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 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 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.

scholarly journals Plasticity of early endosomes

1992 ◽  
Vol 103 (2) ◽  
pp. 335-348 ◽  
Author(s):  
R.G. Parton ◽  
P. Schrotz ◽  
C. Bucci ◽  
J. Gruenberg
Keyword(s):  
Fluid Phase ◽  
Early Endosome ◽  
Cross Linking ◽  
High Plasticity ◽  
Late Endosomes ◽  
Low Concentrations ◽  
Cell Surface Biotinylation ◽  
Early Endosomes

We observed that the structural organization of early endosomes was significantly modified after cell surface biotinylation followed by incubation in the presence of low concentrations of avidin. Under these conditions early endosomes increased in size to form structures which extended over several micrometers and which had an intra-luminal content with a characteristic electron-dense appearance. The modified early endosomes were not formed when either avidin or biotinylation was omitted, suggesting that they resulted from the cross-linking of internalized biotinylated proteins by avidin. Accumulation of a fluid-phase tracer was increased after the avidin-biotin treatment (145% after 45 min). Both recycling and transport to the late endosomes still occurred, albeit to a somewhat lower extent than in control cells. Quantitative electron microscopy showed that the volume of the endosomal compartment was increased approximately 1.5-fold but that the surface area of the compartment decreased relative to its volume after avidin-biotin treatment. Finally, overexpression of a rab5 mutant, which is known to inhibit early endosome fusion in vitro, prevented the formation of these structures in vivo and caused early endosome fragmentation. Altogether, our data suggest that early endosomes exhibit a high plasticity in vivo. Cross-linking appears to interfere with this dynamic process but does not arrest membrane traffic to/from early endosomes.

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.

Evolutionarily conserved structural and functional roles of the FYVE domain

2007 ◽  
Vol 74 ◽  
pp. 95-105 ◽  
Author(s):  
Akira Hayakawa ◽  
Susan Hayes ◽  
Deborah Leonard ◽  
David Lambright ◽  
Silvia Corvera
Keyword(s):  
Mammalian Cells ◽  
Electrostatic Interactions ◽  
Head Group ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Intact Cells ◽  
C Elegans ◽  
Fyve Domain ◽  
Early Endosomes

The FYVE domain is an approx. 80 amino acid motif that binds to the phosphoinositide PtdIns3P with high specificity and affinity. It is present in 38 predicted gene products within the human genome, but only in 12–13 in Caenorhabditis elegans and Drosophila melanogaster. Eight of these are highly conserved in all three organisms, and they include proteins that have not been characterized in any species. One of these, WDFY2, appears to play an important role in early endocytosis and was revealed in a RNAi (RNA interference) screen in C. elegans. Interestingly, some proteins contain FYVE-like domains in C. elegans and D. melanogaster, but have lost this domain during evolution. One of these is the homologue of Rabatin-5, a protein that, in mammalian cells, binds both Rab5 and Rabex-5, a guanine-nucleotide exchange factor for Rab5. Thus the Rabatin-5 homologue suggests that mechanisms to link PtdIns3P and Rab5 activation developed in evolution. In mammalian cells, these mechanisms are apparent in the existence of proteins that bind PtdIns3P and Rab GTPases, such as EEA1, Rabenosyn-5 and Rabip4′. Despite the comparable ability to bind to PtdIns3P in vitro, FYVE domains display widely variable abilities to interact with endosomes in intact cells. This variation is due to three distinct properties of FYVE domains conferred by residues that are not involved in PtdIns3P head group recognition: These properties are: (i) the propensity to oligomerize, (ii) the ability to insert into the membrane bilayer, and (iii) differing electrostatic interactions with the bilayer surface. The different binding properties are likely to regulate the extent and duration of the interaction of specific FYVE domain-containing proteins with early endosomes, and thereby their biological function.

scholarly journals Mycobacterium tuberculosisPhagosome Maturation Arrest: Mycobacterial Phosphatidylinositol Analog Phosphatidylinositol Mannoside Stimulates Early Endosomal Fusion

2004 ◽  
Vol 15 (2) ◽  
pp. 751-760 ◽  
Author(s):  
Isabelle Vergne ◽  
Rutilio A. Fratti ◽  
Preston J. Hill ◽  
Jennifer Chua ◽  
John Belisle ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Dependent Manner ◽  
Rab Gtpases ◽  
Phosphatidylinositol 3 Kinase ◽  
Host Membrane ◽  
Absolute Requirement ◽  
Early Endosomes ◽  
Phosphatidylinositol 3

