scholarly journals Characterization of a Fourth Adaptor-related Protein Complex

1999 ◽  
Vol 10 (8) ◽  
pp. 2787-2802 ◽  
Author(s):  
Jennifer Hirst ◽  
Nicholas A. Bright ◽  
Brian Rous ◽  
Margaret S. Robinson
Keyword(s):  
Expressed Sequence Tag ◽  
Protein Complexes ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Cell Types ◽  
Small Gtpase ◽  
Immunogold Electron Microscopy ◽  
Coat Proteins ◽  
Cargo Proteins ◽  
Golgi Network

Adaptor protein complexes (APs) function as vesicle coat components in different membrane traffic pathways; however, there are a number of pathways for which there is still no candidate coat. To find novel coat components related to AP complexes, we have searched the expressed sequence tag database and have identified, cloned, and sequenced a new member of each of the four AP subunit families. We have shown by a combination of coimmunoprecipitation and yeast two-hybrid analysis that these four proteins (ε, β4, μ4, and ς4) are components of a novel adaptor-like heterotetrameric complex, which we are calling AP-4. Immunofluorescence reveals that AP-4 is localized to ∼10–20 discrete dots in the perinuclear region of the cell. This pattern is disrupted by treating the cells with brefeldin A, indicating that, like other coat proteins, the association of AP-4 with membranes is regulated by the small GTPase ARF. Immunogold electron microscopy indicates that AP-4 is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network. The μ4 subunit of the complex specifically interacts with a tyrosine-based sorting signal, indicating that, like the other three AP complexes, AP-4 is involved in the recognition and sorting of cargo proteins with tyrosine-based motifs. AP-4 is of relatively low abundance, but it is expressed ubiquitously, suggesting that it participates in a specialized trafficking pathway but one that is required in all cell types.

scholarly journals Intracellular Localization of Phospholipase D1 in Mammalian Cells

2001 ◽  
Vol 12 (4) ◽  
pp. 943-955 ◽  
Author(s):  
Zachary Freyberg ◽  
David Sweeney ◽  
Anirban Siddhanta ◽  
Sylvain Bourgoin ◽  
Michael Frohman ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Intracellular Localization ◽  
Brefeldin A ◽  
Cell Types ◽  
Immunogold Electron Microscopy ◽  
Cell Fractionation ◽  
Cell Nuclei ◽  
Phospholipase D1 ◽  
Golgi Network

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid. In mammalian cells this reaction has been implicated in the recruitment of coatomer to Golgi membranes and release of nascent secretory vesicles from the trans-Golgi network. These observations suggest that PLD is associated with the Golgi complex; however, to date, because of its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. We have used highly sensitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracellular localization of endogenous PLD1 in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched significantly in the Golgi apparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membrane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated from the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 resulted in displacement of the endogenous enzyme from its perinuclear localization to large vesicular structures. Surprisingly, when the Golgi apparatus collapsed in response to brefeldin A, the nuclear localization of PLD1 was enhanced significantly. Our data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.

scholarly journals An AP-3-dependent mechanism drives synaptic-like microvesicle biogenesis in pancreatic islet β-cells

2010 ◽  
Vol 299 (1) ◽  
pp. E23-E32 ◽  
Author(s):  
Arthur T. Suckow ◽  
Branch Craige ◽  
Victor Faundez ◽  
William J. Cain ◽  
Steven D. Chessler
Keyword(s):  
Synaptic Vesicle ◽  
Pancreatic Islet ◽  
Membrane Trafficking ◽  
Protein Complexes ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Β Cells ◽  
Β Cell ◽  
Subunit Expression ◽  
Bona Fide

Pancreatic islet β-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since β-cell maturation and exocytotic function seem to parallel that of the inhibitory synapse, we predicted that AP-3B-associated vesicles would be present in β-cells. Here, we test the hypothesis that AP-3B is expressed in islets and mediates β-cell SLMV biogenesis. A secondary aim was to test whether the sedimentation properties of INS-1 β-cell microvesicles are identical to those of bona fide SLMVs isolated from PC12 cells. Our results show that the two neuron-specific AP-3 subunits β3B and μ3B are expressed in β-cells, the first time these proteins have been found to be expressed outside the nervous system. We found that β-cell SLMVs share the same sedimentation properties as PC12 SLMVs and contain SV proteins that sort specifically to AP-3B-associated vesicles in the brain. Brefeldin A, a drug that interferes with AP-3-mediated SV biogenesis, inhibits the delivery of AP-3 cargoes to β-cell SLMVs. Consistent with a role for AP-3 in the biogenesis of GABAergic SLMV in β-cells, INS-1 cell VGAT content decreases upon inhibition of AP-3 δ-subunit expression. Our findings suggest that β-cells and neurons share molecules and mechanisms important for mediating the neuron-specific membrane trafficking pathways that underlie synaptic vesicle formation.

