A novel adaptor-related protein complex.

Coat proteins are required for the budding of the transport vesicles that mediate membrane traffic pathways, but for many pathways such proteins pathways, but for many pathways such proteins have not yet been identified. We have raised antibodies against p47, a homologue of the medium chains of the adaptor complexes of clathrin-coated vesicles (Pevsner, J., W. Volknandt, B.R. Wong, and R.H. Scheller. 1994. Gene (Amst.). 146:279-283), to determine whether this protein might be a component of a new type of coat. p47 coimmunoprecipitates with three other proteins: two unknown proteins of 160 and 25 kD, and beta-NAP, a homologue of the beta/beta'-adaptins, indicating that it is a subunit of an adaptor-like heterotetrameric complex. However, p47 is not enriched in preparations of clathrin-coated vesicles. Recruitment of the p47-containing complex onto cell membranes is stimulated by GTP gamma S and blocked by brefeldin A, indicating that, like other coat proteins, its membrane association is regulated by an ARF. The newly recruited complex is localized to non-clathrin-coated buds and vesicles associated with the TGN. Endogenous complex in primary cultures of neuronal cells is also localized to the TGN, and in addition, some complex is associated with the plasma membrane. These results indicate that the complex is a component of a novel type of coat that facilitates the budding of vesicles from the TGN, possibly for transporting newly synthesized proteins to the plasma membrane.

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ADP-ribosylation factor and coatomer couple fusion to vesicle budding

The Journal of Cell Biology ◽

10.1083/jcb.124.4.415 ◽

1994 ◽

Vol 124 (4) ◽

pp. 415-424 ◽

Cited By ~ 77

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.

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Evidence for endocytosis of ROMK potassium channel via clathrin-coated vesicles

AJP Renal Physiology ◽

10.1152/ajprenal.00378.2001 ◽

2002 ◽

Vol 283 (4) ◽

pp. F630-F639 ◽

Cited By ~ 52

Author(s):

Wei-Zhong Zeng ◽

Victor Babich ◽

Bernardo Ortega ◽

Raymond Quigley ◽

Stanley J. White ◽

...

Keyword(s):

Brefeldin A ◽

Fluorescent Imaging ◽

Coated Vesicles ◽

Dominant Negative Mutant ◽

Kidney Cells ◽

Coat Proteins ◽

Madin Darby Canine Kidney ◽

Collecting Ducts ◽

Darby Canine Kidney ◽

Canine Kidney

ROMK channels are present in the cortical collecting ducts of kidney and are responsible for K+secretion in this nephron segment. Recent studies suggest that endocytosis of ROMK channels is important for regulation of K+ secretion in cortical collecting ducts. We investigated the molecular mechanisms for endocytosis of ROMK channels expressed in Xenopus laevis oocytes and cultured Madin-Darby canine kidney cells. When plasma membrane insertion of newly synthesized channel proteins was blocked by incubation with brefeldin A, ROMK currents decreased with a half-time of ∼6 h. Coexpression with the Lys44→Ala dominant-negative mutant dynamin, but not wild-type dynamin, reduced the rate of reduction of ROMK in the presence of brefeldin A. Mutation of Asn371 to Ile in the putative NPXY internalization motif of ROMK1 abolished the effect of the Lys44→Ala dynamin mutant on endocytosis of the channel. Coimmunoprecipitation study and confocal fluorescent imaging revealed that ROMK channels associated with clathrin coat proteins in Madin-Darby canine kidney cells. These results provide compelling evidence for endocytosis of ROMK channels via clathrin-coated vesicles.

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A novel class of clathrin-coated vesicles budding from endosomes.

The Journal of Cell Biology ◽

10.1083/jcb.132.1.21 ◽

1996 ◽

Vol 132 (1) ◽

pp. 21-33 ◽

Cited By ~ 258

Author(s):

W Stoorvogel ◽

V Oorschot ◽

H J Geuze

Keyword(s):