Mycobacterium tuberculosis is a facultative intracellular pathogen that parasitizes macrophages by modulating properties of the Mycobacterium-containing phagosome. Mycobacterial phagosomes do not fuse with late endosomal/lysosomal organelles but retain access to early endosomal contents by an unknown mechanism. We have previously reported that mycobacterial phosphatidylinositol analog lipoarabinomannan (LAM) blocks a trans-Golgi network-to-phagosome phosphatidylinositol 3-kinase-dependent pathway. In this work, we extend our investigations of the effects of mycobacterial phosphoinositides on host membrane trafficking. We present data demonstrating that phosphatidylinositol mannoside (PIM) specifically stimulated homotypic fusion of early endosomes in an ATP-, cytosol-, and N-ethylmaleimide sensitive factor-dependent manner. The fusion showed absolute requirement for small Rab GTPases, and the stimulatory effect of PIM increased upon partial depletion of membrane Rabs with RabGDI. We found that stimulation of early endosomal fusion by PIM was higher when phosphatidylinositol 3-kinase was inhibited by wortmannin. PIM also stimulated in vitro fusion between model phagosomes and early endosomes. Finally, PIM displayed in vivo effects in macrophages by increasing accumulation of plasma membrane-endosomal syntaxin 4 and transferrin receptor on PIM-coated latex bead phagosomes. In addition, inhibition of phagosomal acidification was detected with PIM-coated beads. The effects of PIM, along with the previously reported action of LAM, suggest that M. tuberculosis has evolved a two-prong strategy to modify its intracellular niche: its products block acquisition of late endosomal/lysosomal constituents, while facilitating fusion with early endosomal compartments.

scholarly journals Fusion between Phagosomes, Early and Late Endosomes: A Role for Actin in Fusion between Late, but Not Early Endocytic Organelles

2004 ◽  
Vol 15 (1) ◽  
pp. 345-358 ◽  
Author(s):  
Rune Kjeken ◽  
Morten Egeberg ◽  
Anja Habermann ◽  
Mark Kuehnel ◽  
Pascale Peyron ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
De Novo ◽  
Light And Electron Microscopy ◽  
Early Endosome ◽  
Latex Bead ◽  
Fusion Partner ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Membrane Bound

Actin is implicated in membrane fusion, but the precise mechanisms remain unclear. We showed earlier that membrane organelles catalyze the de novo assembly of F-actin that then facilitates the fusion between latex bead phagosomes and a mixture of early and late endocytic organelles. Here, we correlated the polymerization and organization of F-actin with phagosome and endocytic organelle fusion processes in vitro by using biochemistry and light and electron microscopy. When membrane organelles and cytosol were incubated at 37°C with ATP, cytosolic actin polymerized rapidly and became organized into bundles and networks adjacent to membrane organelles. By 30-min incubation, a gel-like state was formed with little further polymerization of actin thereafter. Also during this time, the bulk of in vitro fusion events occurred between phagosomes/endocytic organelles. The fusion between latex bead phagosomes and late endocytic organelles, or between late endocytic organelles themselves was facilitated by actin, but we failed to detect any effect of perturbing F-actin polymerization on early endosome fusion. Consistent with this, late endosomes, like phagosomes, could nucleate F-actin, whereas early endosomes could not. We propose that actin assembled by phagosomes or late endocytic organelles can provide tracks for fusion-partner organelles to move vectorially toward them, via membrane-bound myosins, to facilitate fusion.

In silico approach for designing a novel recombinant fusion protein as a Candidate Vaccine against HPV

2020 ◽  
Vol 17 ◽  
Author(s):  
Mohsen Sisakht ◽  
Amir Mahmoodzadeh ◽  
Mohammadsaeid Zahedi ◽  
Davood Rostamzadeh ◽  
Amin Moradi Hasan-Abad ◽  
...  
Keyword(s):  
Immune Responses ◽  
Fusion Protein ◽  
In Silico ◽  
Precancerous Lesions ◽  
Candidate Vaccine ◽  
T Cell Epitopes ◽  
Binding Interaction ◽  
Hpv16 E6 ◽  
E7 Proteins

Background: Human papillomavirus (HPV) is the main biological agent causing sexually transmitted diseases (STDs), including precancerous lesions and several types of prevalent cancers. To date, numerous types of vaccines are designed to prevent high-risk HPV. However, their prophylactic effect is not the same and does not clear previous infections. Therefore, there is an urgent need for developing therapeutic vaccines that trigger cell-mediated immune responses for the treatment of HPV. The HPV16 E6 and E7 proteins are ideal targets for vaccine therapy against HPV. Fusion protein vaccines, which include both immunogenic interest protein and an adjuvant for augmenting the immunogenicity effects, are theoretically capable of guarantee the power of the immune system against HPV. Method: A vaccine construct, including HPV16 E6/E7 proteins along with a heat shock protein GP96 (E6/E7-NTGP96 construct), was designed using in silico methods. By the aid of the SWISS-MODEL server, the optimal 3D model of the designed vaccine was selected, followed by physicochemical and molecular parameters were performed using bioinformatics tools. Docking studies were done to evaluate the binding interaction of the vaccine. Allergenicity, immunogenicity, B, and T cell epitopes of the designed construct were predicted. Results: Immunological and structural computational results illustrated that our designed construct is potentially proper for stimulation of cellular and humoral immune responses against HPV. Conclusion: Computational studies showed that the E6/E7-NTGP96 construct is a promising candidate vaccine that needs further in vitro and in vivo evaluations.

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