scholarly journals GLUT4 and Transferrin Receptor Are Differentially Sorted Along the Endocytic Pathway in CHO Cells

1998 ◽  
Vol 140 (3) ◽  
pp. 565-575 ◽  
Author(s):  
Maria L. Wei ◽  
Frank Bonzelius ◽  
Rebecca M. Scully ◽  
Regis B. Kelly ◽  
Gary A. Herman
Keyword(s):  
Cell Surface ◽  
Cho Cells ◽  
Transferrin Receptor ◽  
Synaptic Vesicles ◽  
Glucose Transporter ◽  
Immunofluorescence Microscopy ◽  
Neuroendocrine Cells ◽  
Endocytic Pathway ◽  
Coat Proteins ◽  
Double Labeling

The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750–12754.). In this study, we demonstrate that at 37°C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37°C, cell surface–labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15°C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37°C after accumulating labeled GLUT4 at 15°C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15°C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37°C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15° and 37°C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

scholarly journals Phorbol ester treatment increases the exocytic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation.

1988 ◽  
Vol 106 (4) ◽  
pp. 1061-1066 ◽  
Author(s):  
T E McGraw ◽  
K W Dunn ◽  
F R Maxfield
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Phorbol Ester ◽  
Cho Cells ◽  
Transferrin Receptor ◽  
Phosphorylation Site ◽  
Major Protein ◽  
Kinase C ◽  
Recycling Pathway

In Chinese hamster ovary (CHO) fibroblast cells the protein kinase C activating phorbol ester, phorbol myristate acetate (PMA), stimulates an increase in cell surface transferrin receptor (TR) expression by increasing the exocytic rate of the recycling pathway. The human TR expressed in CHO cells is similarly affected by PMA treatment. A mutant human TR in which the major protein kinase C phosphorylation site, serine 24, has been replaced with the non-phosphorylatable amino acid glycine has been constructed to investigate the role of receptor phosphorylation in the PMA induced up-regulation. The Gly-24-substituted receptor binds, internalizes, and recycles Tf. Furthermore, the altered receptor mediates cellular Fe accumulation from diferric-Tf, thereby fulfilling the receptor's major biological role. The Gly-24 TR behaves identically to the wild-type TR when cells are treated with PMA. Therefore, Ser-24 phosphorylation is not required for the PMA-induced redistribution of the human TR expressed in CHO cells. The increased TR expression on the cell surface after PMA treatment results from an increase in the rate of exocytosis of the recycling receptors. No change in the endocytic rate or the size of the recycling receptor pool was observed. These results indicate that the PMA effect on the TR surface expression may result from a more general perturbation of membrane trafficking rather than a specific modulation of the TR.

scholarly journals The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin-sensitive cells.

1996 ◽  
Vol 134 (3) ◽  
pp. 625-635 ◽  
Author(s):  
S Martin ◽  
J Tellam ◽  
C Livingstone ◽  
J W Slot ◽  
G W Gould ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Transferrin Receptor ◽  
Glucose Transporter ◽  
Intracellular Distribution ◽  
Expression Library ◽  
Recycling Endosomes ◽  
Glut 4 ◽  
Intracellular Compartments ◽  
Intracellular Vesicles

