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 SMAP2, a Novel ARF GTPase-activating Protein, Interacts with Clathrin and Clathrin Assembly Protein and Functions on the AP-1–positive Early Endosome/Trans-Golgi Network

2006 ◽  
Vol 17 (6) ◽  
pp. 2592-2603 ◽  
Author(s):  
Waka Natsume ◽  
Kenji Tanabe ◽  
Shunsuke Kon ◽  
Naomi Yoshida ◽  
Toshio Watanabe ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Adaptor Proteins ◽  
Early Endosome ◽  
Dependent Manner ◽  
Transferrin Receptors ◽  
Gtpase Activating Protein ◽  
Trans Golgi Network ◽  
Early Endosomes ◽  
Golgi Network

We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1–dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking.

scholarly journals Clathrin Hub Expression Affects Early Endosome Distribution with Minimal Impact on Receptor Sorting and Recycling

2001 ◽  
Vol 12 (9) ◽  
pp. 2790-2799 ◽  
Author(s):  
Elizabeth M. Bennett ◽  
Sharron X. Lin ◽  
Mhairi C. Towler ◽  
Frederick R. Maxfield ◽  
Frances M. Brodsky
Keyword(s):  
Plasma Membrane ◽  
Early Endosome ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Endocytic Trafficking ◽  
Minimal Impact ◽  
Recycling Endosomes ◽  
Receptor Mediated Endocytosis ◽  
Early Endosomes ◽  
Endosomal Membranes

Clathrin-coated vesicles execute receptor-mediated endocytosis at the plasma membrane. However, a role for clathrin in later endocytic trafficking processes, such as receptor sorting and recycling or maintaining the organization of the endocytic pathway, has not been thoroughly characterized. The existence of clathrin-coated buds on endosomes suggests that clathrin might mediate later endocytic trafficking events. To investigate the function of clathrin-coated buds on endosomal membranes, endosome function and distribution were analyzed in a HeLa cell line that expresses the dominant-negative clathrin inhibitor Hub in an inducible manner. As expected, Hub expression reduced receptor-mediated endocytosis at the plasma membrane. Hub expression also induced a perinuclear aggregation of early endosome antigen 1-positive early endosomes, such that sorting and recycling endosomes were found tightly concentrated in the perinuclear region. Despite the dramatic redistribution of endosomes, Hub expression did not affect the overall kinetics of receptor sorting or recycling. These data show that clathrin function is necessary to maintain proper cellular distribution of early endosomes but does not play a prominent role in sorting and recycling events. Thus, clathrin's role on endosomal membranes is to influence organelle localization and is distinct from its role in trafficking pathways at the plasma membrane and trans-Golgi network.

scholarly journals Mammalian Late Vacuole Protein Sorting Orthologues Participate in Early Endosomal Fusion and Interact with the Cytoskeleton

2004 ◽  
Vol 15 (3) ◽  
pp. 1197-1210 ◽  
Author(s):  
Simon C. W. Richardson ◽  
Stanley C. Winistorfer ◽  
Viviane Poupon ◽  
J. Paul Luzio ◽  
Robert C. Piper
Keyword(s):  
Protein Sorting ◽  
Early Endosome ◽  
Endocytic Pathway ◽  
Rab Proteins ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Early Endosomes ◽  
Sensitive Factor ◽  
Factor Attachment Protein Receptor

In Saccharomyces cerevisiae, the class C vacuole protein sorting (Vps) proteins, together with Vam2p/Vps41p and Vam6p/Vps39p, form a complex that interacts with soluble N-ethylmaleimide-sensitive factor attachment protein receptor and Rab proteins to “tether” vacuolar membranes before fusion. To determine a role for the corresponding mammalian orthologues, we examined the function, localization, and protein interactions of endogenous mVps11, mVps16, mVps18, mVam2p, and mVam6. We found a significant proportion of these proteins localized to early endosome antigen-1 and transferrin receptor-positive early endosomes in Vero, normal rat kidney, and Chinese hamster ovary cells. Immunoprecipitation experiments showed that mVps18 not only interacted with Syntaxin (Syn)7, vesicle-associated membrane protein 8, and Vti1-b but also with Syn13, Syn6, and the Sec1/Munc18 protein mVps45, which catalyze early endosomal fusion events. Moreover, anti-mVps18 antibodies inhibited early endosome fusion in vitro. Mammalian mVps18 also associated with mVam2 and mVam6 as well as with the microtubule-associated Hook1 protein, an orthologue of the Drosophila Hook protein involved in endosome biogenesis. Using in vitro binding and immunofluorescence experiments, we found that mVam2 and mVam6 also associated with microtubules, whereas mVps18, mVps16, and mVps11 associated with actin filaments. These data indicate that the late Vps proteins function during multiple soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated fusion events throughout the endocytic pathway and that their activity may be coordinated with cytoskeletal function.

