scholarly journals SNARE Function Is Not Involved in Early Endosome Docking

2008 ◽  
Vol 19 (12) ◽  
pp. 5327-5337 ◽  
Author(s):  
Ulf Geumann ◽  
Sina Victoria Barysch ◽  
Peer Hoopmann ◽  
Reinhard Jahn ◽  
Silvio O. Rizzoli
Keyword(s):  
Phosphatidyl Inositol ◽  
Endocytic Pathway ◽  
Rab Gtpases ◽  
Vitro Assay ◽  
Receptor Proteins ◽  
Transport Vesicles ◽  
Early Endosomes ◽  
Sensitive Factor ◽  
Rab Effector

Docking and fusion of transport vesicles constitute elementary steps in intracellular membrane traffic. While docking is thought to be initiated by Rab-effector complexes, fusion is mediated by SNARE (N-ethylmaleimide-sensitive factor [NSF] attachment receptor) proteins. However, it has been recently debated whether SNAREs also play a role in the establishment or maintenance of a stably docked state. To address this question, we have investigated the SNARE dependence of docking and fusion of early endosomes, one of the central sorting compartments in the endocytic pathway. A new, fluorescence-based in vitro assay was developed, which allowed us to investigate fusion and docking in parallel. Similar to homotypic fusion, docking of early endosomes is dependent on the presence of ATP and requires physiological temperatures. Unlike fusion, docking is insensitive to the perturbation of SNARE function by means of soluble SNARE motifs, SNARE-specific Fab fragments, or by a block of NSF activity. In contrast, as expected, docking is strongly reduced by interfering with the synthesis of phosphatidyl inositol (PI)-3 phosphate, with the function of Rab-GTPases, as well as with early endosomal autoantigen 1 (EEA1), an essential tethering factor. We conclude that docking of early endosomes is independent of SNARE function.

scholarly journals Syntaxin 7 and VAMP-7 are SolubleN-Ethylmaleimide–sensitive Factor Attachment Protein Receptors Required for Late Endosome–Lysosome and Homotypic Lysosome Fusion in Alveolar Macrophages

2000 ◽  
Vol 11 (7) ◽  
pp. 2327-2333 ◽  
Author(s):  
Diane McVey Ward ◽  
Jonathan Pevsner ◽  
Matthew A. Scullion ◽  
Michael Vaughn ◽  
Jerry Kaplan
Keyword(s):  
Alveolar Macrophages ◽  
Transmembrane Domain ◽  
Late Endosome ◽  
Endocytic Pathway ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Early Endosomes ◽  
Sensitive Factor ◽  
Protein Receptors

Endocytosis in alveolar macrophages can be reversibly inhibited, permitting the isolation of endocytic vesicles at defined stages of maturation. Using an in vitro fusion assay, we determined that each isolated endosome population was capable of homotypic fusion. All vesicle populations were also capable of heterotypic fusion in a temporally specific manner; early endosomes, isolated 4 min after internalization, could fuse with endosomes isolated 8 min after internalization but not with 12-min endosomes or lysosomes. Lysosomes fuse with 12-min endosomes but not with earlier endosomes. Using homogenous populations of endosomes, we have identified Syntaxin 7 as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) required for late endosome–lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inhibited homotypic late endosome and lysosome fusion as well as heterotypic late endosome–lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no affect on homotypic early endosome fusion. Previous work suggested that human VAMP-7 (vesicle-associated membrane protein-7) was a SNARE required for late endosome–lysosome fusion. A bacterially expressed human VAMP-7 lacking the transmembrane domain inhibited both late endosome–lysosome fusion and homotypic lysosome fusion in vitro. These studies indicate that: 1) fusion along the endocytic pathway is a highly regulated process, and 2) two SNARE molecules, Syntaxin 7 and human VAMP-7, are involved in fusion of vesicles in the late endocytic pathway in alveolar macrophages.

