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 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 Rab5 and Rab11 Are Required for Clathrin-Dependent Endocytosis of Japanese Encephalitis Virus in BHK-21 Cells

2017 ◽  
Vol 91 (19) ◽  
Author(s):  
Chun-Chun Liu ◽  
Yun-Na Zhang ◽  
Zhao-Yao Li ◽  
Jin-Xiu Hou ◽  
Jing Zhou ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Drug Targets ◽  
Encephalitis Virus ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Rab Proteins ◽  
Endosomal Trafficking ◽  
Rab Gtpases

ABSTRACT During infection Japanese encephalitis virus (JEV) generally enters host cells via receptor-mediated clathrin-dependent endocytosis. The trafficking of JEV within endosomes is controlled by Rab GTPases, but which Rab proteins are involved in JEV entry into BHK-21 cells is unknown. In this study, entry and postinternalization of JEV were analyzed using biochemical inhibitors, RNA interference, and dominant negative (DN) mutants. Our data demonstrate that JEV entry into BHK-21 cells depends on clathrin, dynamin, and cholesterol but not on caveolae or macropinocytosis. The effect on JEV infection of dominant negative (DN) mutants of four Rab proteins that regulate endosomal trafficking was examined. Expression of DN Rab5 and DN Rab11, but not DN Rab7 and DN Rab9, significantly inhibited JEV replication. These results were further tested by silencing Rab5 or Rab11 expression before viral infection. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab11 within 15 min after virus entry, suggesting that after internalization JEV moves to early and recycling endosomes before the release of the viral genome. Our findings demonstrate the roles of Rab5 and Rab11 on JEV infection of BHK-21 cells through the endocytic pathway, providing new insights into the life cycle of flaviviruses. IMPORTANCE Although Japanese encephalitis virus (JEV) utilizes different endocytic pathways depending on the cell type being infected, the detailed mechanism of its entry into BHK-21 cells is unknown. Understanding the process of JEV endocytosis and postinternalization will advance our knowledge of JEV infection and pathogenesis as well as provide potential novel drug targets for antiviral intervention. With this objective, we used systematic approaches to dissect this process. The results show that entry of JEV into BHK-21 cells requires a low-pH environment and that the process occurs through dynamin-, actin-, and cholesterol-dependent clathrin-mediated endocytosis that requires Rab5 and Rab11. Our work provides a detailed picture of the entry of JEV into BHK-21 cells and the cellular events that follow.

scholarly journals Membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway

2004 ◽  
Vol 166 (5) ◽  
pp. 645-651 ◽  
Author(s):  
Laurence Abrami ◽  
Margaret Lindsay ◽  
Robert G. Parton ◽  
Stephen H. Leppla ◽  
F. Gisou van der Goot
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Endocytic Pathway ◽  
Cell Surface Receptor ◽  
Membrane Insertion ◽  
Edema Factor ◽  
Early Endosomes ◽  
Surface Receptor ◽  
A Cell ◽  
Anthrax Protective Antigen

The protective antigen (PA) of anthrax toxin binds to a cell surface receptor, undergoes heptamerization, and binds the enzymatic subunits, the lethal factor (LF) and the edema factor (EF). The resulting complex is then endocytosed. Via mechanisms that depend on the vacuolar ATPase and require membrane insertion of PA, LF and EF are ultimately delivered to the cytoplasm where their targets reside. Here, we show that membrane insertion of PA already occurs in early endosomes, possibly only in the multivesicular regions, but that subsequent delivery of LF to the cytoplasm occurs preferentially later in the endocytic pathway and relies on the dynamics of internal vesicles of multivesicular late endosomes.