Plasma Membrane ◽

Microscopic Examination ◽

Transferrin Receptor ◽

Brefeldin A ◽

Coated Vesicles ◽

Integral Membrane Proteins ◽

Transferrin Receptors ◽

Receptor Recycling ◽

Coated Pits ◽

Novel Technique

Clathrin-coated vesicles transport selective integral membrane proteins from the plasma membrane to endosomes and from the TGN to endosomes. Recycling of proteins from endosomes to the plasma membrane occurs via unidentified vesicles. To study this pathway, we used a novel technique that allows for the immunoelectron microscopic examination of transferrin receptor-containing endosomes in nonsectioned cells. Endosomes were identified as separate discontinuous tubular-vesicular entities. Each endosome was decorated, mainly on the tubules, with many clathrin-coated buds. Endosome-associated clathrin-coated buds were discerned from plasma membrane-derived clathrin-coated vesicles by three criteria: size (60 nm and 100 nm, respectively), continuity with endosomes, and the lack of labeling for alpha-adaptin. They were also distinguished from TGN-derived clathrin-coated vesicles by their location at the periphery of the cell, size, and the lack of labeling for gamma-adaptin. In the presence of brefeldin A, a large continuous endosomal network was formed. Transferrin receptor recycling as well as the formation of clathrin-coated pits at endosomes was inhibited in the presence of brefeldin A. Together with the localization of transferrin receptors at endosome-associated buds, this indicates that a novel class of clathrin-coated vesicles serves an exit pathway from endosomes. The target organelles for endosome-derived clathrin-coated vesicles remain, however, to be identified.

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ADP-Ribosylation Factor 1 (ARF1) Regulates Recruitment of the AP-3 Adaptor Complex to Membranes

The Journal of Cell Biology ◽

10.1083/jcb.142.2.391 ◽

1998 ◽

Vol 142 (2) ◽

pp. 391-402 ◽

Cited By ~ 145

Author(s):

Chean Eng Ooi ◽

Esteban C. Dell'Angelica ◽

Juan S. Bonifacino

Keyword(s):

Membrane Trafficking ◽

Brefeldin A ◽

Membrane Association ◽

Coat Proteins ◽

Nucleotide Exchange ◽

Membrane Recruitment ◽

Adp Ribosylation ◽

Adp Ribosylation Factor

Small GTP-binding proteins such as ADP- ribosylation factor 1 (ARF1) and Sar1p regulate the membrane association of coat proteins involved in intracellular membrane trafficking. ARF1 controls the clathrin coat adaptor AP-1 and the nonclathrin coat COPI, whereas Sar1p controls the nonclathrin coat COPII. In this study, we demonstrate that membrane association of the recently described AP-3 adaptor is regulated by ARF1. Association of AP-3 with membranes in vitro was enhanced by GTPγS and inhibited by brefeldin A (BFA), an inhibitor of ARF1 guanine nucleotide exchange. In addition, recombinant myristoylated ARF1 promoted association of AP-3 with membranes. The role of ARF1 in vivo was examined by assessing AP-3 subcellular localization when the intracellular level of ARF1-GTP was altered through overexpression of dominant ARF1 mutants or ARF1- GTPase-activating protein (GAP). Lowering ARF1-GTP levels resulted in redistribution of AP-3 from punctate membrane-bound structures to the cytosol as seen by immunofluorescence microscopy. In contrast, increasing ARF1-GTP levels prevented redistribution of AP-3 to the cytosol induced by BFA or energy depletion. Similar experiments with mutants of ARF5 and ARF6 showed that these other ARF family members had little or no effect on AP-3. Taken together, our results indicate that membrane recruitment of AP-3 is promoted by ARF1-GTP. This finding suggests that ARF1 is not a regulator of specific coat proteins, but rather is a ubiquitous molecular switch that acts as a transducer of diverse signals influencing coat assembly.

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Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles

The Journal of Cell Biology ◽

10.1083/jcb.201111049 ◽

2012 ◽

Vol 197 (1) ◽

pp. 141-160 ◽

Cited By ~ 107

Author(s):

Georg H.H. Borner ◽

Robin Antrobus ◽

Jennifer Hirst ◽

Gary S. Bhumbra ◽

Patrycja Kozik ◽

...

Keyword(s):

Mass Spectrometry ◽

Isotope Labeling ◽

Principal Component ◽

Coated Vesicles ◽

Coated Vesicle ◽

Coat Proteins ◽

Proteomic Profiling ◽

Transport Vesicles ◽

Associated Proteins ◽

Sirna Knockdown

Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a “profiling” cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions.

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Disruption of endoplasmic reticulum to Golgi transport leads to the accumulation of large aggregates containing beta-COP in pancreatic acinar cells.

