The delivery of endocytosed cargo to lysosomes

2009 ◽  
Vol 37 (5) ◽  
pp. 1019-1021 ◽  
Author(s):  
J. Paul Luzio ◽  
Michael D.J. Parkinson ◽  
Sally R. Gray ◽  
Nicholas A. Bright
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Late Endosome ◽  
Snare Complex ◽  
Coated Pits ◽  
Free System ◽  
Endosomal Sorting ◽  
Protein Receptor ◽  
Late Endosomes ◽  
Early Endosomes

In mammalian cells, endocytosed cargo that is internalized through clathrin-coated pits/vesicles passes through early endosomes and then to late endosomes, before delivery to lysosomes for degradation by proteases. Late endosomes are MVBs (multivesicular bodies) with ubiquitinated membrane proteins destined for lysosomal degradation being sorted into their luminal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery. Cargo is delivered from late endosomes to lysosomes by kissing and direct fusion. These processes have been studied in live cell experiments and a cell-free system. Late endosome–lysosome fusion is preceded by tethering that probably requires mammalian orthologues of the yeast HOPS (homotypic fusion and vacuole protein sorting) complex. Heterotypic late endosome–lysosome membrane fusion is mediated by a trans-SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex comprising Syntaxin7, Vti1b, Syntaxin8 and VAMP7 (vesicle-associated membrane protein 7). This differs from the trans-SNARE complex required for homotypic late endosome fusion in which VAMP8 replaces VAMP7. VAMP7 is also required for lysosome fusion with the plasma membrane and its retrieval from the plasma membrane to lysosomes is mediated by its folded N-terminal longin domain. Co-ordinated interaction of the ESCRT, HOPS and SNARE complexes is required for cargo delivery to lysosomes.

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.

Endosomal system of Paramecium: coated pits to early endosomes

1992 ◽  
Vol 101 (2) ◽  
pp. 449-461 ◽  
Author(s):  
R.D. Allen ◽  
C.C. Schroeder ◽  
A.K. Fok
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Fluid Phase ◽  
Early Endosome ◽  
Coated Vesicles ◽  
Striking Similarity ◽  
Coated Pits ◽  
Early Endosomes ◽  
Membrane Components ◽  
Endosomal System

A detailed morphological and tracer study of endocytosis via coated pits in Paramecium multimicronucleatum was undertaken to compare endocytic processes in a free-living protozoon with similar processes in higher organisms. Permanent pits at the cell surface enlarge, become coated and give rise to coated vesicles (188 +/− 41 nm in diameter) that enclose fluid-phase markers such as horseradish peroxidase (HRP). Both the pits and vesicles are labeled by the immunogold technique when a monoclonal antibody (mAb) raised against the plasma membrane of this cell is applied to cryosections. The HRP is delivered to an early endosome compartment, which also shares the plasma membrane antigen. The early endosome, as shown in quick-freeze deep-etch replicas of chemically unfixed cells, is a definitive non-reticular compartment composed of many individual flattened cisternal units of 0.2 to 0.7 microns diameter, each potentially bearing one or more approximately 80-nm-wide coated evaginations. These coated evaginations on the early endosomes contain HRP but are not labeled by the mAb. The coated evaginations pinch off to form a second group of coated vesicles (90 +/− 17 nm in diameter), which can be differentiated from those formed from coated pits by their smaller size, absence of plasma membrane antigen and their location somewhat deeper into the cytoplasm. This study shows a striking similarity between protozoons and mammalian cells in their overall early endosomal machinery and in the ability of early endosomes to sort cargo from plasma membrane components. The vesicles identified in this study form two distinct populations of putative shuttle vesicles, pre-endosomal (large) and early endosome-derived vesicles (small), which facilitate incoming and outgoing traffic from the early endosomes.

scholarly journals Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana

2013 ◽  
Vol 24 (4) ◽  
pp. 510-520 ◽  
Author(s):  
Matyáš Fendrych ◽  
Lukáš Synek ◽  
Tamara Pečenková ◽  
Edita Janková Drdová ◽  
Juraj Sekereš ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Complex Dynamics ◽  
Active Regions ◽  
Snare Complex ◽  
Epifluorescence Microscopy ◽  
Rab Gtpases ◽  
Exocyst Complex ◽  
Protein Receptor ◽  
Outer Domain

The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6–green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.

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 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 A novel mass assay to quantify the bioactive lipid PtdIns3P in various biological samples

2012 ◽  
Vol 447 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Gaëtan Chicanne ◽  
Sonia Severin ◽  
Cécile Boscheron ◽  
Anne-Dominique Terrisse ◽  
Marie-Pierre Gratacap ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Liquid Chromatography ◽  
Biological Samples ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Human Platelets ◽  
Bhk Cells ◽  
Early Endosomes ◽  
Important Player ◽  
Mass Level

PtdIns3P is recognized as an important player in the control of the endocytotic pathway and in autophagy. Recent data also suggest that PtdIns3P contributes to molecular mechanisms taking place at the plasma membrane and at the midbody during cytokinesis. This lipid is present in low amounts in mammalian cells and remains difficult to quantify either by traditional techniques based on radiolabelling followed by HPLC to separate the different phosphatidylinositol monophosphates, or by high-sensitive liquid chromatography coupled to MS, which is still under development. In the present study, we describe a mass assay to quantify this lipid from various biological samples using the recombinant PtdIns3P 5-kinase, PIKfyve. Using this assay, we show an increase in the mass level of PtdIns3P in mouse and human platelets following stimulation, loss of this lipid in Vps34-deficient yeasts and its relative enrichment in early endosomes isolated from BHK cells.