Insulin stimulates glucose transport in adipocytes by translocation of the glucose transporter (GLUT-4) from an intracellular site to the cell surface. We have characterized different synaptobrevin/vesicle-associated membrane protein (VAMP) homologues in adipocytes and studied their intracellular distribution with respect to GLUT-4. VAMP-1, VAMP-2, and cellubrevin cDNAs were isolated from a 3T3-L1 adipocyte expression library. VAMP-2 and cellubrevin were: (a) the most abundant isoforms in adipocytes, (b) detectable in all insulin responsive tissues, (c) translocated to the cell surface in response to insulin, and (d) found in immunoadsorbed GLUT-4 vesicles. To further define their intracellular distribution, 3T3-L1 adipocytes were incubated with a transferrin/HRP conjugate (Tf/HRP) and endosomes ablated following addition of DAB and H2O2. While this resulted in ablation of > 90% of the transferrin receptor (TfR) and cellubrevin found in intracellular membranes, 60% of GLUT-4 and 90% of VAMP-2 was not ablated. Immuno-EM on intracellular vesicles from adipocytes revealed that VAMP-2 was colocalized with GLUT-4, whereas only partial colocalization was observed between GLUT-4 and cellubrevin. These studies show that two different v-SNAREs, cellubrevin and VAMP-2, are partially segregated in different intracellular compartments in adipocytes, implying that they may define separate classes of secretory vesicles in these cells. We conclude that a proportion of GLUT-4 is found in recycling endosomes in nonstimulated adipocytes together with cellubrevin and the transferrin receptor. In addition, GLUT-4 and VAMP-2 are selectively enriched in a postendocytic compartment. Further study is required to elucidate the function of this latter compartment in insulin-responsive cells.

scholarly journals Dominant-Interfering Hsc70 Mutants Disrupt Multiple Stages of the Clathrin-Coated Vesicle Cycle in Vivo

2001 ◽  
Vol 152 (3) ◽  
pp. 607-620 ◽  
Author(s):  
Sherri L. Newmyer ◽  
Sandra L. Schmid
Keyword(s):  
Cell Surface ◽  
Transferrin Receptor ◽  
Initial Rate ◽  
Coated Vesicle ◽  
Coat Proteins ◽  
Heat Shock Cognate Protein ◽  
Vesicle Cycle ◽  
Cognate Protein ◽  
Multiple Stages

Within the clathrin-coated vesicle (CCV) cycle, coat assembly drives the internalization of receptors from the cell surface and disassembly allows for the processing of internalized ligands. The heat shock cognate protein, hsc70, has been implicated in regulating coat disassembly. We find that in cells overexpressing ATPase-deficient hsc70 mutants, uncoating of CCVs is inhibited in vivo, and the majority of unassembled cytosolic clathrin shifts to an assembled pool that cofractionates with AP1 and AP2. Surprisingly, this assembled pool of coat proteins accumulates in the absence of cargo receptors, suggesting that disruption of hsc70 activity may cause misassembly of empty clathrin cages. The strongest effect of overexpression of hsc70 mutants is a block in transferrin receptor (TfnR) recycling, which cannot be accounted for by the degree of inhibition of uncoating of endocytic CCVs. These results suggest that hsc70 participates in multiple transport and/or sorting events between endosomal compartments. Additionally, the mutant-expressing cells are defective at internalizing transferrin. In the most potent case, the initial rate of uptake is inhibited 10-fold, and TfnR levels double at the cell surface. Our findings demonstrate that hsc70 indeed regulates coat disassembly and also suggest that this chaperone broadly modulates clathrin dynamics throughout the CCV cycle.

scholarly journals The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus

1992 ◽  
Vol 117 (4) ◽  
pp. 729-743 ◽  
Author(s):  
RC Piper ◽  
C Tai ◽  
JW Slot ◽  
CS Hahn ◽  
CM Rice ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transport ◽  
Cho Cells ◽  
Sindbis Virus ◽  
Glucose Transporter ◽  
Intracellular Compartment ◽  
Glut 1 ◽  
Glut 4 ◽  
Intracellular Sequestration