scholarly journals AP-4 vesicles contribute to spatial control of autophagy via RUSC-dependent peripheral delivery of ATG9A

2017 ◽  
Author(s):  
Alexandra K. Davies ◽  
Daniel N. Itzhak ◽  
James R. Edgar ◽  
Tara L. Archuleta ◽  
Jennifer Hirst ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Close Association ◽  
Adaptor Protein ◽  
Cell Types ◽  
Subcellular Localisation ◽  
Cell Periphery ◽  
Accessory Proteins ◽  
Unknown Aetiology ◽  
Spatial Control ◽  
Cargo Proteins

AbstractAdaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including ‘Dynamic Organellar Maps’, to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the TGN to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the “ATG9A reservoir” required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.

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.

scholarly journals Regulation of Clathrin-Mediated Endocytosis

2018 ◽  
Vol 87 (1) ◽  
pp. 871-896 ◽  
Author(s):  
Marcel Mettlen ◽  
Ping-Hung Chen ◽  
Saipraveen Srinivasan ◽  
Gaudenz Danuser ◽  
Sandra L. Schmid
Keyword(s):  
Conformational Changes ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Environmental Changes ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Endocytic Pathway ◽  
Coat Proteins ◽  
Membrane Composition ◽  
Coated Pits

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in mammalian cells. It is responsible for the uptake of transmembrane receptors and transporters, for remodeling plasma membrane composition in response to environmental changes, and for regulating cell surface signaling. CME occurs via the assembly and maturation of clathrin-coated pits that concentrate cargo as they invaginate and pinch off to form clathrin-coated vesicles. In addition to the major coat proteins, clathrin triskelia and adaptor protein complexes, CME requires a myriad of endocytic accessory proteins and phosphatidylinositol lipids. CME is regulated at multiple steps—initiation, cargo selection, maturation, and fission—and is monitored by an endocytic checkpoint that induces disassembly of defective pits. Regulation occurs via posttranslational modifications, allosteric conformational changes, and isoform and splice-variant differences among components of the CME machinery, including the GTPase dynamin. This review summarizes recent findings on the regulation of CME and the evolution of this complex process.

scholarly journals Adaptor protein 2–mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

2012 ◽  
Vol 23 (12) ◽  
pp. 2339-2351 ◽  
Author(s):  
Yogikala Prabhu ◽  
Patricia V. Burgos ◽  
Christina Schindler ◽  
Ginny G. Farías ◽  
Javier G. Magadán ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amyloid Precursor Protein ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Proteolytic Processing ◽  
Precursor Protein ◽  
Amyloid Precursor Protein Processing ◽  
Intermediate Compartment ◽  
Golgi Network

The β-site amyloid precursor protein (APP)–cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER–Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway.

scholarly journals Ent3p and Ent5p Exhibit Cargo-specific Functions in Trafficking Proteins between the Trans-Golgi Network and the Endosomes in Yeast

2007 ◽  
Vol 18 (5) ◽  
pp. 1803-1815 ◽  
Author(s):  
Alenka Čopič ◽  
Trevor L. Starr ◽  
Randy Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Surface ◽  
Protein Transport ◽  
Adaptor Protein ◽  
Additional Function ◽  
Trans Golgi Network ◽  
Cargo Proteins ◽  
Clathrin Adaptor ◽  
Dependent Transport ◽  
Golgi Network

