scholarly journals Rab22a Regulates the Sorting of Transferrin to Recycling Endosomes

2006 ◽  
Vol 26 (7) ◽  
pp. 2595-2614 ◽  
Author(s):  
Javier G. Magadán ◽  
M. Alejandro Barbieri ◽  
Rosana Mesa ◽  
Philip D. Stahl ◽  
Luis S. Mayorga
Keyword(s):  
Transferrin Receptor ◽  
Brefeldin A ◽  
Small Gtpases ◽  
Endocytic Pathway ◽  
Recycling Endosomes ◽  
Endogenous Protein ◽  
Plasma Membrane Receptor ◽  
Endocytic Compartments ◽  
Transferrin Uptake ◽  
Interfering Rna

ABSTRACT Rab22a is a member of the Rab family of small GTPases that localizes in the endocytic pathway. In CHO cells, expression of canine Rab22a (cRab22a) causes a dramatic enlargement of early endocytic compartments. We wondered whether transferrin recycling is altered in these cells. Expression of the wild-type protein and a GTP hydrolysis-deficient mutant led to the redistribution of transferrin receptor to large cRab22a-positive structures in the periphery of the cell and to a significant decrease in the plasma membrane receptor. Kinetic analysis of transferrin uptake indicates that internalization and early recycling were not affected by cRab22a expression. However, recycling from large cRab22a-positive compartments was strongly inhibited. A similar effect on transferrin transport was observed when human but not canine Rab22a was expressed in HeLa cells. After internalization for short periods of time (5 to 8 min) or at a reduced temperature (16°C), transferrin localized with endogenous Rab22a in small vesicles that did not tubulate with brefeldin A, suggesting that the endogenous protein is present in early/sorting endosomes. Rab22a depletion by small interfering RNA disorganized the perinuclear recycling center and strongly inhibited transferrin recycling. We speculate that Rab22a controls the transport of the transferrin receptor from sorting to recycling endosomes.

Molecular cloning and subcellular localization of three GTP-binding proteins of the rab subfamily

1993 ◽  
Vol 106 (4) ◽  
pp. 1249-1261 ◽  
Author(s):  
V.M. Olkkonen ◽  
P. Dupree ◽  
I. Killisch ◽  
A. Lutcke ◽  
M. Zerial ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Semliki Forest Virus ◽  
Small Gtpases ◽  
Endocytic Pathway ◽  
Mrna Levels ◽  
Vector Systems ◽  
Golgi Region ◽  
Endogenous Protein ◽  
Mouse Tissues ◽  
Intracellular Membrane Transport

Small GTPases of the rab subfamily are involved in regulation of intracellular membrane transport events. We recently used a PCR approach to isolate short cDNA fragments of a number of novel rab sequences. These PCR fragments have not been used with cDNA library screening and PCR-based techniques to clone the cDNAs encoding three of these proteins, rab12, rab22, and rab24. By northern blot analysis, the messages were found to be present in a wide variety of mouse tissues. However, quantitative differences in the mRNA levels between the tissues were detected. We determined the subcellular localization of the GTPases by expressing the c-myc epitope-tagged proteins with the Semliki Forest virus and the vaccinia T7 vector systems. Transiently expressed rab12 was localized to the Golgi complex. This localization was confirmed using a polyclonal anti-peptide antibody detecting the endogenous protein in BHK cells. rab22 expressed from the cDNA was localized to endosomal compartments and to the plasma membrane. After longer periods of expression, the protein was found on abnormally large perinuclear endosomal structures, suggesting that it is a potent regulator of events in the endocytic pathway. Finally, rab24 was found in the endoplasmic reticulum/cis-Golgi region and on late endosomal structures. The localization of rab24 may indicate its involvement in autophagy-related processes.

scholarly journals Regulation of transferrin receptor recycling by protein phosphorylation

1994 ◽  
Vol 303 (2) ◽  
pp. 647-655 ◽  
Author(s):  
J R Beauchamp ◽  
P G Woodman
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Cell Lines ◽  
Okadaic Acid ◽  
Transferrin Receptor ◽  
Kinase Inhibitors ◽  
K562 Cells ◽  
Protein Kinase Inhibitors ◽  
Endocytic Pathway ◽  
Transferrin Uptake

The effect of the protein phosphatase inhibitor okadaic acid on transferrin receptor internalization and recycling was examined in HeLa and K562 cells. Okadaic acid inhibited receptor uptake by more than 85% in both cell lines, whereas it affected transferrin recycling to differing degrees: recycling in HeLa cells was inhibited by greater than 90%, compared with only 65% in K562 cells. Okadaic acid also caused a marked redistribution of receptors in each cell line, which was accounted for by the difference in the extent to which transferrin uptake and recycling were inhibited. These effects were most likely mediated by a protein kinase, as they were delayed by 10-15 min and could be suppressed by prior incubation with certain protein kinase inhibitors. In addition, it was found that specific kinase inhibitors affected basal rates of transferrin uptake and recycling, although the extent of these effects differed between cell lines. Together, these results suggest that a complex pattern of protein phosphorylation influences the flux of the endocytic pathway in interphase cells.

