scholarly journals Di-Leucine Signals Mediate Targeting of Tyrosinase and Synaptotagmin to Synaptic-like Microvesicles within PC12 Cells

1999 ◽  
Vol 10 (11) ◽  
pp. 3979-3990 ◽  
Author(s):  
Anastasiya D. Blagoveshchenskaya ◽  
Eric W. Hewitt ◽  
Daniel F. Cutler
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Protein Traffic ◽  
C Terminus ◽  
Targeting Signal

One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRP-tyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents.

scholarly journals Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner

2012 ◽  
Vol 23 (11) ◽  
pp. 2156-2169 ◽  
Author(s):  
Eloy Bejarano ◽  
Henrique Girao ◽  
Andrea Yuste ◽  
Bindi Patel ◽  
Carla Marques ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Connexin 43 ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Sequence Of Events ◽  
Functional Consequences ◽  
Autophagic Degradation ◽  
The One

Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins.

Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane

2013 ◽  
Vol 450 (1) ◽  
pp. 73-83 ◽  
Author(s):  
Sonja Huser ◽  
Gregor Suri ◽  
Pascal Crottet ◽  
Martin Spiess
Keyword(s):  
Plasma Membrane ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Trans Golgi Network ◽  
Src Homology 3 ◽  
Amphiphysin 1 ◽  
Src Homology ◽  
Golgi Network

The assembly of clathrin/AP (adaptor protein)-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than that of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, the association of AP-1 with protein-free liposomes had been shown to require phosphoinositides, Arf1 (ADP-ribosylation factor 1)–GTP and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed that AP-1 stabilization on liposomes depends on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 (Src homology 3) domain, but requires interaction of the WDLW motif with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines co-localize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on interaction of clathrin and the adaptor sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by co-immunoprecipitation after cross-linking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network.

scholarly journals A novel Netrin-1–sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching

2014 ◽  
Vol 205 (2) ◽  
pp. 217-232 ◽  
Author(s):  
Cortney C. Winkle ◽  
Leslie M. McClain ◽  
Juli G. Valtschanoff ◽  
Charles S. Park ◽  
Christopher Maglione ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cortical Neurons ◽  
Direct Interaction ◽  
Vesicle Fusion ◽  
Dependent Manner ◽  
Axon Branching ◽  
Spatial Regulation ◽  
Novel Model

Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1–sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.

scholarly journals In Vitro Formation of Recycling Vesicles from Endosomes Requires Adaptor Protein-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5

2004 ◽  
Vol 15 (11) ◽  
pp. 4990-5000 ◽  
Author(s):  
Adriana Pagano ◽  
Pascal Crottet ◽  
Cristina Prescianotto-Baschong ◽  
Martin Spiess
Keyword(s):  
Brefeldin A ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Vesicle Formation ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Vitro Assay ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and γ-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.

scholarly journals Brefeldin A-dependent Membrane Tubule Formation Reconstituted In Vitro Is Driven by a Cell Cycle–regulated Microtubule Motor

2000 ◽  
Vol 11 (3) ◽  
pp. 941-955 ◽  
Author(s):  
Alasdair M. Robertson ◽  
Victoria J. Allan
Keyword(s):  
Cell Cycle ◽  
Cultured Cells ◽  
Brefeldin A ◽  
Binding Activity ◽  
Membrane Dynamics ◽  
Dependent Manner ◽  
Tubule Formation ◽  
Early Endosomes

Treatment of cultured cells with brefeldin A (BFA) induces the formation of extensive membrane tubules from the Golgi apparatus,trans-Golgi network, and early endosomes in a microtubule-dependent manner. We have reconstituted this transport process in vitro using Xenopus egg cytosol and a rat liver Golgi-enriched membrane fraction. The presence of BFA results in the formation of an intricate, interconnected tubular membrane network, a process that, as in vivo, is inhibited by nocodazole, the H1 anti-kinesin monoclonal antibody, and by membrane pretreatment with guanosine 5′-O-(3-thiotriphosphate). Surprisingly, membrane tubule formation is not due to the action of conventional kinesin or any of the other motors implicated in Golgi membrane dynamics. Two candidate motors of ∼100 and ∼130 kDa have been identified using the H1 antibody, both of which exhibit motor properties in a biochemical assay. Finally, BFA-induced membrane tubule formation does not occur in metaphase cytosol, and because membrane binding of both candidate motors is not altered after incubation in metaphase compared with interphase cytosol, these results suggest that either the ATPase or microtubule-binding activity of the relevant motor is cell cycle regulated.

scholarly journals Identification of PCIF1, a POZ Domain Protein That Inhibits PDX-1 (MODY4) Transcriptional Activity