The phosphoinositide-binding proteins Ent3p and Ent5p are required for protein transport from the trans-Golgi network (TGN) to the vacuole in Saccharomyces cerevisiae. Both proteins interact with the monomeric clathrin adaptor Gga2p, but Ent5p also interacts with the clathrin adaptor protein 1 (AP-1) complex, which facilitates retention of proteins such as Chs3p at the TGN. When both ENT3 and ENT5 are mutated, Chs3p is diverted from an intracellular reservoir to the cell surface. However, Ent3p and Ent5p are not required for the function of AP-1, but rather they seem to act in parallel with AP-1 to retain proteins such as Chs3p at the TGN. They have all the properties of clathrin adaptors, because they can both bind to clathrin and to cargo proteins. Like AP-1, Ent5p binds to Chs3p, whereas Ent3p facilitates the interaction between Gga2p and the endosomal syntaxin Pep12p. Thus, Ent3p has an additional function in Gga-dependent transport to the late endosome. Ent3p also facilitates the association between Gga2p and clathrin; however, Ent5p can partially substitute for this function. We conclude that the clathrin adaptors AP-1, Ent3p, Ent5p, and the Ggas cooperate in different ways to sort proteins between the TGN and the endosomes.

scholarly journals Commonly used trafficking blocks disrupt ARF1 activation and the localization and function of specific Golgi proteins

2018 ◽  
Vol 29 (8) ◽  
pp. 937-947 ◽  
Author(s):  
Catherine E. Gilbert ◽  
Elizabeth Sztul ◽  
Carolyn E. Machamer
Keyword(s):  
Protein Trafficking ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Small Gtpase ◽  
Cold Temperature ◽  
Nucleotide Exchange ◽  
Specific Subset ◽  
Nucleotide Exchange Factor ◽  
Cargo Proteins ◽  
And Function

ADP-ribosylation factor (ARF) proteins are key regulators of the secretory pathway. ARF1, through interacting with its effectors, regulates protein trafficking by facilitating numerous events at the Golgi. One unique ARF1 effector is golgin-160, which promotes the trafficking of only a specific subset of cargo proteins through the Golgi. While studying this role of golgin-160, we discovered that commonly used cold temperature blocks utilized to synchronize cargo trafficking (20 and 16°C) caused golgin-160 dispersal from Golgi membranes. Here, we show that the loss of golgin-160 localization correlates with a decrease in the levels of activated ARF1, and that golgin-160 dispersal can be prevented by expression of a GTP-locked ARF1 mutant. Overexpression of the ARF1 activator Golgi brefeldin A–resistant guanine nucleotide exchange factor 1 (GBF1) did not prevent golgin-160 dispersal, suggesting that GBF1 may be nonfunctional at lower temperatures. We further discovered that several other Golgi resident proteins had altered localization at lower temperatures, including proteins recruited by ARF-like GTPase 1 (ARL1), a small GTPase that also became dispersed in the cold. Although cold temperature blocks are useful for synchronizing cargo trafficking through the Golgi, our data indicate that caution must be taken when interpreting results from these assays.

scholarly journals Mechanism of Constitutive Export from the Golgi: Bulk Flow via the Formation, Protrusion, and En Bloc Cleavage of large trans-Golgi Network Tubular Domains

2003 ◽  
Vol 14 (11) ◽  
pp. 4470-4485 ◽  
Author(s):  
Elena V. Polishchuk ◽  
Alessio Di Pentima ◽  
Alberto Luini ◽  
Roman S. Polishchuk
Keyword(s):  
Three Dimensional ◽  
Bulk Flow ◽  
Dimensional Structure ◽  
Coat Proteins ◽  
En Bloc ◽  
Trans Golgi Network ◽  
Membrane Fission ◽  
Large Structures ◽  
Cargo Proteins ◽  
Golgi Network

Transport of constitutive cargo proteins from the Golgi complex to the plasma membrane (PM) is known to be mediated by large tubular-saccular carriers moving along microtubules. However, the process by which these large structures emerge from the trans-Golgi network (TGN) remains unclear. Here, we address the question of the formation of Golgi-to-PM carriers (GPCs) by using a suitable cluster of morphological techniques, providing an integrated view of their dynamics and three-dimensional structure. Our results indicate that exit from the TGN of a constitutive traffic marker, the VSVG protein, occurs by bulk flow and is a three-step process. First, the formation of a tubular-reticular TGN domain (GPC precursor) that includes PM-directed proteins and excludes other cargo and Golgi-resident proteins. Notably, this step does not require membrane fusion. Second, the docking of this preformed domain on microtubules and its kinesin-mediated extrusion. Finally, the detachment of the extruded domain by membrane fission. The formation of GPCs does not involve cargo concentration and is not associated with the presence of known coat proteins on GPC precursors. In summary, export from the Golgi occurs via the formation, protrusion and en bloc cleavage of specialized TGN tubular-saccular domains.

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