
...  
Keyword(s):  
Growth Defect ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Specific Suppression ◽  
Golgi Transport ◽  
New Gene ◽  
Insight Into ◽  
Suppressor Screen

Abstract The BET3 gene in the yeast Saccharomyces cerevisiae encodes a 22-kD hydrophilic protein that is required for vesicular transport between the ER and Golgi complex. To gain insight into the role of Bet3p, we screened for genes that suppress the growth defect of the temperature-sensitive bet3 mutant at 34°. This high copy suppressor screen resulted in the isolation of a new gene, called BET5. BET5 encodes an essential 18-kD hydrophilic protein that in high copy allows growth of the bet3-1 mutant, but not other ER accumulating mutants. This strong and specific suppression is consistent with the fact that Bet3p and Bet5p are members of the same complex. Using PCR mutagenesis, we generated a temperature-sensitive mutation in BET5 (bet5-1) that blocks the transport of carboxypeptidase Y to the vacuole and prevents secretion of the yeast pheromone α-factor at 37°. The precursor forms of these proteins that accumulate in this mutant are indicative of a block in membrane traffic between the ER and Golgi apparatus. High copy suppressors of the bet5-1 mutant include several genes whose products are required for ER-to-Golgi transport (BET1, SEC22, USO1 and DSS4) and the maintenance of the Golgi (ANP1). These findings support the hypothesis that Bet5p acts in conjunction with Bet3p to mediate a late stage in ER-to-Golgi transport. The identification of mammalian homologues of Bet3p and Bet5p implies that the Bet3p/Bet5p complex is highly conserved in evolution.

scholarly journals p230 is associated with vesicles budding from the trans-Golgi network

1996 ◽  
Vol 109 (12) ◽  
pp. 2811-2821 ◽  
Author(s):  
P.A. Gleeson ◽  
T.J. Anderson ◽  
J.L. Stow ◽  
G. Griffiths ◽  
B.H. Toh ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Immunogold Labeling ◽  
Coated Vesicles ◽  
Specific Marker ◽  
Very High Frequency ◽  
Trans Golgi Network ◽  
Golgi Membranes ◽  
Vesicle Biogenesis ◽  
Heptad Repeats ◽  
Golgi Network

Transport vesicle formation requires the association of cytosolic proteins with the membrane. We have previously described a brefeldin-A sensitive, hydrophilic protein (p230), containing a very high frequency of heptad repeats, found in the cytosol and associated with Golgi membranes. We show here that p230 is localised on the trans-Golgi network, by immunogold labeling of HeLa cell cryosections using alpha 2,6 sialyltransferase as a compartment-specific marker. The role of G protein activators on the binding of p230 to Golgi membranes and in vesicle biogenesis has been investigated. Treatment of streptolysin-O permeabilised HeLa cells with either GTP gamma S or AlF4- resulted in accumulation of p230 on Golgi membranes. Furthermore, immunolabeling of isolated Golgi membranes treated with AlF4-, to induce the accumulation of vesicles, showed that p230 is predominantly localised to the cytoplasmic surface of trans-Golgi network-derived budding structures and small coated vesicles. p230-labeled vesicles have a thin (approximately 10 nm) electron dense cytoplasmic coat and could be readily distinguished from clathrin-coated vesicles. Dual immunogold labeling of perforated cells, or of cryosections of treated Golgi membranes, revealed that p230 and the trans-Golgi network-associated p200, which we show here to be distinct molecules, appear to be localised on separate populations of vesicles budding from the trans-Golgi network. These results strongly suggest the presence of distinct populations of non-clathrin coated vesicles derived from the trans-Golgi network. As p230 recycles between the cytosol and buds/vesicles of TGN membranes, a process regulated by G proteins, we propose that p230 is involved in the biogenesis of a specific population of non-clathrin coated vesicles.

scholarly journals ATP- and Cytosol-dependent Release of Adaptor Proteins from Clathrin-coated Vesicles: A Dual Role for Hsc70

1998 ◽  
Vol 9 (8) ◽  
pp. 2217-2229 ◽  
Author(s):  
Lisa A. Hannan ◽  
Sherri L. Newmyer ◽  
Sandra L. Schmid
Keyword(s):  
Plasma Membrane ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Dual Role ◽  
Vesicular Trafficking ◽  
Coated Vesicles ◽  
Golgi Network ◽  
Cytosolic Factor

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.

Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules in living cells

1999 ◽  
Vol 112 (1) ◽  
pp. 21-33 ◽  
Author(s):  
D. Toomre ◽  
P. Keller ◽  
J. White ◽  
J.C. Olivo ◽  
K. Simons
Keyword(s):  
Plasma Membrane ◽  
Protein Trafficking ◽  
Membrane Traffic ◽  
Living Cells ◽  
Tubular Structures ◽  
Trans Golgi Network ◽  
Vesicular Stomatitis ◽  
Golgi Network ◽  
Color Visualization

The mechanisms and carriers responsible for exocytic protein trafficking between the trans-Golgi network (TGN) and the plasma membrane remain unclear. To investigate the dynamics of TGN-to-plasma membrane traffic and role of the cytoskeleton in these processes we transfected cells with a GFP-fusion protein, vesicular stomatitis virus G protein tagged with GFP (VSVG3-GFP). After using temperature shifts to block VSVG3-GFP in the endoplasmic reticulum and subsequently accumulate it in the TGN, dynamics of TGN-to-plasma membrane transport were visualized in real time by confocal and video microscopy. Both small vesicles (<250 nm) and larger vesicular-tubular structures (>1.5 microm long) are used as transport containers (TCs). These TCs rapidly moved out of the Golgi along curvilinear paths with average speeds of approximately 0.7 micrometer/second. Automatic computer tracking objectively determined the dynamics of different carriers. Fission and fusion of TCs were observed, suggesting that these late exocytic processes are highly interactive. To directly determine the role of microtubules in post-Golgi traffic, rhodamine-tubulin was microinjected and both labeled cargo and microtubules were simultaneously visualized in living cells. These studies demonstrated that exocytic cargo moves along microtubule tracks and reveals that carriers are capable of switching between tracks.

scholarly journals Polarization of myelinating Schwann cell surface membranes: role of microtubules and the trans-Golgi network

1995 ◽  
Vol 15 (3) ◽  
pp. 1797-1807 ◽  
Author(s):  
BD Trapp ◽  
GJ Kidd ◽  
P Hauer ◽  
E Mulrenin ◽  
CA Haney ◽  
...  
Keyword(s):  
Schwann Cell ◽  
Cell Surface ◽  
Trans Golgi Network ◽  
Myelinating Schwann Cell ◽  
Golgi Network
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