GLUT-4 is the major facilitative glucose transporter isoform in tissues that exhibit insulin-stimulated glucose transport. Insulin regulates glucose transport by the rapid translocation of GLUT-4 from an intracellular compartment to the plasma membrane. A critical feature of this process is the efficient exclusion of GLUT-4 from the plasma membrane in the absence of insulin. To identify the amino acid domains of GLUT-4 which confer intracellular sequestration, we analyzed the subcellular distribution of chimeric glucose transporters comprised of GLUT-4 and a homologous isoform, GLUT-1, which is found predominantly at the cell surface. These chimeric transporters were transiently expressed in CHO cells using a double subgenomic recombinant Sindbis virus vector. We have found that wild-type GLUT-4 is targeted to an intracellular compartment in CHO cells which is morphologically similar to that observed in adipocytes and muscle cells. Sindbis virus-produced GLUT-1 was predominantly expressed at the cell surface. Substitution of the GLUT-4 amino-terminal region with that of GLUT-1 abolished the efficient intracellular sequestration of GLUT-4. Conversely, substitution of the NH2 terminus of GLUT-1 with that of GLUT-4 resulted in marked intracellular sequestration of GLUT-1. These data indicate that the NH2-terminus of GLUT-4 is both necessary and sufficient for intracellular sequestration.

scholarly journals GLUT4 Retention in Adipocytes Requires Two Intracellular Insulin-regulated Transport Steps

2002 ◽  
Vol 13 (7) ◽  
pp. 2421-2435 ◽  
Author(s):  
Anja Zeigerer ◽  
Michael A. Lampson ◽  
Ola Karylowski ◽  
David D. Sabatini ◽  
Milton Adesnik ◽  
...  
Keyword(s):  
Cell Surface ◽  
Transferrin Receptor ◽  
Glucose Transporter ◽  
Trafficking Pathway ◽  
Endosomal Recycling ◽  
Step Model ◽  
Recycling Pathway ◽  
Endosomal Pathway ◽  
Endosomal System ◽  
Regulated Trafficking

Insulin regulates glucose uptake into fat and muscle by modulating the distribution of the GLUT4 glucose transporter between the surface and interior of cells. The GLUT4 trafficking pathway overlaps with the general endocytic recycling pathway, but the degree and functional significance of the overlap are not known. In this study of intact adipocytes, we demonstrate, by using a compartment-specific fluorescence-quenching assay, that GLUT4 is equally distributed between two intracellular pools: the transferrin receptor-containing endosomes and a specialized compartment that excludes the transferrin receptor. These pools of GLUT4 are in dynamic communication with one another and with the cell surface. Insulin-induced redistribution of GLUT4 to the surface requires mobilization of both pools. These data establish a role for the general endosomal system in the specialized, insulin-regulated trafficking of GLUT4. Trafficking through the general endosomal system is regulated by rab11. Herein, we show that rab11 is required for the transport of GLUT4 from endosomes to the specialized compartment and for the insulin-induced translocation to the cell surface, emphasizing the importance of the general endosomal pathway in the specialized trafficking of GLUT4. Based on these findings we propose a two-step model for GLUT4 trafficking in which the general endosomal recycling compartment plays a specialized role in the insulin-regulated traffic of GLUT4. This compartment-based model provides the framework for understanding insulin-regulated trafficking at a molecular level.

scholarly journals Synaptic-like Microvesicles of Neuroendocrine Cells Originate from a Novel Compartment That Is Continuous with the Plasma Membrane and Devoid of Transferrin Receptor

1997 ◽  
Vol 137 (2) ◽  
pp. 445-458 ◽  
Author(s):  
Anne Schmidt ◽  
Matthew J. Hannah ◽  
Wieland B. Huttner
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Transferrin Receptor ◽  
Synaptic Vesicles ◽  
Membrane System ◽  
Neuroendocrine Cells ◽  
Immunogold Electron Microscopy ◽  
Cell Surface Biotinylation ◽  
Digitonin Permeabilization ◽  
Triton X

We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18°C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18°C with the membraneimpermeant, cleavable sulfo-NHS-SS–biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37°C originated exclusively from the membranes containing the MesNaaccessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37°C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18° or 37°C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18°C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.

scholarly journals GLUT-4 NH2 terminus contains a phenylalanine-based targeting motif that regulates intracellular sequestration.

