scholarly journals Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes

2011 ◽  
Vol 22 (12) ◽  
pp. 2068-2082 ◽  
Author(s):  
Karine Laulagnier ◽  
Nicole L. Schieber ◽  
Tanja Maritzen ◽  
Volker Haucke ◽  
Robert G. Parton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Cytosolic Calcium ◽  
Endocytic Pathway ◽  
Secretory Process ◽  
Microtubule Depolymerization ◽  
Lysosome Related Organelles ◽  
Endocytic Compartments

Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

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 Chicken Erythroid AE1 Anion Exchangers Associate with the Cytoskeleton During Recycling to the Golgi

1999 ◽  
Vol 10 (2) ◽  
pp. 455-469 ◽  
Author(s):  
Sourav Ghosh ◽  
Kathleen H. Cox ◽  
John V. Cox
Keyword(s):  
Plasma Membrane ◽  
Ammonium Chloride ◽  
Anion Exchanger ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Endocytic Pathway ◽  
Erythroid Cells ◽  
Anion Exchangers ◽  
Membrane Compartment ◽  
Chloride Treatment

Chicken erythroid AE1 anion exchangers receive endoglycosidase F (endo F)-sensitive sugar modifications in their initial transit through the secretory pathway. After delivery to the plasma membrane, anion exchangers are internalized and recycled to the Golgi where they acquire additional N-linked modifications that are resistant to endo F. During recycling, some of the anion exchangers become detergent insoluble. The acquisition of detergent insolubility correlates with the association of the anion exchanger with cytoskeletal ankyrin. Reagents that inhibit different steps in the endocytic pathway, including 0.4 M sucrose, ammonium chloride, and brefeldin A, block the acquisition of endo F-resistant sugars and the acquisition of detergent insolubility by newly synthesized anion exchangers. The inhibitory effects of ammonium chloride on anion exchanger processing are rapidly reversible. Furthermore, AE1 anion exchangers become detergent insoluble more rapidly than they acquire endo F-resistant modifications in cells recovering from an ammonium chloride block. This suggests that the cytoskeletal association of the recycling anion exchangers occurs after release from the compartment where they accumulate due to ammonium chloride treatment, and prior to their transit through the Golgi. The recycling pool of newly synthesized anion exchangers is reflected in the steady-state distribution of the polypeptide. In addition to plasma membrane staining, anion exchanger antibodies stain a perinuclear compartment in erythroid cells. This perinuclear AE1-containing compartment is also stained by ankyrin antibodies and partially overlaps the membrane compartment stained by NBD C6-ceramide, a Golgi marker. Detergent extraction of erythroid cells in situ has suggested that a substantial fraction of the perinuclear pool of AE1 is cytoskeletal associated. The demonstration that erythroid anion exchangers interact with elements of the cytoskeleton during recycling to the Golgi suggests the cytoskeleton may be involved in the post-Golgi trafficking of this membrane transporter.

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 Eps15 Is Recruited to the Plasma Membrane upon Epidermal Growth Factor Receptor Activation and Localizes to Components of the Endocytic Pathway during Receptor Internalization

1999 ◽  
Vol 10 (2) ◽  
pp. 417-434 ◽  
Author(s):  
Maria Rosaria Torrisi ◽  
Lavinia Vittoria Lotti ◽  
Francesca Belleudi ◽  
Roberto Gradini ◽  
Anna Elisabetta Salcini ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Receptor Activation ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Epidermal Growth

Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment, suggesting sustained phosphorylation in endocytic compartments. Our results are consistent with a model in which eps15 undergoes cycles of association:dissociation with membranes and suggest multiple roles for this protein in the endocytic pathway.

scholarly journals p120-Catenin Inhibits VE-Cadherin Internalization through a Rho-independent Mechanism

2009 ◽  
Vol 20 (7) ◽  
pp. 1970-1980 ◽  
Author(s):  
Christine M. Chiasson ◽  
Kristin B. Wittich ◽  
Peter A. Vincent ◽  
Victor Faundez ◽  
Andrew P. Kowalczyk
Keyword(s):  
Plasma Membrane ◽  
Rho Gtpase ◽  
Retention Mechanism ◽  
Endocytic Pathway ◽  
Membrane Domains ◽  
P120 Catenin ◽  
Rhoa Activity ◽  
Endocytic Machinery ◽  
Endocytic Compartments ◽  
The Relationship