Biochemical analysis of distinct Rab5- and Rab11-positive endosomes along the transferrin pathway

1999 ◽  
Vol 112 (24) ◽  
pp. 4773-4783 ◽  
Author(s):  
M. Trischler ◽  
W. Stoorvogel ◽  
O. Ullrich
Keyword(s):  
Intracellular Transport ◽  
Protein Composition ◽  
Endocytic Pathway ◽  
Rab Gtpases ◽  
Vitro Assay ◽  
Recycling Endosomes ◽  
Morphological Differences ◽  
And Function ◽  
Cellular Compartments

Rab GTPases are associated with distinct cellular compartments and function as specific regulators of intracellular transport. In the endocytic pathway, it is well documented that Rab5 regulates transport from plasma membrane to early (sorting) endosomes. In contrast, little is known about the precise localization and function of Rab4 and Rab11, which are believed to control endocytic recycling. In the present study we have analysed the protein composition of Rab5- and Rab11-carrying endosomes to gain further insight into the compartmental organization of the endocytic and recycling pathway. Endosome populations of this transport route were purified by immunoadsorption from endosome-enriched subcellular fractions using antibodies directed against the cytoplasmic tail of the transferrin receptor, Rab5 or Rab11. Endocytosed transferrin moved sequentially through compartments that could be immunoadsorbed with anti-Rab5 and anti-Rab11, consistent with the theory that Rab5 and Rab11 localise to sorting and recycling endosomes, respectively. These compartments exhibited morphological differences, as determined by electron microscopy. Although their overall protein compositions were very similar, some proteins were found to be selectively enriched. While Rab4 was present on all endosome populations, Rab5 and Rab11 were strikingly segregated. Furthermore, the Rab11-positive endosomes were rich in annexin II, actin and the t-SNARE syntaxin 13, compared to Rab5-containing endosomes. In an in vitro assay, the Rab5 effector protein EEA1 was preferentially recruited by Rab5-positive endosomes. Taken together, our data suggest an organization of the transferrin pathway into distinct Rab5- and Rab11-positive compartments.

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 Live Salmonella Recruits N-Ethylmaleimide–Sensitive Fusion Protein on Phagosomal Membrane and Promotes Fusion with Early Endosome

2000 ◽  
Vol 148 (4) ◽  
pp. 741-754 ◽  
Author(s):  
Konark Mukherjee ◽  
Shadab A. Siddiqi ◽  
Shehla Hashim ◽  
Manoj Raje ◽  
Sandip K. Basu ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Intracellular Trafficking ◽  
Early Endosome ◽  
Atp Depletion ◽  
Rab Gtpases ◽  
Early Endosomes ◽  
Sensitive Factor ◽  
Phagosomal Membrane

To understand intracellular trafficking modulations by live Salmonella, we investigated the characteristics of in vitro fusion between endosomes and phagosomes containing live (LSP) or dead Salmonella (DSP). We observed that fusion of both DSP and LSP were time, temperature and cytosol dependent. GTPγS and treatment of the phagosomes with Rab-GDI inhibited fusion, indicating involvement of Rab-GTPases. LSP were rich in rab5, α-SNAP, and NSF, while DSP mainly contained rab7. Fusion of endosomes with DSP was inhibited by ATP depletion, N-ethylmaleimide (NEM) treatment, and in NEM-sensitive factor (NSF)–depleted cytosol. In contrast, fusion of endosomes with LSP was not inhibited by ATP depletion or NEM treatment, and occurred in NSF-depleted cytosol. However, ATPγS inhibited both fusion events. Fusion of NEM-treated LSP with endosomes was abrogated in NSF- depleted cytosol and was restored by adding purified NSF, whereas no fusion occurred with NEM-treated DSP, indicating that NSF recruitment is dependent on continuous signals from live Salmonella. Binding of NSF with LSP required prior presence of rab5 on the phagosome. We have also shown that rab5 specifically binds with Sop E, a protein from Salmonella. Our results indicate that live Salmonella help binding of rab5 on the phagosomes, possibly activate the SNARE which leads to further recruitment of α-SNAP for subsequent binding with NSF to promote fusion of the LSP with early endosomes and inhibition of their transport to lysosomes.

scholarly journals A syntaxin 10–SNARE complex distinguishes two distinct transport routes from endosomes to the trans-Golgi in human cells

