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

2002 ◽  
Vol 13 (1) ◽  
pp. 169-182 ◽  
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.

scholarly journals Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump.

1995 ◽  
Vol 130 (4) ◽  
pp. 821-834 ◽  
Author(s):  
A W van Weert ◽  
K W Dunn ◽  
H J Gueze ◽  
F R Maxfield ◽  
W Stoorvogel
Keyword(s):  
Plasma Membrane ◽  
Proton Pump ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Immunogold Electron Microscopy ◽  
Cell Fractionation ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endosomal Ph ◽  
Vacuolar Proton Pump

Endocytosed proteins are sorted in early endosomes to be recycled to the plasma membrane or transported further into the degradative pathway. We studied the role of endosomes acidification on the endocytic trafficking of the transferrin receptor (TfR) as a representative for the recycling pathway, the cation-dependent mannose 6-phosphate receptor (MPR) as a prototype for transport to late endosomes, and fluid-phase endocytosed HRP as a marker for transport to lysosomes. Toward this purpose, bafilomycin A1 (Baf), a specific inhibitor of the vacuolar proton pump, was used to inhibit acidification of the vacuolar system. Microspectrofluorometric measurement of the pH of fluorescein-rhodamine-conjugated transferrin (Tf)-containing endocytic compartments in living cells revealed elevated endosomal pH values (pH > 7.0) within 2 min after addition of Baf. Although recycling of endocytosed Tf to the plasma membrane continued in the presence of Baf, recycled Tf did not dissociate from its receptor, indicating failure of Fe3+ release due to a neutral endosomal pH. In the presence of Baf, the rates of internalization and recycling of Tf were reduced by a factor of 1.40 +/- 0.08 and 1.57 +/- 0.25, respectively. Consequently, little if any in TfR expression at the cell surface was measured during Baf treatment. Sorting between endocytosed TfR and MPR was analyzed by the HRP-catalyzed 3,3'-diaminobenzidine cross-linking technique, using transferrin conjugated to HRP to label the endocytic pathway of the TfR. In the absence of Baf, endocytosed surface 125I-labeled MPR was sorted from the TfR pathway starting at 10 min after uptake, reaching a plateau of 40% after 45 min. In the presence of Baf, sorting was initiated after 20 min of uptake, reaching approximately 40% after 60 min. Transport of fluid-phase endocytosed HRP to late endosomes and lysosomes was measured using cell fractionation and immunogold electron microscopy. Baf did not interfere with transport of HRP to MPR-labeled late endosomes, but nearly completely abrogated transport to cathepsin D-labeled lysosomes. From these results, we conclude that trafficking through early and late endosomes, but not to lysosomes, continued upon inactivation of the vacuolar proton pump.

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

scholarly journals Rab5-mediated endosome formation is regulated at the trans-Golgi network

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Makoto Nagano ◽  
Junko Y. Toshima ◽  
Daria Elisabeth Siekhaus ◽  
Jiro Toshima
Keyword(s):  
Plasma Membrane ◽  
Early Endosome ◽  
Vesicle Transport ◽  
Nucleotide Exchange ◽  
Trafficking Pathway ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Critical Location ◽  
Golgi Network

AbstractEarly endosomes, also called sorting endosomes, are known to mature into late endosomes via the Rab5-mediated endolysosomal trafficking pathway. Thus, early endosome existence is thought to be maintained by the continual fusion of transport vesicles from the plasma membrane and the trans-Golgi network (TGN). Here we show instead that endocytosis is dispensable and post-Golgi vesicle transport is crucial for the formation of endosomes and the subsequent endolysosomal traffic regulated by yeast Rab5 Vps21p. Fittingly, all three proteins required for endosomal nucleotide exchange on Vps21p are first recruited to the TGN before transport to the endosome, namely the GEF Vps9p and the epsin-related adaptors Ent3/5p. The TGN recruitment of these components is distinctly controlled, with Vps9p appearing to require the Arf1p GTPase, and the Rab11s, Ypt31p/32p. These results provide a different view of endosome formation and identify the TGN as a critical location for regulating progress through the endolysosomal trafficking pathway.

scholarly journals A Myosin I Is Involved in Membrane Recycling from Early Endosomes

2000 ◽  
Vol 150 (5) ◽  
pp. 1013-1026 ◽  
Author(s):  
Eva M. Neuhaus ◽  
Thierry Soldati
Keyword(s):  
Plasma Membrane ◽  
Fluid Phase ◽  
Surface Proteins ◽  
Endocytic Pathway ◽  
Membrane Recycling ◽  
Myosin I ◽  
Early Endosomes ◽  
Butanedione Monoxime ◽  
Microscopy Method ◽  
Membrane Components

