Rab22a affects the morphology and function of the endocytic pathway

2001 ◽  
Vol 114 (22) ◽  
pp. 4041-4049 ◽  
Author(s):  
Rosana Mesa ◽  
Cristina Salomón ◽  
Marcelo Roggero ◽  
Philip D. Stahl ◽  
Luis S. Mayorga
Keyword(s):  
Fluorescent Protein ◽  
Fluid Phase ◽  
Small Gtpases ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Rab Proteins ◽  
Rab Protein ◽  
Late Endosomes ◽  
And Function ◽  
Negative Mutant

Soon after endocytosis, internalized material is sorted along different pathways in a process that requires the coordinated activity of several Rab proteins. Although abundant information is available about the subcellular distribution and function of some of the endocytosis-specific Rabs (e.g. Rab5 and Rab4), very little is known about some other members of this family of proteins. To unveil some of the properties of Rab22a, one of the less studied endosome-associated small GTPases, we have expressed the protein tagged with the green fluorescent protein in CHO cells. The results indicate that Rab22a associates with early and late endosomes (labeled by a 5 minute rhodamine-transferrin uptake and the cation-independent mannose 6-phosphate receptor, respectively) but not with lysosomes (labeled by 1 hour rhodamine horseradish peroxidase uptake followed by 1 hour chase). Overexpression of the protein causes a prominent morphological enlargement of the early and late endosomes. Two mutants were generated by site-directed mutagenesis, a negative mutant (Rab22aS19N, with reduced affinity for GTP) and a constitutively active mutant (Rab22aQ64L, with reduced endogenous GTPase activity). The distribution of the negative mutant was mostly cytosolic, whereas the positive mutant associated with early and late endosomes and, interestingly also with lysosomes and autophagosomes (labeled with monodansylcadaverine). Cells expressing Rab22a wild type and Rab22aS19N displayed decreased endocytosis of a fluid phase marker. Conversely, overexpression of Rab22aQ64L, which strongly affects the morphology of endosomes, did not inhibit bulk endocytosis. Our results show that Rab22a has a unique distribution along the endocytic pathway that is not shared by any other Rab protein, and that it strongly affects the morphology and function of endosomes.

Trafficking of interleukin 2 and transferrin in endosomal fractions of T lymphocytes

1994 ◽  
Vol 107 (5) ◽  
pp. 1289-1295 ◽  
Author(s):  
V. Duprez ◽  
M. Smoljanovic ◽  
M. Lieb ◽  
A. Dautry-Varsat
Keyword(s):  
Horseradish Peroxidase ◽  
Interleukin 2 ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Recycling Endosomes ◽  
High Affinity ◽  
Human T Cell ◽  
Late Endosomes ◽  
Discontinuous Sucrose Gradient ◽  
Acidic Organelles

The T lymphocyte growth factor interleukin 2 binds to surface high-affinity receptors and is rapidly internalized and degraded in acidic organelles. The alpha and beta chains of high-affinity interleukin 2 receptors are internalized together with interleukin 2. To identify the intracellular pathway followed by interleukin 2, we have compared the subcellular distribution of interleukin 2, transferrin and a fluid-phase marker, horseradish peroxidase, in the human T cell line IARC 301.5. Transferrin was used as a marker of early and recycling endosomes, and horseradish peroxidase to probe for the whole endocytic pathway. Fractionation of intracellular organelles on a discontinuous sucrose gradient showed that internalized interleukin 2 is initially mostly found in compartments with similar densities to transferrin, e.g. early and recycling endosomes. The kinetics of entry and exit of interleukin 2 from such organelles was much slower than that of transferrin. Later on, interleukin 2 is predominantly found in dense lysosome-containing fractions. Very little, if any, interleukin 2 was found in fractions corresponding to late endosomes containing horseradish peroxidase. These results suggest that, after endocytosis, interleukin 2 enters early or recycling endosomes before it reaches dense lysosomes.

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 Rab GDI: a solubilizing and recycling factor for rab9 protein.

1993 ◽  
Vol 4 (4) ◽  
pp. 425-434 ◽  
Author(s):  
T Soldati ◽  
M A Riederer ◽  
S R Pfeffer
Keyword(s):  
Complex Formation ◽  
Intracellular Transport ◽  
Rab Proteins ◽  
Rab Protein ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Geranylgeranyl Transferase ◽  
Carboxy Terminal ◽  
Golgi Network