scholarly journals Discovery of a vezatin-like protein for dynein-mediated early endosome transport

2015 ◽  
Vol 26 (21) ◽  
pp. 3816-3827 ◽  
Author(s):  
Xuanli Yao ◽  
Herbert N. Arst ◽  
Xiangfeng Wang ◽  
Xin Xiang
Keyword(s):  
Aspergillus Nidulans ◽  
Genetic Study ◽  
Cytoplasmic Dynein ◽  
Early Endosome ◽  
Dependent Manner ◽  
C Terminus ◽  
Early Endosomes ◽  
Abnormal Accumulation ◽  
Hyphal Tip

Early endosomes are transported bidirectionally by cytoplasmic dynein and kinesin-3, but how the movements are regulated in vivo remains unclear. Here our forward genetic study led to the discovery of VezA, a vezatin-like protein in Aspergillus nidulans, as a factor critical for early endosome distribution. Loss of vezA causes an abnormal accumulation of early endosomes at the hyphal tip, where microtubule plus ends are located. This abnormal accumulation depends on kinesin-3 and is due to a decrease in the frequency but not the speed of dynein-mediated early endosome movement. VezA-GFP signals are enriched at the hypha tip in an actin-dependent manner but are not obviously associated with early endosomes, thus differing from the early endosome association of the cargo adapter HookA (Hook in A. nidulans). On loss of VezA, HookA associates normally with early endosomes, but the interaction between dynein-dynactin and the early-endosome-bound HookA is significantly decreased. However, VezA is not required for linking dynein-dynactin to the cytosolic ∆C-HookA, lacking the cargo-binding C-terminus. These results identify VezA as a novel regulator required for the interaction between dynein and the Hook-bound early endosomes in vivo.

scholarly journals Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
Author(s):  
Fabien Lefèbvre ◽  
Valérie Prouzet-Mauléon ◽  
Michel Hugues ◽  
Marc Crouzet ◽  
Aurélie Vieillemard ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Rho Gtpase ◽  
Secretory Vesicles ◽  
Gtpase Activating Protein ◽  
Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

scholarly journals Early Endosomes Are Required for Major Histocompatiblity Complex Class II Transport to Peptide-loading Compartments

1999 ◽  
Vol 10 (9) ◽  
pp. 2891-2904 ◽  
Author(s):  
Valérie Brachet ◽  
Gérard Péhau-Arnaudet ◽  
Catherine Desaymard ◽  
Graça Raposo ◽  
Sebastian Amigorena
Keyword(s):  
Mhc Class Ii ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Cross Linking ◽  
Histocompatibility Complex ◽  
Early Endosomes ◽  
Peptide Loading ◽  
Golgi Network ◽  
Mhc Class Ii Molecules

Antigen presentation to CD4+ T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19°C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor–containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most αβIi complexes accumulated in tubules and vesicles devoid of γ-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to endosomes.

scholarly journals Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes

1992 ◽  
Vol 103 (4) ◽  
pp. 1139-1152
Author(s):  
J.W. Kok ◽  
K. Hoekstra ◽  
S. Eskelinen ◽  
D. Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Recycling Pathway ◽  
Golgi Network ◽  
Extensive Involvement

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.