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 Endocytic Trafficking Routes of Wild Type and ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator

2004 ◽  
Vol 15 (6) ◽  
pp. 2684-2696 ◽  
Author(s):  
Martina Gentzsch ◽  
Xiu-Bao Chang ◽  
Liying Cui ◽  
Yufeng Wu ◽  
Victor V. Ozols ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Secretory Pathway ◽  
Endocytic Pathway ◽  
Rab Gtpases ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Early Endosomes ◽  
Δf508 Cftr ◽  
Cystic Fibrosis Transmembrane

Intracellular trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) is a focus of attention because it is defective in most patients with cystic fibrosis. ΔF508 CFTR, which does not mature conformationally, normally does not exit the endoplasmic reticulum, but if induced to do so at reduced temperature is short-lived at the surface. We used external epitope-tagged constructs to elucidate the itinerary and kinetics of wild type and ΔF508 CFTR in the endocytic pathway and visualized movement of CFTR from the surface to intracellular compartments. Modulation of different endocytic steps with low temperature (16°C) block, protease inhibitors, and overexpression of wild type and mutant Rab GTPases revealed that surface CFTR enters several different routes, including a Rab5-dependent initial step to early endosomes, then either Rab11-dependent recycling back to the surface or Rab7-regulated movement to late endosomes or alternatively Rab9-mediated transit to the trans-Golgi network. Without any of these modulations ΔF508 CFTR rapidly disappears from and does not return to the cell surface, confirming that its altered structure is detected in the distal as well as proximal secretory pathway. Importantly, however, the mutant protein can be rescued at the plasma membrane by Rab11 overexpression, proteasome inhibitors, or inhibition of Rab5-dependent endocytosis.

scholarly journals Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex

2001 ◽  
Vol 12 (7) ◽  
pp. 2219-2228 ◽  
Author(s):  
Roger Lippé ◽  
Marta Miaczynska ◽  
Vladimir Rybin ◽  
Anja Runge ◽  
Marino Zerial
Keyword(s):  
Complex Formation ◽  
Rho Gtpases ◽  
Endocytic Pathway ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Physical Association ◽  
Nucleotide Exchange Factor ◽  
Early Endosomes ◽  
Gtpase Activation

Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases.

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.

scholarly journals Rab7: A Key to Lysosome Biogenesis

2000 ◽  
Vol 11 (2) ◽  
pp. 467-480 ◽  
Author(s):  
Cecilia Bucci ◽  
Peter Thomsen ◽  
Paolo Nicoziani ◽  
Janice McCarthy ◽  
Bo van Deurs
Keyword(s):  
Fluorescent Protein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Mutant Proteins ◽  
Lysosome Biogenesis ◽  
Late Endosomes ◽  
Early Endosomes ◽  
The Individual

The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)–tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein–1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and thetrans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment.

scholarly journals A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium

2007 ◽  
Vol 176 (3) ◽  
pp. 263-268 ◽  
Author(s):  
Adam C. Smith ◽  
Won Do Heo ◽  
Virginie Braun ◽  
Xiuju Jiang ◽  
Chloe Macrae ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Dominant Negative ◽  
Rab Gtpases ◽  
Wild Type ◽  
Intracellular Growth ◽  
Non Invasive ◽  
Serovar Typhimurium ◽  
Guanosine Triphosphatase ◽  
Kinetics Of

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome–lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.

scholarly journals Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments.

1993 ◽  
Vol 177 (3) ◽  
pp. 583-596 ◽  
Author(s):  
P Romagnoli ◽  
C Layet ◽  
J Yewdell ◽  
O Bakke ◽  
R N Germain
Keyword(s):  
Major Histocompatibility Complex ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Invariant Chain ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endocytic Compartments

Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the characteristics of late endosomes/prelysosomes. The Ii chains in these late endocytic vesicles have undergone proteolytic cleavage in the lumenal region postulated to control MHC class II peptide binding. These data indicate that the association of class II with Ii results in initial movement to early endosomes. At high levels of Ii expression, egress to later endocytic compartments is delayed and class II-Ii complexes accumulate together with endocytosed material. At lower levels of Ii expression, class II-Ii complexes are found primarily in late endosomes/prelysosomes. These data provide evidence that the route of class II transport to the site of antigen processing and loading involves movement through early endosomes to late endosomes/prelysosomes. Our results also reveal an unexpected ability of intact Ii to modify the structure and function of the early endosomal compartment, which may play a role in regulating this processing pathway.

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