1993 ◽  
Vol 121 (6) ◽  
pp. 1221-1232 ◽  
Author(s):  
R C Piper ◽  
C Tai ◽  
P Kulesza ◽  
S Pang ◽  
D Warnock ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Cho Cells ◽  
Sindbis Virus ◽  
Glucose Transporter ◽  
Expression System ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Glut 4 ◽  
Intracellular Sequestration

Expression of chimeras, composed of portions of two different glucose transporter isoforms (GLUT-1 and GLUT-4), in CHO cells had indicated that the cytoplasmic NH2 terminus of GLUT-4 contains important targeting information that mediates intracellular sequestration of this isoform (Piper, R. C., C. Tai, J. W. Slot, C. S. Hahn, C. M. Rice, H. Huang, D. E. James. 1992. J. Cell Biol. 117:729-743). In the present studies, the amino acid constituents of the GLUT-4 NH2-terminal targeting domain have been identified. GLUT-4 constructs containing NH2-terminal deletions or alanine substitutions within the NH2 terminus were expressed in CHO cells using a Sindbis virus expression system. Deletion of eight amino acids from the GLUT-4 NH2 terminus or substituting alanine for phenylalanine at position 5 in GLUT-4 resulted in a marked accumulation of the transporter at the plasma membrane. Mutations at other amino acids surrounding Phe5 also caused increased cell surface expression of GLUT-4 but not to the same extent as the Phe5 mutation. GLUT-4 was also localized to clathrin lattices and this colocalization was abolished when either the first 13 amino acids were deleted or when Phe5 was changed to alanine. To ascertain whether the targeting information within the GLUT-4 NH2-terminal targeting domain could function independently of the glucose transporter structure this domain was inserted into the cytoplasmic tail of the H1 subunit of the asialoglycoprotein receptor. H1 with the GLUT-4 NH2 terminus was predominantly localized to an intracellular compartment similar to GLUT-4 and was sequestered more from the cell surface than was the wild-type H1 protein. It is concluded that the NH2 terminus of GLUT-4 contains a phenylalanine-based targeting motif that mediates intracellular sequestration at least in part by facilitating interaction of the transporter with endocytic machinery located at the cell surface.

scholarly journals Multiple endosomal recycling pathways in rat adipose cells

1998 ◽  
Vol 331 (3) ◽  
pp. 829-835 ◽  
Author(s):  
Konstantin V. KANDROR ◽  
Paul F. PILCH
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Insulin Receptor ◽  
Transferrin Receptor ◽  
Glucose Transporter ◽  
Sedimentation Coefficient ◽  
Membrane Vesicles ◽  
Average Diameter ◽  
Subcellular Fractionation ◽  
Adipose Cells

Adipose and skeletal-muscle cells can translocate several membrane proteins from intracellular compartment(s) to the cell surface in an insulin-dependent fashion. Among these proteins is Glut4, a physiologically important glucose transporter which mediates insulin's effect on blood glucose clearance. Under basal conditions, Glut4 is localized in uniform, intracellular membrane vesicles with an average diameter of 50–70 nm and a sedimentation coefficient of 100–120 S. The nature of this compartment and its trafficking pathway to the plasma membrane is still unresolved. We show here that, in addition to Glut4, the aminopeptidase gp160 or insulin-responsive aminopeptidase (‘IRAP’), sortilin, and an acutely recycling population of the insulin-like growth factor-II/mannose 6-phosphate receptor, this compartment includes 60% of the intracellular population of the transferrin receptor. We used subcellular fractionation, cell-surface biotinylation, and radioactive-ligand (125I-transferrin) uptake to demonstrate that the transferrin receptor recycles between this compartment and the plasma membrane in response to insulin along with Glut4 and other protein components of these vesicles. The co-localization of Glut4 and several endosomal markers in the terminally differentiated fat-cells during several stages of their cycling pathways suggests that the ‘Glut4 pathway ’ may derive from the hormone-insensitive endosomes of undifferentiated preadipocytes. The insulin receptor is excluded from Glut4-containing vesicles in both insulin-stimulated and unstimulated adipocytes, and thus it is likely to traffic independently from Glut4 through different intracellular compartments. Our data show that, in adipose cells, the ligand-dependent recycling pathway of the insulin receptor is structurally separated from the ligand-independent pathway of the transferrin receptor, and that Glut4 is specifically targetted to the latter.