Molecular Biology of the Cell ◽

10.1091/mbc.4.4.413 ◽

1993 ◽

Vol 4 (4) ◽

pp. 413-424 ◽

Cited By ~ 24

Author(s):

L C Hendricks ◽

M McCaffery ◽

G E Palade ◽

M G Farquhar

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Brefeldin A ◽

Acinar Cells ◽

Atp Depletion ◽

Pancreatic Acinar Cells ◽

Coat Proteins ◽

Golgi Membranes ◽

Golgi Transport ◽

Transport Vesicles

When transport between the rough endoplasmic reticulum (ER) and Golgi complex is blocked by Brefeldin A (BFA) treatment or ATP depletion, the Golgi apparatus and associated transport vesicles undergo a dramatic reorganization. Because recent studies suggest that coat proteins such as beta-COP play an important role in the maintenance of the Golgi complex, we have used immunocytochemistry to determine the distribution of beta-COP in pancreatic acinar cells (PAC) in which ER to Golgi transport was blocked by BFA treatment or ATP depletion. In controls, beta-COP was associated with Golgi cisternae and transport vesicles as expected. Upon BFA treatment, PAC Golgi cisternae are dismantled and replaced by clusters of remnant vesicles surrounded by typical ER transitional elements that are generally assumed to represent the exit site of vesicular carriers for ER to Golgi transport. In BFA-treated PAC, beta-COP was concentrated in large (0.5-1.0 micron) aggregates closely associated with remnant Golgi membranes. In addition to typical ER transitional elements, we detected a new type of transitional element that consists of specialized regions of rough ER (RER) with ribosome-free ends that touched or extended into the beta-COP containing aggregates. In ATP-depleted PAC, beta-COP was not detected on Golgi membranes but was concentrated in similar large aggregates found on the cis side of the Golgi stacks. The data indicate that upon arrest of ER to Golgi transport by either BFA treatment or energy depletion, beta-COP dissociates from PAC Golgi membranes and accumulates as large aggregates closely associated with specialized ER elements. The latter may correspond to either the site of entry or exit for vesicles recycling between the Golgi and the RER.

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Targeting and mistargeting of plasma membrane adaptors in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.123.5.1093 ◽

1993 ◽

Vol 123 (5) ◽

pp. 1093-1105 ◽

Cited By ~ 61

Author(s):

M N Seaman ◽

C L Ball ◽

M S Robinson

Keyword(s):

Plasma Membrane ◽

Brefeldin A ◽

Coated Vesicle ◽

Double Labeling ◽

In Vitro System ◽

Specific Antibodies ◽

Excess Calcium ◽

Gamma S ◽

Intracellular Sequestration

Targeting and recruitment of the plasma membrane (PM) clathrin-coated vesicle adaptor complexes has been studied using an in vitro system based on permeabilized acceptor cells and donor cytosol. Through the use of species- and/or tissue-specific antibodies, only newly recruited exogenous PM adaptors are visualized. Targeting of PM adaptors can be switched from the plasma membrane to a perinuclear compartment by GTP gamma S or excess calcium. Prior treatment with brefeldin A prevents GTP gamma S-induced mistargeting. Double-labeling immunofluorescence and immunogold EM indicate that the perinuclear PM adaptor binding compartment is late endosomal. We propose that receptors for PM adaptors cycle between the plasma membrane and an endosomal storage compartment. Normally the receptors would be switched on only at the plasma membrane, but both GTP gamma S and calcium are capable of reversing this switch. Intracellular sequestration of PM adaptor receptors may provide the cell with a mechanism for up-regulating endocytosis following a burst of exocytosis.

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Dynamin-dependent Transferrin Receptor Recycling by Endosome-derived Clathrin-coated Vesicles

Molecular Biology of the Cell ◽

10.1091/mbc.01-07-0380 ◽

2002 ◽

Vol 13 (1) ◽

pp. 169-182 ◽

Cited By ~ 152

Author(s):

Ellen M. van Dam ◽

Willem Stoorvogel

Keyword(s):

Plasma Membrane ◽

Brefeldin A ◽

Fluid Phase ◽

Coated Vesicles ◽

Temperature Sensitive ◽

Dependent Manner ◽

Coated Pits ◽

Recycling Endosomes ◽

Early Endosomes ◽

Golgi Network

Previously we described clathrin-coated buds on tubular early endosomes that are distinct from those at the plasma membrane and the trans-Golgi network. Here we show that these clathrin-coated buds, like plasma membrane clathrin-coated pits, contain endogenous dynamin-2. To study the itinerary that is served by endosome-derived clathrin-coated vesicles, we used cells that overexpressed a temperature-sensitive mutant of dynamin-1 (dynamin-1G273D) or, as a control, dynamin-1 wild type. In dynamin-1G273D–expressing cells, 29–36% of endocytosed transferrin failed to recycle at the nonpermissive temperature and remained associated with tubular recycling endosomes. Sorting of endocytosed transferrin from fluid-phase endocytosed markers in early endosome antigen 1-labeled sorting endosomes was not inhibited. Dynamin-1G273D associated with accumulated clathrin-coated buds on extended tubular recycling endosomes. Brefeldin A interfered with the assembly of clathrin coats on endosomes and reduced the extent of transferrin recycling in control cells but did not further affect recycling by dynamin-1G273D–expressing cells. Together, these data indicate that the pathway from recycling endosomes to the plasma membrane is mediated, at least in part, by endosome-derived clathrin-coated vesicles in a dynamin-dependent manner.