scholarly journals Ordering the Final Events in Yeast Exocytosis

2000 ◽  
Vol 151 (2) ◽  
pp. 439-452 ◽  
Author(s):  
Eric Grote ◽  
Chavela M. Carr ◽  
Peter J. Novick
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Late Stage ◽  
Binding Protein ◽  
Secretory Vesicle ◽  
Snare Complex ◽  
Wild Type ◽  
Attachment Protein ◽  
Complex Assembly ◽  
Protein Receptor

In yeast, assembly of exocytic soluble N-ethylmaleimide–sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs Ssop and Sec9p occurs at a late stage of the exocytic reaction. Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion. By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly. In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane. Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.

scholarly journals Syntaxin 8 has two functionally distinct di-leucine-based motifs

2008 ◽  
Vol 13 (1) ◽  
Author(s):  
Kazuo Kasai ◽  
Kei Suga ◽  
Tetsuro Izumi ◽  
Kimio Akagawa
Keyword(s):  
Plasma Membrane ◽  
Intracellular Localization ◽  
Cytoplasmic Domain ◽  
Snare Complex ◽  
Transport Pathways ◽  
First Report ◽  
Late Endosomes

AbstractSyntaxin 8 has been shown to form the SNARE complex with syntaxin 7, vti1b and endobrevin. These have been shown to function as the machinery for the homotypic fusion of late endosomes. Recently, we showed that syntaxins 7 and 8 cycle through the plasma membrane, and that the di-leucine-based motifs in the cytoplasmic domain of syntaxins 7 and 8 respectively function in their endocytic and exocytic processes. However, we could not elucidate the mechanism by which syntaxin 8 cycles through the plasma membrane. In this study, we constructed several different syntaxin 8 molecules by mutating putative di-leucine-based motifs, and analyzed their intracellular localization and trafficking. We found a di-leucine-based motif in the cytoplasmic domain of syntaxin 8. It is similar to that of syntaxin 7, and functions in its endocytosis. These results suggest that in the cytoplasmic domain, syntaxin 8 has two functionally distinct di-leucine-based motifs that act independently in its endocytic and exocytic processes. This is the first report on two di-leucine-based motifs in the same molecule acting independently in distinct transport pathways.

scholarly journals The Tetraspanin CD63/lamp3 Cycles between Endocytic and Secretory Compartments in Human Endothelial Cells

2000 ◽  
Vol 11 (5) ◽  
pp. 1829-1843 ◽  
Author(s):  
Toshihide Kobayashi ◽  
Ulrich M. Vischer ◽  
Corinne Rosnoblet ◽  
Cécile Lebrand ◽  
Margaret Lindsay ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Late Endosome ◽  
Human Endothelial Cells ◽  
Late Endosomes ◽  
Lysobisphosphatidic Acid ◽  
Type C ◽  
Niemann Pick ◽  
Tetraspanin Cd63

In the present study, we show that in human endothelial cells the tetraspanin CD63/lamp3 distributes predominantly to the internal membranes of multivesicular–multilamellar late endosomes, which contain the unique lipid lysobisphosphatidic acid. Some CD63/lamp3 is also present in Weibel–Palade bodies, the characteristic secretory organelle of these cells. We find that CD63/lamp3 molecules can be transported from late endosomes to Weibel–Palade bodies and thus that CD63/lamp3 cycles between endocytic and biosynthetic compartments; however, movement of CD63/lamp3 is much slower than that of P-selectin, which is known to cycle between plasma membrane and Weibel–Palade bodies. When cells are treated with U18666A, a drug that mimics the Niemann-Pick type C syndrome, both proteins accumulate in late endosomes and fail to reach Weibel–Palade bodies efficiently, suggesting that P-selectin, like CD63/lamp3, cycles via late endosomes. Our data suggest that CD63/lamp3 partitions preferentially within late endosome internal membranes, thus causing its accumulation, and that this mechanism contributes to CD63/lamp3 retention in late endosomes; however, our data also indicate that the protein can eventually escape from these internal membranes and recycle toward Weibel–Palade bodies to be reused. Our observations thus uncover the existence of a selective trafficking route from late endosomes to Weibel–Palade bodies.

scholarly journals De novo formation of an early endosome during Rab5 to Rab7 transition

2020 ◽  
Author(s):  
Frode Miltzow Skjeldal ◽  
Linda Hofstad Haugen ◽  
Duarte Mateus ◽  
Dominik Frei ◽  
Oddmund Bakke
Keyword(s):  
De Novo ◽  
Early Endosome ◽  
Late Endosome ◽  
Second Phase ◽  
Specific Domain ◽  
Endosomal Membrane ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Main Determinants ◽  
Insight Into

AbstractRab5 and Rab7a are the main determinants of early and late endosomes and are important regulators of endosomal progression. The transport from early endosomes to late endosome seems to be regulated through an endosomal maturation switch where Rab5 is exchanged with Rab7a on the same endosome. Here we provide new insight into the mechanism of endosomal maturation where we have discovered a stepwise Rab5 detachment, sequentially regulated by Rab7a. The initial detachment of Rab5 is Rab7a independent and demonstrate a diffusion-like exchange between cytosol and endosomal membrane, and the second phase is slower where Rab5 converges into a specific domain that specifically detaches as a Rab5 indigenous endosome. Consequently, we show that early endosomal maturation regulated through the Rab5 to Rab7a switch induce the formation of a new fully functional early endosome. Progression through a stepwise early endosomal maturation regulates the direction of the transport and concomitantly regulates the homeostasis of early endosomes.

Sign in / Sign up

Export Citation Format

Share Document