scholarly journals The GTP/GDP Cycling of Rho GTPase TCL Is an Essential Regulator of the Early Endocytic Pathway

2003 ◽  
Vol 14 (12) ◽  
pp. 4846-4856 ◽  
Author(s):  
Marion de Toledo ◽  
Francesca Senic-Matuglia ◽  
Jean Salamero ◽  
Gilles Uze ◽  
Franck Comunale ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Early Endosome ◽  
Effector Proteins ◽  
Endocytic Pathway ◽  
Actin Dynamics ◽  
Recycling Endosomes ◽  
Downstream Effector ◽  
C Terminus ◽  
Interfering Rna

Rho GTPases are key regulators of actin dynamics. We report that the Rho GTPase TCL, which is closely related to Cdc42 and TC10, localizes to the plasma membrane and the early/sorting endosomes in HeLa cells, suggesting a role in the early endocytic pathway. Receptor-dependent internalization of transferrin (Tf) is unaffected by suppression of endogenous TCL by small interfering RNA treatment. However, Tf accumulates in Rab5-positive uncoated endocytic vesicles and fails to reach the early endosome antigen-1–positive early endosomal compartments and the pericentriolar recycling endosomes. Moreover, Tf release upon TCL knockdown is significantly slower. Conversely, in the presence of dominant active TCL, internalized Tf accumulates in early endosome antigen-1–positive early/sorting endosomes and not in perinuclear recycling endosomes. Tf recycles directly from the early/sorting endosomes and it is normally released by the cells. The same phenotype is generated by replacing the C terminus of dominant active Cdc42 and TC10 with that of TCL, indicating that all three proteins share downstream effector proteins. Thus, TCL is essential for clathrin-dependent endocytosed receptors to enter the early/sorting endosomes. Furthermore, the active GTPase favors direct recycling from early/sorting endosomes without accumulating in the perinuclear recycling endosomes.

scholarly journals Potential sites of PI-3 kinase function in the endocytic pathway revealed by the PI-3 kinase inhibitor, wortmannin.

1996 ◽  
Vol 132 (4) ◽  
pp. 595-605 ◽  
Author(s):  
H Shpetner ◽  
M Joly ◽  
D Hartley ◽  
S Corvera
Keyword(s):  
Mammalian Cells ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Lucifer Yellow ◽  
Brefeldin A ◽  
Endocytic Pathway ◽  
Subsequent Degradation ◽  
Pre Treatment ◽  
Transferrin Uptake

Previously we have shown that PDGF receptor mutants that do not bind PI-3 kinase internalize after ligand binding, but fail to downregulate and degrade. To define further the role of PI-3 kinase in trafficking processes in mammalian cells, we have investigated the effects of a potent inhibitor of PI-3 kinase activity, wortmannin. At nanomolar concentrations, wortmannin inhibited both the transfer of PDGF receptors from peripheral compartments to juxtanuclear vesicles, and their subsequent degradation. In contrast, the delivery of soluble phase markers to lysosomes, assessed by the accumulation of Lucifer yellow (LY) in perinuclear vesicles after 120 min of incubation, was not blocked by wortmannin. Furthermore, wortmannin did not affect the rate of transferrin uptake, and caused only a small decrease in its rate of recycling. Thus, the effects of wortmannin on PDGFr trafficking are much more pronounced than its effects on other endocytic events. Unexpectedly, wortmannin also caused a striking effect on the morphology of endosomal compartments, marked by tubulation and enlargement of endosomes containing transferrin or LY. This effect was somewhat similar to that produced by brefeldin A, and was also blocked by pre-treatment of cells with aluminum fluoride (AlF4-). These results suggest two sites in the endocytic pathway where PI-3 kinase activity may be required: (a) to sort PDGF receptors from peripheral compartments to the lysosomal degradative pathway; and (b) to regulate the structure of endosomes containing lysosomally directed and recycling molecules. This latter function could be mediated through the activation of AlFt4-)-sensitive GTP-binding proteins downstream of PI-3 kinase.

scholarly journals The Annexin 2/S100A10 Complex Controls the Distribution of Transferrin Receptor-containing Recycling Endosomes