Geometry-based mechanisms have been proposed to account for the sorting of membranes and fluid phase in the endocytic pathway, yet little is known about the involvement of the actin–myosin cytoskeleton. Here, we demonstrate that Dictyostelium discoideum myosin IB functions in the recycling of plasma membrane components from endosomes back to the cell surface. Cells lacking MyoB (myoA−/B−, and myoB− cells) and wild-type cells treated with the myosin inhibitor butanedione monoxime accumulated a plasma membrane marker and biotinylated surface proteins on intracellular endocytic vacuoles. An assay based on reversible biotinylation of plasma membrane proteins demonstrated that recycling of membrane components is severely impaired in myoA/B null cells. In addition, MyoB was specifically found on magnetically purified early pinosomes. Using a rapid-freezing cryoelectron microscopy method, we observed an increased number of small vesicles tethered to relatively early endocytic vacuoles in myoA−/B− cells, but not to later endosomes and lysosomes. This accumulation of vesicles suggests that the defects in membrane recycling result from a disordered morphology of the sorting compartment.

scholarly journals Chimeric Forms of Furin and Tgn38 Are Transported from the Plasma Membrane to the Trans-Golgi Network via Distinct Endosomal Pathways

1999 ◽  
Vol 146 (2) ◽  
pp. 345-360 ◽  
Author(s):  
William G. Mallet ◽  
Frederick R. Maxfield
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Chimeric Proteins ◽  
Endocytic Recycling ◽  
Single Pass ◽  
Trans Golgi Network ◽  
Late Endosomes ◽  
Recycling Pathway ◽  
Endosomal Transport ◽  
Golgi Network

Furin and TGN38 are membrane proteins that cycle between the plasma membrane and the trans-Golgi network (TGN), each maintaining a predominant distribution in the TGN. We have used chimeric proteins with an extracellular Tac domain and the cytoplasmic domain of TGN38 or furin to study the trafficking of these proteins in endosomes. Previously, we demonstrated that the postendocytic trafficking of Tac-TGN38 to the TGN is via the endocytic recycling pathway (Ghosh, R.N., W.G. Mallet, T.T. Soe, T.E. McGraw, and F.R. Maxfield. 1998. J. Cell Biol. 142:923–936). Here we show that internalized Tac-furin is delivered to the TGN through late endosomes, bypassing the endocytic recycling compartment. The transport of Tac-furin from late endosomes to the TGN appears to proceed via an efficient, single-pass mechanism. Delivery of Tac-furin but not Tac-TGN38 to the TGN is blocked by nocodazole, and the two pathways are also differentially affected by wortmannin. These studies demonstrate the existence of two independent pathways for endosomal transport of proteins to the TGN from the plasma membrane.

End13p/Vps4p is required for efficient transport from early to late endosomes in Saccharomyces cerevisiae

2001 ◽  
Vol 114 (10) ◽  
pp. 1935-1947 ◽  
Author(s):  
R. Zahn ◽  
B.J. Stevenson ◽  
S. Schroder-Kohne ◽  
B. Zanolari ◽  
H. Riezman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Dyes ◽  
Membrane Receptors ◽  
Endocytic Pathway ◽  
Aaa Atpase ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Plasma Membrane Receptors ◽  
Vacuolar Protein ◽  
Mutant Cells

end13-1 was isolated in a screen for endocytosis mutants and has been shown to have a post-internalisation defect in endocytic transport as well as a defect in vacuolar protein sorting (Vps(-) phenotype), leading to secretion of newly synthesised vacuolar proteins. Here we demonstrate that END13 is identical to VPS4, encoding an AAA (ATPase associated with a variety of cellular activities)-family ATPase. We also report that the end13-1 mutation is a serine 335 to phenylalanine substitution in the AAA-ATPase domain of End13p/Vps4p. It has been reported that mutant cells lacking End13p/Vps4p (end13(vps4)((Dgr;)) accumulate endocytosed marker dyes, plasma membrane receptors and newly synthesised vacuolar hydrolase precursors in an endosomal compartment adjacent to the vacuole (prevacuolar compartment, or PVC). We find, however, that the end13 mutants have defects in transport of endocytosed fluorescent dyes, plasma membrane receptors and ligands from small peripherally located early endosomes to larger late endosomes, which are often located adjacent to the vacuole. Our results indicate that End13p/Vps4p may play an important role in multiple steps of membrane traffic through the endocytic pathway.

The PDZ-interaction of the intestinal anion exchanger downregulated in adenoma (DRA; SLC26A3) facilitates its movement into Rab11a-positive recycling endosomes

2013 ◽  
Vol 304 (11) ◽  
pp. G980-G990 ◽  
Author(s):  
S. Lissner ◽  
C.-J. Hsieh ◽  
L. Nold ◽  
K. Bannert ◽  
P. Bodammer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Expression ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Dominant Negative Mutant ◽  
Recycling Endosomes ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Recycling Pathway