Rab proteins are thought to function in the processes by which transport vesicles identify and/or fuse with their respective target membranes. The bulk of these proteins are membrane associated, but a measurable fraction can be found in the cytosol. The cytosolic forms of rab3A, rab11, and Sec4 occur as equimolar complexes with a class of proteins termed "GDIs," or "GDP dissociation inhibitors." We show here that the cytosolic form of rab9, a protein required for transport between late endosomes and the trans Golgi network, also occurs as a complex with a GDI-like protein, with an apparent mass of approximately 80 kD. Complex formation could be reconstituted in vitro using recombinant rab9 protein, cytosol, ATP, and geranylgeranyl diphosphate, and was shown to require an intact rab9 carboxy terminus, as well as rab9 geranylgeranylation. Monoprenylation was sufficient for complex formation because a mutant rab9 protein bearing the carboxy terminal sequence, CLLL, was prenylated in vitro by geranylgeranyl transferase I and was efficiently incorporated into 80-kD complexes. Purified, prenylated rab9 could also assemble into 80-kD complexes by addition of purified, rab3A GDI. Finally, rab3A-GDI had the capacity to solubilize rab9GDP, but not rab9GTP, from cytoplasmic membranes. These findings support the proposal that GDI proteins serve to recycle rab proteins from their target membranes after completion of a rab protein-mediated, catalytic cycle. Thus GDI proteins have the potential to regulate the availability of specific intracellular transport factors.

scholarly journals Annexin A8 Regulates Late Endosome Organization and Function

2008 ◽  
Vol 19 (12) ◽  
pp. 5267-5278 ◽  
Author(s):  
Verena Goebeler ◽  
Michaela Poeter ◽  
Dagmar Zeuschner ◽  
Volker Gerke ◽  
Ursula Rescher
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Actin Filaments ◽  
Late Endosome ◽  
Endocytic Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Late Endosomes ◽  
Endosomal Membranes ◽  
Epidermal Growth ◽  
And Function

Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. Late endosomes, organelles of the degradative lysosomal route, seem to require associated actin filaments for proper localization and function. We show here that the F-actin and phospholipid binding protein annexin A8 is associated specifically with late endosomes. Altering intracellular annexin A8 levels drastically affected the morphology and intracellular distribution of late endosomes. Trafficking through the degradative pathway was delayed in the absence of annexin A8, resulting in attenuated ligand-induced degradation of the epidermal growth factor receptor and prolonged epidermal growth factor-induced activation of mitogen-activated protein kinase. Depletion of annexin A8 reduced the association of late endosomal membranes with actin filaments. These results indicate that the defective cargo transport through the late endocytic pathway and the imbalanced signaling of activated receptors observed in the absence of annexin A8 results from the disturbed association of late endosomal membranes with the actin network, resulting in impaired actin-based late endosome motility.

scholarly journals Fusion of Lysosomes with Late Endosomes Produces a Hybrid Organelle of Intermediate Density and Is NSF Dependent

1998 ◽  
Vol 140 (3) ◽  
pp. 591-601 ◽  
Author(s):  
Barbara M. Mullock ◽  
Nicholas A. Bright ◽  
Clare W. Fearon ◽  
Sally R. Gray ◽  
J. Luzio
Keyword(s):  
Rat Liver ◽  
Gradient Centrifugation ◽  
Rab Protein ◽  
Late Endosomes ◽  
A Cell ◽  
Enzyme Formation ◽  
And Function ◽  
Gdp Dissociation Inhibitor ◽  
Brain Cytosol ◽  
Direct Fusion

Using a cell-free content mixing assay containing rat liver endosomes and lysosomes in the presence of pig brain cytosol, we demonstrated that after incubation at 37°C, late endosome–lysosome hybrid organelles were formed, which could be isolated by density gradient centrifugation. ImmunoEM showed that the hybrids contained both an endocytosed marker and a lysosomal enzyme. Formation of the hybrid organelles appeared not to require vesicular transport between late endosomes and lysosomes but occurred as a result of direct fusion. Hybrid organelles with similar properties were isolated directly from rat liver homogenates and thus were not an artifact of cell-free incubations. Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide–sensitive factor– dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein. We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes. The consequences of this fusion to the maintenance and function of lysosomes are discussed.

scholarly journals Dual Prenylation Is Required for Rab Protein Localization and Function

2003 ◽  
Vol 14 (5) ◽  
pp. 1852-1867 ◽  
Author(s):  
Monica Calero ◽  
Catherine Z. Chen ◽  
Wenyan Zhu ◽  
Nena Winand ◽  
Karyn A. Havas ◽  
...  
Keyword(s):  
Protein Localization ◽  
Sole Source ◽  
Small Gtpase ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Rab Protein ◽  
Correct Function ◽  
Stable Association ◽  
And Function ◽  
Organelle Membrane

The majority of Rab proteins are posttranslationally modified with two geranylgeranyl lipid moieties that enable their stable association with membranes. In this study, we present evidence to demonstrate that there is a specific lipid requirement for Rab protein localization and function. Substitution of different prenyl anchors on Rab GTPases does not lead to correct function. In the case of YPT1 and SEC4, two essential Rab genes in Saccharomyces cerevisiae, alternative lipid tails cannot support life when present as the sole source of YPT1 and SEC4. Furthermore, our data suggest that double geranyl-geranyl groups are required for Rab proteins to correctly localize to their characteristic organelle membrane. We have identified a factor, Yip1p that specifically binds the di-geranylgeranylated Rab and does not interact with mono-prenylated Rab proteins. This is the first demonstration that the double prenylation modification of Rab proteins is an important feature in the function of this small GTPase family and adds specific prenylation to the already known determinants of Rab localization.