2008 ◽  
Vol 180 (1) ◽  
pp. 159-172 ◽  
Author(s):  
Ian G. Ganley ◽  
Eric Espinosa ◽  
Suzanne R. Pfeffer
Keyword(s):  
Human Cells ◽  
Endocytic Pathway ◽  
Snare Complex ◽  
Rab Gtpases ◽  
Protein Receptor ◽  
Transport Vesicles ◽  
Early Endosomes ◽  
Target Membrane ◽  
Guanosine Triphosphatase ◽  
Dependent Transport

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the Golgi after delivering lysosomal enzymes to the endocytic pathway. This process requires Rab9 guanosine triphosphatase (GTPase) and the putative tether GCC185. We show in human cells that a soluble NSF attachment protein receptor (SNARE) complex comprised of syntaxin 10 (STX10), STX16, Vti1a, and VAMP3 is required for this MPR transport but not for the STX6-dependent transport of TGN46 or cholera toxin from early endosomes to the Golgi. Depletion of STX10 leads to MPR missorting and hypersecretion of hexosaminidase. Mouse and rat cells lack STX10 and, thus, must use a different target membrane SNARE for this process. GCC185 binds directly to STX16 and is competed by Rab6. These data support a model in which the GCC185 tether helps Rab9-bearing transport vesicles deliver their cargo to the trans-Golgi and suggest that Rab GTPases can regulate SNARE–tether interactions. Importantly, our data provide a clear molecular distinction between the transport of MPRs and TGN46 to the trans-Golgi.

NSF is required for transport from early to late endosomes

1997 ◽  
Vol 110 (17) ◽  
pp. 2079-2087 ◽  
Author(s):  
L.J. Robinson ◽  
F. Aniento ◽  
J. Gruenberg
Keyword(s):  
Membrane Trafficking ◽  
Protein Transport ◽  
Degradation Pathway ◽  
Biochemical Properties ◽  
Endocytic Pathway ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Sensitive Factor ◽  
Endosomal Membranes

Protein transport between early and late endosomes is a major membrane trafficking pathway in the cell followed by many proteins, including all down-regulated receptors. Yet, little is known at the molecular level about the mechanisms regulating membrane interactions in the endocytic pathway beyond early endosomes. In this study, we have used an in vitro transport assay to study the biochemical properties of endosome docking/fusion events. Our data demonstrate that N-ethylmaleimide (NEM) sensitive factor (NSF) and its soluble associated proteins (SNAPs) are required for transport from early to late endosomes, as well as at all other steps of endosomal membrane transport. We also find that these proteins are enriched on endosomal membranes. In addition, our studies suggest that besides NSF/SNAPs, another NEM-sensitive component may also be involved in docking/fusion at this late stage of the pathway. Finally, we find that, in contrast to Golgi membranes, NSF association to both early and late endosomal membranes occurs via an ATP-independent mechanism, indicating that the binding properties of endosomal and biosynthetic NSF are different. Our data thus show that NSF/SNAPs, perhaps together with another NEM-sensitive factor, are part of the basic molecular machinery which controls docking/fusion events during transport from early to late endosomes, along the lysosomal degradation pathway.

scholarly journals Accumulation of rab4GTP in the Cytoplasm and Association with the Peptidyl-Prolyl Isomerase Pin1 during Mitosis

2000 ◽  
Vol 11 (7) ◽  
pp. 2201-2211 ◽  
Author(s):  
Lisya Gerez ◽  
Karin Mohrmann ◽  
Marcel van Raak ◽  
Mandy Jongeneelen ◽  
Xiao Zhen Zhou ◽  
...  
Keyword(s):  
Membrane Transport ◽  
Subcellular Fractionation ◽  
Effector Proteins ◽  
Endocytic Pathway ◽  
Prolyl Isomerase ◽  
Peptidyl Prolyl Isomerase ◽  
Transport Vesicles ◽  
Early Endosomes ◽  
Gtpase Cycle ◽  
Mitotic Cells