p120-catenin is a cytoplasmic binding partner of cadherins and functions as a set point for cadherin expression by preventing cadherin endocytosis, and degradation. p120 is known to regulate cell motility and invasiveness by inhibiting RhoA activity. However, the relationship between these functions of p120 is not understood. Here, we provide evidence that p120 functions as part of a plasma membrane retention mechanism for VE-cadherin by preventing the recruitment of VE-cadherin into membrane domains enriched in components of the endocytic machinery, including clathrin and the adaptor complex AP-2. The mechanism by which p120 regulates VE-cadherin entry into endocytic compartments is dependent on p120's interaction with the cadherin juxtamembrane domain, but occurs independently of p120's prevention of Rho GTPase activity. These findings clarify the mechanism for p120's function in stabilizing VE-cadherin at the plasma membrane and demonstrate a novel role for p120 in modulating the availability of cadherins for entry into a clathrin-dependent endocytic pathway.

scholarly journals Retrograde lipid traffic in yeast: identification of two distinct pathways for internalization of fluorescent-labeled phosphatidylcholine from the plasma membrane.

1993 ◽  
Vol 123 (6) ◽  
pp. 1403-1419 ◽  
Author(s):  
L S Kean ◽  
R S Fuller ◽  
J W Nichols
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Nuclear Envelope ◽  
Secretory Pathway ◽  
Acyl Chain ◽  
Energy Charge ◽  
Endocytic Pathway ◽  
Atp Depletion ◽  
Yeast Saccharomyces Cerevisiae ◽  
Flip Flop

Digital, video-enhanced fluorescence microscopy and spectrofluorometry were used to follow the internalization into the yeast Saccharomyces cerevisiae of phosphatidylcholine molecules labeled on one acyl chain with the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). Two pathways were found: (1) transport by endocytosis to the vacuole and (2) transport by a non-endocytic pathway to the nuclear envelope and mitochondria. The endocytic pathway was inhibited at low temperature (< 2 degrees C) and by ATP depletion. Mutations in secretory (SEC) genes that are necessary for membrane traffic through the secretory pathway (including SEC1, SEC2, SEC4, SEC6, SEC7, SEC12, SEC14, SEC17, SEC18, and SEC21) almost completely blocked endocytic uptake. In contrast, mutations in the SEC63, SEC65, or SEC11 genes, required for translocation of nascent secretory polypeptides into the ER or signal peptide processing in the ER, only slightly reduced endocytic uptake. Phospholipid endocytosis was also independent of the gene encoding the clathrin heavy chain, CHC1. The correlation of biochemical analysis with fluorescence microscopy indicated that the fluorescent phosphatidylcholine was degraded in the vacuole and that degradation was, at least in part, dependent on the vacuolar proteolytic cascade. The non-endocytic route functioned with a lower cellular energy charge (ATP levels 80% reduced) and was largely independent of the SEC genes. Non-endocytic transport of NBD-phosphatidylcholine to the nuclear envelope and mitochondria was inhibited by pretreatment of cells with the sulfhydryl reagents N-ethylmaleimide and p-chloromercuribenzenesulfonic acid, suggesting the existence of protein-mediated transmembrane transfer (flip-flop) of phosphatidylcholine across the yeast plasma membrane. These data establish a link between lipid movement during secretion and endocytosis in yeast and suggest that phospholipids may also gain access to intracellular organelles through non-endocytic, protein-mediated events.

scholarly journals BLOC-1 Complex Deficiency Alters the Targeting of Adaptor Protein Complex-3 Cargoes

2006 ◽  
Vol 17 (9) ◽  
pp. 4014-4026 ◽  
Author(s):  
G. Salazar ◽  
B. Craige ◽  
M. L. Styers ◽  
K. A. Newell-Litwa ◽  
M. M. Doucette ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Cellular Mechanism ◽  
Type Ii ◽  
Mouse Mutants ◽  
Late Endosomes ◽  
Complex Deficiency ◽  
Lysosome Related Organelles ◽  
Phosphatidylinositol 4