scholarly journals The Role of Intraorganellar Ca2+In Late Endosome–Lysosome Heterotypic Fusion and in the Reformation of Lysosomes from Hybrid Organelles

2000 ◽  
Vol 149 (5) ◽  
pp. 1053-1062 ◽  
Author(s):  
Paul R. Pryor ◽  
Barbara M. Mullock ◽  
Nicholas A. Bright ◽  
Sally R. Gray ◽  
J. Paul Luzio
Keyword(s):  
Dynamic Equilibrium ◽  
Late Endosome ◽  
Endocytic Pathway ◽  
Free System ◽  
Rab Protein ◽  
Late Endosomes ◽  
The Reformation ◽  
A Cell ◽  
Gdp Dissociation Inhibitor

We have investigated the requirement for Ca2+ in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca2+. Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca2+ reduced the amount of fusion. The requirement for Ca2+ for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome–lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca2+ plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.

scholarly journals Bioinformatic and Comparative Localization of Rab Proteins Reveals Functional Insights into the Uncharacterized GTPases Ypt10p and Ypt11p

2006 ◽  
Vol 26 (19) ◽  
pp. 7299-7317 ◽  
Author(s):  
Stéphanie Buvelot Frei ◽  
Peter B. Rahl ◽  
Maria Nussbaum ◽  
Benjamin J. Briggs ◽  
Monica Calero ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Ectopic Expression ◽  
Rab Proteins ◽  
Sequence Classification ◽  
Rab Protein ◽  
Subcellular Membrane ◽  
Green Fluorescent ◽  
Expression In Mammalian Cells

ABSTRACT A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising multiple Rab orthologs from diverse species. We compared the locations of individual yeast Rab proteins with their localizations following ectopic expression in mammalian cells. Our results suggest that green fluorescent protein-tagged Rab proteins maintain localizations across large evolutionary distances and that the major known player in the Rab localization pathway, mammalian Rab-GDI, is able to function in yeast. These findings enable us to provide insight into novel gene functions and classify the uncharacterized Rab proteins Ypt10p (YBR264C) as being involved in endocytic function and Ypt11p (YNL304W) as being localized to the endoplasmic reticulum, where we demonstrate it is required for organelle inheritance.

Traffic to the endocytic compartment of plants

1989 ◽  
Vol 47 ◽  
pp. 754-755
Author(s):  
L. R. Griffing ◽  
R. D. Record ◽  
H. H. Mollenhauer
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Fluid Phase ◽  
Multivesicular Body ◽  
Endocytic Pathway ◽  
Cationized Ferritin ◽  
Plant Protoplasts ◽  
Whole Cells ◽  
Membrane Bound ◽  
And Function

The endocytic pathway of plants has been identified and partially characterized using nonspecific membrane-bound and fluid phase probes . The function of endocytosis in plants is, however, unknown. We shall describe how ultrastructural histochemistry, immunocytochemical analyses and fluorescence imaging have been used to explore the physiology and function of the endocytic pathway in plant protoplasts and whole cells.Cationized ferritin (CF) can be used as a marker of plasma membrane uptake in plant protoplasts. Several different organelles become labeled upon exposure of protoplasts to CF: clathrin-coated vesicles (CV), the partially coated reticulum (PCR), the Golgi complex (GC), the multivesicular body (MVB), and the vacuole (V). These organelles also participate in the pathways of secretion and delivery of protein to the lysosome (vacuole). What are the sites of overlap/divergence among the secretory, endocytic and lysosomal pathways in these cells?

Early endocytic Rabs: functional prediction to functional characterization.

2005 ◽  
Vol 72 ◽  
pp. 99-108 ◽  
Author(s):  
Jeremy C Simpson ◽  
Arwyn T Jones
Keyword(s):  
Membrane Trafficking ◽  
Functional Characterization ◽  
Specific Interaction ◽  
Small Gtpases ◽  
Endocytic Pathway ◽  
Functional Prediction ◽  
Final Destination ◽  
Similarities And Differences ◽  
And Function ◽  
Endocytic Pathways

Endocytic pathways are highly dynamic gateways for molecules to enter cells. Functionality and specificity is in part controlled by a number of small GTPases called Rabs. In defined cellular locations, Rabs mediate multiple functions in membrane trafficking via their specific interaction with organelle membranes and a host of affector and effector molecules. On endocytic pathways, Rabs have been shown to control the formation of vesicles on the plasma membrane and the downstream delivery of internalized molecules to a number of cellular locations. As numerous Rabs are located to endocytic pathways, an internalized molecule may traverse a number of Rab specific substations or subdomains en route to its final destination. Rabs 5, 21 and 22 have all been localized to the early endocytic pathway and have been shown to share a number of characteristics to merit their segregation into a single functional endocytic group. In this review, we compare experiments that describe similarities and differences in endosome morphology and function that is mediated by their expression in cells.

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