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A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits.

The Journal of Cell Biology ◽

10.1083/jcb.125.6.1239 ◽

1994 ◽

Vol 125 (6) ◽

pp. 1239-1250 ◽

Cited By ~ 169

Author(s):

S L Shyng ◽

J E Heuser ◽

D A Harris

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Prion Protein ◽

Transmembrane Protein ◽

Primary Cultures ◽

Neuroblastoma Cells ◽

Cellular Prion Protein ◽

Coated Vesicles ◽

Coated Pits ◽

Hypertonic Medium

The cellular prion protein (PrPc) is a glycolipid-anchored, cell surface protein of unknown function, a posttranslationally modified isoform of which PrPSc is involved in the pathogenesis of Creutzfeldt-Jakob disease, scrapie, and other spongiform encephalopathies. We have shown previously that chPrP, a chicken homologue of mammalian PrPC, constitutively cycles between the cell surface and an endocytic compartment, with a transit time of approximately 60 min in cultured neuroblastoma cells. We now report that endocytosis of chPrP is mediated by clathrin-coated pits. Immunogold labeling of neuroblastoma cells demonstrates that the concentration of chPrP within 0.05 microns of coated pits is 3-5 times higher than over other areas of the plasma membrane. Moreover, gold particles can be seen within coated vesicles and deeply invaginated coated pits that are in the process of pinching off from the plasma membrane. ChPrP is also localized to coated pits in primary cultures of neurons and glia, and is found in coated vesicles purified from chicken brain. Finally, internalization of chPrP is reduced by 70% after neuroblastoma cells are incubated in hypertonic medium, a treatment that inhibits endocytosis by disrupting clathrin lattices. Caveolae, plasmalemmal invaginations in which several other glycolipid-anchored proteins are concentrated, are not seen in neuroblastoma cells analyzed by thin-section or deep-etch electron microscopy. Moreover, these cells do not express detectable levels of caveolin, a caveolar coat protein. Since chPrP lacks a cytoplasmic domain that could interact directly with the intracellular components of clathrin-coated pits, we propose that the polypeptide chain of chPrP associates with the extracellular domain of a transmembrane protein that contains a coated pit internalization signal.

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Trafficking of an Acylated Cytosolic Protein: Newly Synthesized p56lck Travels to the Plasma Membrane via the Exocytic Pathway

The Journal of Cell Biology ◽

10.1083/jcb.145.3.457 ◽

1999 ◽

Vol 145 (3) ◽

pp. 457-468 ◽

Cited By ~ 41

Author(s):

Marie-José J.E. Bijlmakers ◽

Mark Marsh

Keyword(s):

T Cells ◽

Plasma Membrane ◽

T Cell ◽

Cell Surface ◽

Brefeldin A ◽

Binding Kinetics ◽

Membrane Association ◽

Cytosolic Side ◽

Acylated Proteins ◽

Exocytic Pathway

The Src-related tyrosine kinase p56lck (Lck) is primarily expressed in T lymphocytes where it localizes to the cytosolic side of the plasma membrane and associates with the T cell coreceptors CD4 and CD8. As a model for acylated proteins, we studied how this localization of Lck is achieved. We followed newly synthesized Lck by pulse–chase analysis and found that membrane association of Lck starts soon after synthesis, but is not complete until at least 30–45 min later. Membrane-binding kinetics are similar in CD4/CD8-positive and CD4/CD8-negative cells. In CD4-positive T cells, the interaction with CD4 rapidly follows membrane association of Lck. Studying the route via which Lck travels from its site of synthesis to the plasma membrane, we found that: CD4 associates with Lck within 10 min of synthesis, long before CD4 has reached the plasma membrane; Lck associates with intracellular CD4 early after synthesis and with cell surface CD4 at later times; and transport of CD4-bound Lck to the plasma membrane is inhibited by Brefeldin A. These data indicate that the initial association of newly synthesized Lck with CD4, and therefore with membranes, occurs on intracellular membranes of the exocytic pathway. From this location Lck is transported to the plasma membrane.

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