Transport through the endocytic pathway is inhibited during mitosis. The mechanism responsible for this inhibition is not understood. Rab4 might be one of the proteins involved as it regulates transport through early endosomes, is phosphorylated by p34cdc2 kinase, and is translocated from early endosomes to the cytoplasm during mitosis. We investigated the perturbation of the rab4 GTPase cycle during mitosis. Newly synthesized rab4 was less efficiently targeted to membranes during mitosis. By subcellular fractionation of mitotic cells, we found a large increase of cytosolic rab4 in the active GTP-form, an increase not associated with the cytosolic rabGDP chaperone GDI. Instead, phosphorylated rab4 is in a complex with the peptidyl-prolyl isomerase Pin1 during mitosis, but not during interphase. Our results show that less efficient recruitment of rab4 to membranes and a bypass of the normal GDI-mediated retrieval of rab4GDP from early endosomes reduce the amount of rab4GTP on membranes during mitosis. We propose that phosphorylation of rab4 inhibits both the recruitment of rab4 effector proteins to early endosomes and the docking of rab4-containing transport vesicles. This mechanism might contribute to the inhibition of endocytic membrane transport during mitosis.

Lysosome proteins are redistributed during expression of a GTP-hydrolysis-defective rab5a

2001 ◽  
Vol 114 (24) ◽  
pp. 4499-4508 ◽  
Author(s):  
Jennifer L. Rosenfeld ◽  
Robert H. Moore ◽  
K.-Peter Zimmer ◽  
Estrella Alpizar-Foster ◽  
Wenping Dai ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Endocytic Pathway ◽  
Cell Surface Receptor ◽  
Hek293 Cells ◽  
Rab Gtpases ◽  
Protein Distribution ◽  
Lysosome Biogenesis ◽  
Early Endosomes ◽  
Gradient Fractionation ◽  
Texas Red

The functioning of the endocytic pathway is influenced by a distinct set of rab GTPases, including rab5a, which regulates homotypic fusion of early endosomes. Expression of a dominant active, GTPase-defective rab5a accelerates endosome fusion, causing the formation of a greatly enlarged endocytic compartment. Here we present evidence that rab5a also regulates trafficking between endosomes and lysosomes and may play a role in lysosome biogenesis. The GTPase defective rab5aQ79L mutant was inducibly expressed as an EGFP fusion in HEK293 cells, and the distribution of lysosome proteins and endocytic markers then assessed by deconvolution fluorescence microscopy. During expression of EGFP-rab5aQ79L, the lysosome proteins LAMP-1, LAMP-2 and cathepsin D were found in dilated EGFP-rab5aQ79L-positive vesicles, which also rapidly labeled with transferrin Texas Red. Exogenous tracers that normally traffic to lysosomes after prolonged chase (dextran Texas Red and DiI-LDL) also accumulated in these vesicles. Dextran Texas Red preloaded into lysosomes localized with subsequently expressed EGFP-rab5a Q79L, suggesting the existence of lysosome to endosome traffic. Cells expressing EGFP-rab5a wt or the dominant negative EGFP-rab5aS34N did not exhibit these abnormalities. Despite the dramatic alterations in lysosome protein distribution caused by expression of EGFP-rab5a Q79L, there was little change in the endocytosis or recycling of a cell-surface receptor (β2-adrenergic receptor). However, there was a deficiency of dense β-hexosaminidase-containing lysosomes in cells expressing EGFP-rab5aQ79L, as assessed by Percoll gradient fractionation. These results suggest that expression of a GTPase-defective rab5a affects lysosome biogenesis by alteration of traffic between lysosomes and endosomes.

scholarly journals Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.

1996 ◽  
Vol 7 (3) ◽  
pp. 355-367 ◽  
Author(s):  
D J Spiro ◽  
W Boll ◽  
T Kirchhausen ◽  
M Wessling-Resnick
Keyword(s):  
Transferrin Receptor ◽  
Kinase Inhibitor ◽  
Morphological Changes ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Phosphatidylinositol 3 Kinase ◽  
Vitro Assay ◽  
Phosphatidylinositol 3

Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.

Sign in / Sign up

Export Citation Format

Share Document