2003 ◽  
Vol 14 (12) ◽  
pp. 4896-4908 ◽  
Author(s):  
Nicole Zobiack ◽  
Ursula Rescher ◽  
Carsten Ludwig ◽  
Dagmar Zeuschner ◽  
Volker Gerke
Keyword(s):  
Transferrin Receptor ◽  
S100 Protein ◽  
Lipid Binding ◽  
Recycling Endosomes ◽  
Morphological Alterations ◽  
Complex Functions ◽  
Annexin 2 ◽  
Endosomal Membranes ◽  
Transient Loss ◽  
Transferrin Uptake

The Ca2+- and lipid-binding protein annexin 2, which resides in a tight heterotetrameric complex with the S100 protein S100A10 (p11), has been implicated in the structural organization and dynamics of endosomal membranes. To elucidate the function of annexin 2 and S100A10 in endosome organization and trafficking, we used RNA-mediated interference to specifically suppress annexin 2 and S100A10 expression. Down-regulation of both proteins perturbed the distribution of transferrin receptor- and rab11-positive recycling endosomes but did not affect uptake into sorting endosomes. The phenotype was highly specific and could be rescued by reexpression of the N-terminal annexin 2 domain or S100A10 in annexin 2- or S100A10-depleted cells, respectively. Whole-mount immunoelectron microscopy of the aberrantly localized recycling endosomes in annexin 2/S100A10 down-regulated cells revealed extensively bent tubules and an increased number of endosome-associated clathrin-positive buds. Despite these morphological alterations, the kinetics of transferrin uptake and recycling was not affected to a significant extent, indicating that the proper positioning of recycling endosomes is not a rate-limiting step in transferrin recycling. The phenotype generated by this transient loss-of-protein approach shows for the first time that the annexin 2/S100A10 complex functions in the intracellular positioning of recycling endosomes and that both subunits are required for this activity.

scholarly journals Direct Pathway from Early/Recycling Endosomes to the Golgi Apparatus Revealed through the Study of Shiga Toxin B-fragment Transport

1998 ◽  
Vol 143 (4) ◽  
pp. 973-990 ◽  
Author(s):  
Frédéric Mallard ◽  
Claude Antony ◽  
Danièle Tenza ◽  
Jean Salamero ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Shiga Toxin ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Endocytic Pathway ◽  
Toxin B ◽  
Recycling Endosomes ◽  
Ultrastructural Studies ◽  
Late Endosomes ◽  
Rapid Kinetics

Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37°C, ultrastructural studies on cryosections failed to detect B-fragment–specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor–containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.

scholarly journals Proximal tubule transferrin uptake is modulated by cellular iron and mediated by apical membrane megalin–cubilin complex and transferrin receptor 1

2019 ◽  
Vol 294 (17) ◽  
pp. 7025-7036 ◽  
Author(s):  
Craig P. Smith ◽  
Wing-Kee Lee ◽  
Matthew Haley ◽  
Søren B. Poulsen ◽  
Frank Thévenod ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Transferrin Receptor ◽  
Apical Membrane ◽  
Cellular Iron ◽  
Transferrin Receptor 1 ◽  
Transferrin Uptake

scholarly journals Sequestration of cholesterol within the host late endocytic pathway restricts liver-stage Plasmodium development

2017 ◽  
Vol 28 (6) ◽  
pp. 726-735 ◽  
Author(s):  
Wiebke Petersen ◽  
Werner Stenzel ◽  
Olivier Silvie ◽  
Judith Blanz ◽  
Paul Saftig ◽  
...  
Keyword(s):  
Parasitophorous Vacuole ◽  
Parasite Burden ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Parasite Development ◽  
Liver Stage ◽  
Niemann Pick ◽  
Development Analysis ◽  
Endocytic Compartments

While lysosomes are degradative compartments and one of the defenses against invading pathogens, they are also hubs of metabolic activity. Late endocytic compartments accumulate around Plasmodium berghei liver-stage parasites during development, and whether this is a host defense strategy or active recruitment by the parasites is unknown. In support of the latter hypothesis, we observed that the recruitment of host late endosomes (LEs) and lysosomes is reduced in uis4− parasites, which lack a parasitophorous vacuole membrane protein and arrest during liver-stage development. Analysis of parasite development in host cells deficient for late endosomal or lysosomal proteins revealed that the Niemann–Pick type C (NPC) proteins, which are involved in cholesterol export from LEs, and the lysosome-associated membrane proteins (LAMP) 1 and 2 are important for robust liver-stage P. berghei growth. Using the compound U18666A, which leads to cholesterol sequestration in LEs similar to that seen in NPC- and LAMP-deficient cells, we show that the restriction of parasite growth depends on cholesterol sequestration and that targeting this process can reduce parasite burden in vivo. Taken together, these data reveal that proper LE and lysosome function positively contributes to liver-stage Plasmodium development.

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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