Electroneutral NaCl absorption in the ileum and colon is mediated by downregulated in adenoma (DRA) (Cl-/HCO3- exchanger; SLC26A3) and Na+/H+ exchanger 3 (NHE3, SLC9A3). Surface expression of transport proteins undergoes basal and regulated recycling by endo- and exocytosis. Expression and activity of DRA in the plasma membrane depend on intact lipid rafts, phosphatidylinositol 3-kinase (PI3-kinase), and the PDZ interaction of DRA. However, it is unknown how the PDZ interaction of DRA affects its trafficking to the cell surface. Therefore, the (re)cycling pathway of DRA was investigated in HEK cells stably expressing enhanced green fluorescent protein (EGFP)-DRA or EGFP-DRA-ETKFminus (a mutant lacking the PDZ interaction motif). Early, late, and recycling endosomes were immunoisolated by precipitating stably transfected mCherry-hemagglutinin (HA)-Rab5a, -7a, or -11a. EGFP-DRA and EGFP-DRA-ETKFminus were equally present in early endosomes. In recycling endosomes, wild-type DRA was preferentially present, whereas, in late endosomes, DRA-ETKF-minus dominated. Correspondingly, EGFP-DRA colocalized with mCherry-HA-Rab11a in recycling endosomes, whereas EGFP-DRA-ETKFminus colocalized with mCherry-HA-Rab7a in late endosomes. Functionally, this different distribution was reflected by a shorter half-life of the mutant DRA. Transient expression of dominant-negative Rab11aS25N inhibited the activity (-17%, P < 0.05) and the cell surface expression of DRA (-30%, P < 0.05). Transient transfection of Rab4a or its dominant-negative mutant Rab4aS22N was without effect and thus excluded participation of the rapid recycling pathway. Taken together, the PDZ interaction of DRA facilitates its movement into Rab11a-positive recycling endosomes, from where it is inserted in the plasma membrane. A scenario emerges where specific PDZ adaptor proteins are present along several compartments of the endocytosis-recycling pathway.

scholarly journals Entry of Human Parechovirus 1

2001 ◽  
Vol 75 (4) ◽  
pp. 1958-1967 ◽  
Author(s):  
Päivi Joki-Korpela ◽  
Varpu Marjomäki ◽  
Camilla Krogerus ◽  
Jyrki Heino ◽  
Timo Hyypiä
Keyword(s):  
Host Cell ◽  
Preliminary Evidence ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Human Parechovirus ◽  
Double Labeling ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Infected Cells ◽  
Golgi Network

ABSTRACT Human parechovirus 1 (HPEV-1) is a prototype member of parechoviruses, a recently established picornavirus genus. Although there is preliminary evidence that HPEV-1 recognizes αVintegrins as cellular receptors, our understanding of early events during HPEV-1 infection is still very limited. The aim of this study was to clarify the entry mechanisms of HPEV-1, including the attachment of the virus onto the host cell surface and subsequent internalization. In blocking experiments with monoclonal antibodies against different receptor candidates, antibodies against αV and β3 integrin subunits, in particular in combination, appeared to be the most efficient ones in preventing the HPEV-1 infection. To find out whether HPEV-1 uses clathrin-coated vesicles or other routes for the entry into the host cell, we carried out double-labeling experiments of virus-infected cells with anti-HPEV-1 antibodies and antibodies against known markers of the clathrin and the caveolin routes. At the early phase of infection (5 min postinfection [p.i.]) HPEV-1 colocalized with EEA1 (early endosomes), and later, after 30 min p.i., it colocalized with mannose-6-phosphate receptor (late endosomes), whereas no colocalization with caveolin-1 was observed. The data indicate that HPEV-1 utilizes the clathrin-dependent endocytic pathway for entry into the host cells. Interestingly, endocytosed HPEV-1 capsid proteins were observed in the endoplasmic reticulum and cis-Golgi network 30 to 60 min p.i. Depolymerization of microtubules with nocodazole inhibited translocation of the virus to the late endosomes but did not block HPEV-1 replication, suggesting that the RNA genome may be released early during the entry process.

scholarly journals ARF6 controls post-endocytic recycling through its downstream exocyst complex effector

2003 ◽  
Vol 163 (5) ◽  
pp. 1111-1121 ◽  
Author(s):  
Magali Prigent ◽  
Thierry Dubois ◽  
Graça Raposo ◽  
Valérie Derrien ◽  
Danièle Tenza ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Endocytic Pathway ◽  
Membrane Remodeling ◽  
Recycling Endosomes ◽  
Vesicle Tethering ◽  
Exocyst Complex ◽  
A Subunit ◽  
Recycling Pathway ◽  
Golgi Network ◽  
Adp Ribosylation Factor

The small guanosine triphosphate (GTP)–binding protein ADP-ribosylation factor (ARF) 6 regulates membrane recycling to regions of plasma membrane remodeling via the endocytic pathway. Here, we show that GTP–bound ARF6 interacts with Sec10, a subunit of the exocyst complex involved in docking of vesicles with the plasma membrane. We found that Sec10 localization in the perinuclear region is not restricted to the trans-Golgi network, but extends to recycling endosomes. In addition, we report that depletion of Sec5 exocyst subunit or dominant inhibition of Sec10 affects the function and the morphology of the recycling pathway. Sec10 is found to redistribute to ruffling areas of the plasma membrane in cells expressing GTP-ARF6, whereas dominant inhibition of Sec10 interferes with ARF6-induced cell spreading. Our paper suggests that ARF6 specifies delivery and insertion of recycling membranes to regions of dynamic reorganization of the plasma membrane through interaction with the vesicle-tethering exocyst complex.

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