SFV infection in CHO cells: cell-type specific restrictions to productive virus entry at the cell surface

1997 ◽  
Vol 110 (1) ◽  
pp. 95-103 ◽  
Author(s):  
M. Marsh ◽  
R. Bron
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Semliki Forest Virus ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Endocytic Pathway ◽  
Baby Hamster Kidney

Alphaviruses, such as Semliki Forest virus, normally enter cells by penetration from acidic organelles of the endocytic pathway. The virions are internalised intact from the cell surface before undergoing acid-induced fusion in endosomes. To investigate the possibility that endocytosis might play a role in delivering virions to specific sites for replication, we compared SFV infection of baby hamster kidney (BHK) cells and Chinese hamster ovary (CHO) cells following either normal virus fusion in endosomes or experimentally-induced fusion at the cell surface. Whereas baby hamster kidney cells were infected efficiently following fusion in endosomes or at the plasma membrane, Chinese hamster ovary cells were only infected following fusion from endocytic organelles. Virions fused at the plasma membrane of CHO cells failed to initiate viral RNA and protein synthesis. Similar results were observed when CHO cells were challenged with a rhabdovirus, vesicular stomatitis virus. These data suggest that in certain cell types a barrier, other than the plasma membrane, can prevent infection by alpha- and rhabdoviruses fused at the cell surface. Moreover, they suggest the endocytic pathway provides a mechanism for bringing viral particles to a site, or sites, in the cell where replication can proceed.

scholarly journals Exofacial epitope-tagged glucose transporter chimeras reveal COOH-terminal sequences governing cellular localization.

1993 ◽  
Vol 123 (1) ◽  
pp. 127-135 ◽  
Author(s):  
M P Czech ◽  
A Chawla ◽  
C W Woon ◽  
J Buxton ◽  
M Armoni ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cho Cells ◽  
Cellular Localization ◽  
Glucose Transporter ◽  
Intracellular Localization ◽  
Similar Data ◽  
Amino Acid Residues ◽  
Structural Elements ◽  
Residue Substitution ◽  
Glucose Transporter Glut4

The insulin-regulated adipocyte/skeletal muscle glucose transporter (GLUT4) displays a characteristic steady-state intracellular localization under basal conditions, whereas the erythrocyte/brain transporter isoform (GLUT1) distributes mostly to the cell surface. To identify possible structural elements in these transporter proteins that determine their cellular localization, GLUT1/GLUT4 chimera cDNA constructs that contain the hemagglutinin epitope YPYDVPDYA (HA) in their major exofacial loops were engineered. Binding of monoclonal anti-HA antibody to non-permeabilized COS-7 cells expressing HA-tagged transporter chimeras revealed that expression of transporters on the cell surface was strongly influenced by their cytoplasmic COOH-terminal domain. This method also revealed a less marked, but significant effect on cellular localization of amino acid residues between transporter exofacial and middle loops. The subcellular distribution of expressed chimeras was confirmed by immunofluorescence microscopy of permeabilized COS-7 cells. Thus, HA-tagged native GLUT4 was concentrated in the perinuclear region, whereas a chimera containing the COOH-terminal 29 residues of GLUT1 substituted onto GLUT4 distributed to the plasma membrane, as did native GLUT1. Furthermore, a chimera composed of GLUT1 with a GLUT4 COOH-terminal 30-residue substitution exhibited a predominantly intracellular localization. Similar data was obtained in CHO cells stably expressing these chimeras. Taken together, these results define the unique COOH-terminal cytoplasmic sequences of the GLUT1 and GLUT4 glucose transporters as important determinants of cellular localization in COS-7 and CHO cells.

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