2004 ◽  
Vol 24 (10) ◽  
pp. 4372-4383 ◽  
Author(s):  
Aihua Liu ◽  
Biva M. Desai ◽  
Doris A. Stoffers
Keyword(s):  
Late Onset ◽  
Dependent Manner ◽  
Gene Promoters ◽  
Transactivation Domain ◽  
Β Cells ◽  
Β Cell ◽  
C Terminus ◽  
Domain Protein

ABSTRACT Hox factors are evolutionarily conserved homeodomain-containing transcription factors that activate and repress gene expression in a precise temporally and spatially regulated manner during development and differentiation. Pancreatic-duodenal homeobox 1 (PDX-1) is a Hox-type protein that is a critical requirement for normal pancreas development and for proper differentiation of the endocrine pancreas. In humans, PDX-1 gene mutation causes pancreatic agenesis and early- and late-onset type 2 diabetes. PDX-1 consists of an N-terminal transactivation domain, a homeodomain responsible for DNA binding and nuclear localization, and a conserved C terminus that is mutated in human diabetes but whose function is poorly understood. We have identified a novel POZ domain protein, PDX-1 C terminus-interacting factor 1 (PCIF1)/SPOP, that interacts with PDX-1 both in vitro and in vivo. PCIF1 is localized to the nucleus in a speckled pattern, and coexpression of PDX-1 alters the subnuclear distribution of PCIF1. Functionally, PCIF1 inhibits PDX-1 transactivation of established target gene promoters in a specific and dose-dependent manner that requires critical amino acids in the PDX-1 C terminus. PCIF1 is expressed in adult pancreatic insulin-producing β cells, and overexpression of PCIF1 inhibits the rat insulin 1 and rat insulin 2 promoters in the MIN6 insulinoma β cell line. The coexpression of PCIF1 with PDX-1 in β cells and the ability of PCIF1 to repress PDX-1 transactivation suggest that modulation of PDX-1 function by PCIF1 may regulate normal β cell differentiation.

scholarly journals Fat-regulated adaptor protein Dlish binds the growth suppressor Expanded and controls its stability and ubiquitination

2019 ◽  
Vol 116 (4) ◽  
pp. 1319-1324 ◽  
Author(s):  
Xing Wang ◽  
Yifei Zhang ◽  
Seth S. Blair
Keyword(s):  
Ubiquitin Ligase ◽  
Hippo Pathway ◽  
E3 Ubiquitin Ligase ◽  
Growth Control ◽  
Adaptor Protein ◽  
Domain Growth ◽  
C Terminus ◽  
The Hippo Pathway

The Drosophila protocadherin Fat controls organ size through the Hippo pathway, but the biochemical links to the Hippo pathway components are still poorly defined. We previously identified Dlish, an SH3 domain protein that physically interacts with Fat and the type XX myosin Dachs, and showed that Fat’s regulation of Dlish levels and activity helps limit Dachs-mediated inhibition of Hippo pathway activity. We here characterize a parallel growth control pathway downstream of Fat and Dlish. Using immunoprecipitation and mass spectrometry to search for Dlish partners, we find that Dlish binds the FERM domain growth repressor Expanded (Ex); Dlish SH3 domains directly bind sites in the Ex C terminus. We further show that, in vivo, Dlish reduces the subapical accumulation of Ex, and that loss of Dlish blocks the destabilization of Ex caused by loss of Fat. Moreover, Dlish can bind the F-box E3 ubiquitin ligase Slimb and promote Slimb-mediated ubiquitination of Expanded in vitro. Both the in vitro and in vivo effects of Dlish on Ex require Slimb, strongly suggesting that Dlish destabilizes Ex by helping recruit Slimb-containing E3 ubiquitin ligase complexes to Ex.

scholarly journals Neuroendocrine Synaptic Vesicles Are Formed In Vitro by Both Clathrin-dependent and Clathrin-independent Pathways

1998 ◽  
Vol 143 (4) ◽  
pp. 947-955 ◽  
Author(s):  
Gongyi Shi ◽  
Victor Faúndez ◽  
Jack Roos ◽  
Esteban C. Dell'Angelica ◽  
Regis B. Kelly
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Synaptic Vesicles ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Interacting Proteins ◽  
Small Molecular Weight ◽  
Adp Ribosylation Factor ◽  
Dependent Pathway