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 The TRE17 Oncogene Encodes a Component of a Novel Effector Pathway for Rho GTPases Cdc42 and Rac1 and Stimulates Actin Remodeling

2003 ◽  
Vol 23 (6) ◽  
pp. 2151-2161 ◽  
Author(s):  
Jeffrey M. Masuda-Robens ◽  
Sara N. Kutney ◽  
Hongwei Qi ◽  
Margaret M. Chou
Keyword(s):  
Plasma Membrane ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Actin Remodeling ◽  
Membrane Recruitment ◽  
Truncation Mutant ◽  
Effector Pathway

ABSTRACT The Rho family GTPases Cdc42 and Rac1 play fundamental roles in transformation and actin remodeling. Here, we demonstrate that the TRE17 oncogene encodes a component of a novel effector pathway for these GTPases. TRE17 coprecipitated specifically with the active forms of Cdc42 and Rac1 in vivo. Furthermore, the subcellular localization of TRE17 was dramatically regulated by these GTPases and mitogens. Under serum-starved conditions, TRE17 localized predominantly to filamentous structures within the cell. Epidermal growth factor (EGF) induced relocalization of TRE17 to the plasma membrane in a Cdc42-/Rac1-dependent manner. Coexpression of activated alleles of Cdc42 or Rac1 also caused complete redistribution of TRE17 to the plasma membrane, where it partially colocalized with the GTPases in filopodia and ruffles, respectively. Membrane recruitment of TRE17 by EGF or the GTPases was dependent on actin polymerization. Finally, we found that a C-terminal truncation mutant of TRE17 induced the accumulation of cortical actin, mimicking the effects of activated Cdc42. Together, these results identify TRE17 as part of a novel effector complex for Cdc42 and Rac1, potentially contributing to their effects on actin remodeling. The present study provides insights into the regulation and cellular function of this previously uncharacterized oncogene.

scholarly journals c-Cbl Mediates Ubiquitination, Degradation, and Down-regulation of Human Protease-activated Receptor 2

2005 ◽  
Vol 280 (16) ◽  
pp. 16076-16087 ◽  
Author(s):  
Claire Jacob ◽  
Graeme S. Cottrell ◽  
Daphne Gehringer ◽  
Fabien Schmidlin ◽  
Eileen F. Grady ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ring Finger ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Wild Type ◽  
Lysosomal Degradation ◽  
Proteolytic Activation ◽  
Gpcr Signaling ◽  
Early Endosomes ◽  
Protease Activated Receptor 2

Mechanisms that arrest G-protein-coupled receptor (GPCR) signaling prevent uncontrolled stimulation that could cause disease. Although uncoupling from heterotrimeric G-proteins, which transiently arrests signaling, is well described, little is known about the mechanisms that permanently arrest signaling. Here we reported on the mechanisms that terminate signaling by protease-activated receptor 2 (PAR2), which mediated the proinflammatory and nociceptive actions of proteases. Given its irreversible mechanism of proteolytic activation, PAR2is a model to study the permanent arrest of GPCR signaling. By immunoprecipitation and immunoblotting, we observed that activated PAR2was mono-ubiquitinated. Immunofluorescence indicated that activated PAR2translocated from the plasma membrane to early endosomes and lysosomes where it was degraded, as determined by immunoblotting. Mutant PAR2lacking intracellular lysine residues (PAR2Δ14K/R) was expressed at the plasma membrane and signaled normally but was not ubiquitinated. Activated PAR2Δ14K/R internalized but was retained in early endosomes and avoided lysosomal degradation. Activation of wild type PAR2stimulated tyrosine phosphorylation of the ubiquitin-protein isopeptide ligase c-Cbl and promoted its interaction with PAR2at the plasma membrane and in endosomes in an Src-dependent manner. Dominant negative c-Cbl lacking the ring finger domain inhibited PAR2ubiquitination and induced retention in early endosomes, thereby impeding lysosomal degradation. Although wild type PAR2was degraded, and recovery of agonist responses required synthesis of new receptors, lysine mutation and dominant negative c-Cbl impeded receptor ubiquitination and degradation and allowed PAR2to recycle and continue to signal. Thus, c-Cbl mediated ubiquitination and lysosomal degradation of PAR2to irrevocably terminate signaling by this and perhaps other GPCRs.

Sign in / Sign up

Export Citation Format

Share Document