Mutational analyses have revealed many genes that are required for proper biogenesis of lysosomes and lysosome-related organelles. The proteins encoded by these genes assemble into five distinct complexes (AP-3, BLOC-1-3, and HOPS) that either sort membrane proteins or interact with SNAREs. Several of these seemingly distinct complexes cause similar phenotypic defects when they are rendered defective by mutation, but the underlying cellular mechanism is not understood. Here, we show that the BLOC-1 complex resides on microvesicles that also contain AP-3 subunits and membrane proteins that are known AP-3 cargoes. Mouse mutants that cause BLOC-1 or AP-3 deficiencies affected the targeting of LAMP1, phosphatidylinositol-4-kinase type II alpha, and VAMP7-TI. VAMP7-TI is an R-SNARE involved in vesicle fusion with late endosomes/lysosomes, and its cellular levels were selectively decreased in cells that were either AP-3- or BLOC-1–deficient. Furthermore, BLOC-1 deficiency selectively altered the subcellular distribution of VAMP7-TI cognate SNAREs. These results indicate that the BLOC-1 and AP-3 protein complexes affect the targeting of SNARE and non-SNARE AP-3 cargoes and suggest a function of the BLOC-1 complex in membrane protein sorting.

scholarly journals The BLOC-1 complex promotes endosomal maturation by recruiting the Rab5 GTPase-activating protein Msb3

2013 ◽  
Vol 201 (1) ◽  
pp. 97-111 ◽  
Author(s):  
Arun T. John Peter ◽  
Jens Lachmann ◽  
Meenakshi Rana ◽  
Madeleine Bunge ◽  
Margarita Cabrera ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Sorting ◽  
Early Endosome ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Gtpase Activating Protein ◽  
Early Endosomes ◽  
Lysosome Related Organelles

Membrane microcompartments of the early endosomes serve as a sorting and signaling platform, where receptors are either recycled back to the plasma membrane or forwarded to the lysosome for destruction. In metazoan cells, three complexes, termed BLOC-1 to -3, mediate protein sorting from the early endosome to lysosomes and lysosome-related organelles. We now demonstrate that BLOC-1 is an endosomal Rab-GAP (GTPase-activating protein) adapter complex in yeast. The yeast BLOC-1 consisted of six subunits, which localized interdependently to the endosomes in a Rab5/Vps21-dependent manner. In the absence of BLOC-1 subunits, the balance between recycling and degradation of selected cargoes was impaired. Additionally, our data show that BLOC-1 is both a Vps21 effector and an adapter for its GAP Msb3. BLOC-1 and Msb3 interacted in vivo, and both mutants resulted in a redistribution of active Vps21 to the vacuole surface. We thus conclude that BLOC-1 controls the lifetime of active Rab5/Vps21 and thus endosomal maturation along the endocytic pathway.

scholarly journals The kinetic aspects of intracellular fluorescence labeling with TMA-DPH support the maturation model for endocytosis in L929 cells.

1994 ◽  
Vol 125 (4) ◽  
pp. 783-794 ◽  
Author(s):  
D Illinger ◽  
J G Kuhry
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Intensity ◽  
Release Kinetics ◽  
Aqueous Media ◽  
Endocytic Pathway ◽  
Fluorescence Labeling ◽  
Cell Periphery ◽  
Membrane Composition ◽  
Endocytic Compartments ◽  
Intracellular Fluorescence

TMA-DPH (1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene), a hydrophobic fluorescent membrane probe, interacts with living cells by instantaneous incorporation into the plasma membrane, where it becomes fluorescent. It then follows the intracellular constitutive membrane traffic and acts as a bulk membrane marker of the endocytic pathway (Illinger, D., P. Poindron, P. Fonteneau, M. Modolell, and J. G. Kuhry. 1990. Biochim. Biophys. Acta. 1030:73-81; Illinger, D., P. Poindron, and J. G. Kuhry. 1991. Biol. Cell. 73:131-138). As such, TMA-DPH displays particular properties mainly due to partition between membranes and aqueous media. From these properties, original arguments can be inferred in favor of the maturation model for the endocytic pathway, against that of pre-existing compartments, in L929 cultured mouse fibroblasts. (a) TMA-DPH labeling is seen to progress from the cell periphery to perinuclear regions during endocytosis without any noticeable loss in fluorescence intensity; with a vesicle shuttle model this evolution would be accompanied by probe dilution with a decrease in the overall intracellular fluorescence intensity, and the labeling of the inner (late) compartments could in no way become more intense than that of the peripheral (early) ones. (b) From TMA-DPH fluorescence anisotropy assays, it is concluded that membrane fluidity is the same in the successive endocytic compartments as in the plasma membrane, which probably denotes a similar phospholipidic membrane composition, as might be expected in the maturation model. (c) TMA-DPH internalization and release kinetics are more easily described with the maturation model.

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