In the neuroendocrine cell line, PC12, synaptic vesicles can be generated from endosomes by a sorting and vesiculation process that requires the heterotetrameric adaptor protein AP3 and a small molecular weight GTPase of the ADP ribosylation factor (ARF) family. We have now discovered a second pathway that sorts the synaptic vesicle-associated membrane protein (VAMP) into similarly sized vesicles. For this pathway the plasma membrane is the precursor rather than endosomes. Both pathways require cytosol and ATP and are inhibited by GTPγS. The second pathway, however, uses AP2 instead of AP3 and is brefeldin A insensitive. The AP2-dependent pathway is inhibited by depletion of clathrin or by inhibitors of clathrin binding, whereas the AP3 pathway is not. The VAMP-containing, plasma membrane–derived vesicles can be readily separated on sucrose gradients from transferrin (Tf)-containing vesicles generated by incubating Tf-labeled plasma membrane preparations at 37°C. Dynamin- interacting proteins are required for the AP2-mediated vesiculation from the plasma membrane, but not from endosomes. Thus, VAMP is sorted into small vesicles by AP3 and ARF1 at endosomes and by AP2 and clathrin at the plasma membrane.

scholarly journals A Complex Web of Signal-dependent Trafficking Underlies the Triorganellar Distribution of P-Selectin in Neuroendocrine PC12 Cells

1999 ◽  
Vol 145 (7) ◽  
pp. 1419-1433 ◽  
Author(s):  
Anastasiya D. Blagoveshchenskaya ◽  
Eric W. Hewitt ◽  
Daniel F. Cutler
Keyword(s):  
Plasma Membrane ◽  
Horseradish Peroxidase ◽  
Pc12 Cells ◽  
Brefeldin A ◽  
Dense Core ◽  
Concomitant Increase ◽  
Targeting Signal ◽  
Lysosomal Targeting ◽  
Dense Core Granules ◽  
Lumenal Domain

By analyzing the trafficking of HRP–P-selectin chimeras in which the lumenal domain of P-selectin was replaced with horseradish peroxidase, we determined the sequences needed for targeting to synaptic-like microvesicles (SLMV), dense core granules (DCG), and lysosomes in neuroendocrine PC12 cells. Within the cytoplasmic domain of P-selectin, Tyr777 is needed for the appearance of P-selectin in immature and mature DCG, as well as for targeting to SLMV. The latter destination also requires additional sequences (Leu768 and 786DPSP789) which are responsible for movement through endosomes en route to the SLMV. Leu768 also mediates transfer from early transferrin (Trn)-positive endosomes to the lysosomes; i.e., operates as a lysosomal targeting signal. Furthermore, SLMV targeting of HRP–P-selectin chimeras, but not the endogenous SLMV protein synaptophysin/p38, previously shown to be delivered to SLMV directly from the plasma membrane, is a Brefeldin A–sensitive process. Together, these data are consistent with a model of SLMV biogenesis which involves an endosomal intermediate in PC12 cells. In addition, we have discovered that impairment of SLMV or DCG targeting results in a concomitant increase in lysosomal delivery, illustrating the entwined relationships between routes leading to regulated secretory organelles (RSO) and to lysosomes.

scholarly journals VEGFR2 induces c-Src signaling and vascular permeability in vivo via the adaptor protein TSAd

2012 ◽  
Vol 209 (7) ◽  
pp. 1363-1377 ◽  
Author(s):  
Zuyue Sun ◽  
Xiujuan Li ◽  
Sara Massena ◽  
Simone Kutschera ◽  
Narendra Padhan ◽  
...  
Keyword(s):  
Vascular Permeability ◽  
Adaptor Protein ◽  
Cell Junctions ◽  
Vegf Receptor ◽  
Dependent Manner ◽  
Src Tyrosine Kinase ◽  
Rous Sarcoma ◽  
Src Homology 2 Domain

Regulation of vascular endothelial (VE) growth factor (VEGF)–induced permeability is critical in physiological and pathological processes. We show that tyrosine phosphorylation of VEGF receptor 2 (VEGFR2) at Y951 facilitates binding of VEGFR2 to the Rous sarcoma (Src) homology 2-domain of T cell–specific adaptor (TSAd), which in turn regulates VEGF-induced activation of the c-Src tyrosine kinase and vascular permeability. c-Src was activated in vivo and in vitro in a VEGF/TSAd-dependent manner, and was regulated via increased phosphorylation at pY418 and reduced phosphorylation at pY527. Tsad silencing blocked VEGF-induced c-Src activation, but did not affect pathways involving phospholipase Cγ, extracellular regulated kinase, and endothelial nitric oxide. VEGF-induced rearrangement of VE–cadherin–positive junctions in endothelial cells isolated from mouse lungs, or in mouse cremaster vessels, was dependent on TSAd expression, and TSAd formed a complex with VE-cadherin, VEGFR2, and c-Src at endothelial junctions. Vessels in tsad−/− mice showed undisturbed flow and pressure, but impaired VEGF-induced permeability, as measured by extravasation of Evans blue, dextran, and microspheres in the skin and the trachea. Histamine-induced extravasation was not affected by TSAd deficiency. We conclude that TSAd is required for VEGF-induced, c-Src-mediated regulation of endothelial cell junctions and for vascular permeability.

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