Internalization Of Transferrin Research Articles

Secretory ribonucleases are internalized by a dynamin-independent endocytic pathway.

Cytosolic internalization is a requirement for the toxicity of secretory ribonucleases. Here, we investigate the mechanism of internalization of Onconase (ONC), a toxic protein, and ribonuclease A (RNase A), a nontoxic homolog. Microscopy studies indicate that both ribonucleases readily bind to the cell surface and are internalized via acidic vesicles. Blocking dynamin-dependent endocytosis prevents transferrin internalization but does not hinder RNase A internalization. ONC and G88R RNase A, which is a toxic variant, demonstrate enhanced cytotoxicity in the absence of clathrin- and dynamin-mediated endocytosis. The cytosolic entry of ribonucleases does not require an acidic environment or transport to the ER and probably occurs from endosomes. Thus, common proteins - secretory ribonucleases - enter the cytosol by a pathway that is distinct from that of other known toxins.

Synaptotagmin III is a critical factor for the formation of the perinuclear endocytic recycling compartment and determination of secretory granules size.

Early endosomes and a perinuclear, Rab-11-positive compartment have been implicated in the recycling of internalized receptors. In this study, we show that synaptotagmin III (Syt III), a member of the Syt family of proteins, is required for the formation and delivery of cargo to the perinuclear endocytic recycling compartment (ERC). We demonstrate that rat basophilic leukemia (RBL-2H3) mast cells endogenously express Syt III, and >70% of this protein colocalizes with early endosomal markers, such as EEA1, annexin II and syntaxin 7, and the remaining protein colocalizes with secretory granule (SG) markers such as beta-hexosaminidase, histamine and serotonin. To study the functional role of Syt III, we stably transfected RBL cells with Syt III antisense cDNA and monitored the route of transferrin (Tfn) internalization in cells that displayed substantially reduced (<90%) levels of Syt III (RBL-Syt III(-)). In these cells, Tfn binding and internalization into early endosomes were unaltered. However, whereas in the mock-transfected cells Tfn was subsequently delivered to the ERC, in the RBL-Syt III(-) cells, Tfn remained associated with dispersed peripheral vesicles and Rab 11 remained cytosolic. Nevertheless, the rates of Tfn internalization and recycling were not affected. RBL-Syt III(-) cells also displayed enlarged SGs, reminiscent of the SGs present in Chediak-Higashi (beige) mice. However, morphometric analyses suggested that granule formation was unaltered and that the calculated unit granule volume is the same in both cell lines. Therefore, our results implicate Syt III as a critical factor for the generation and delivery of internalized cargo to the perinuclear endocytic recycling compartment and suggest a possible link between ERC and recycling from immature SGs during the process of SG maturation.

Cytoplasmic transport of Stat3 by receptor-mediated endocytosis.

Signal transducer and activator of transcription (STAT) proteins are cytoplasmic transcription factors that translocate to the nucleus and regulate gene expression upon activation of cytokine or growth factor receptors. While this translocation event is essential for gene regulation by STATs, their mechanism of transport through the cytoplasm to the nucleus has remained elusive. We now report that cytoplasmic transport of Stat3 is an active process that requires receptor-mediated endocytosis. Stat3 co-localizes with endocytic vesicles in transit from the cell membrane to the perinuclear region in response to growth factor stimulation. Consistent with a role for receptor endocytosis in growth factor signaling, disruption of endocytosis with specific inhibitors blocks Stat3 nuclear translocation and Stat3-dependent gene regulation. These results indicate that receptor-mediated endocytosis may be a general mechanism of transport through the cytoplasm for a subset of cytoplasmic signaling proteins destined for the nucleus.

Receptor-mediated internalization of [ 3H]–neurotensin in synaptosomal preparations from rat neostriatum

Following its binding to somatodendritic receptors, the neuropeptide neurotensin (NT) internalizes via a clathrin-mediated process. In the present study, we investigated whether NT also internalizes presynaptically using synaptosomes from rat neostriatum, a region in which NT1 receptors are virtually all presynaptic. Binding of [ 3H]–NT to striatal synaptosomes in the presence of levocabastine to block NT2 receptors is specific, saturable, and has NT1 binding properties. A significant fraction of the bound radioactivity is resistant to hypertonic acid wash indicating that it is internalized. Internalization of [ 3H]–NT, like that of [ 125I]–transferrin, is blocked by sucrose and low temperature, consistent with endocytosis occurring via a clathrin-dependent pathway. However, contrary to what was reported at the somatodendritic level, neither [ 3H]–NT nor [ 125I]–transferrin internalization in synaptosomes is sensitive to the endocytosis inhibitor phenylarsine oxide. Moreover, treatment of synaptosomes with monensin, which prevents internalized receptors from recycling to the plasma membrane, reduces [ 3H]–NT binding and internalization, suggesting that presynaptic NT1 receptors, in contrast to somatodendritic ones, are recycled back to the plasma membrane. Taken together, these results suggest that NT internalizes in nerve terminals via an endocytic pathway that is related to, but is mechanistically distinct from that responsible for NT internalization in nerve cell bodies.

Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis.

The μ2 subunit of the AP2 complex is known to be phosphorylated in vitro by a copurifying kinase, and it has been demonstrated recently that μ2 phosphorylation is required for transferrin endocytosis (Olusanya, O., P.D. Andrews, J.R. Swedlow, and E. Smythe. 2001. Curr. Biol. 11:896–900). However, the identity of the endogenous kinase responsible for this phosphorylation is unknown. Here we identify and characterize a novel member of the Prk/Ark family of serine/threonine kinases, adaptor-associated kinase (AAK)1. We find that AAK1 copurifies with adaptor protein (AP)2 and that it directly binds the ear domain of α-adaptin in vivo and in vitro. In neuronal cells, AAK1 is enriched at presynaptic terminals, whereas in nonneuronal cells it colocalizes with clathrin and AP2 in clathrin-coated pits and at the leading edge of migrating cells. AAK1 specifically phosphorylates the μ subunit in vitro, and stage-specific assays for endocytosis show that μ phosphorylation by AAK1 results in a decrease in AP2-stimulated transferrin internalization. Together, these results provide strong evidence that AAK1 is the endogenous μ2 kinase and plays a regulatory role in clathrin-mediated endocytosis. These results also lend support to the idea that clathrin-mediated endocytosis is controlled by cycles of phosphorylation/desphosphorylation.

DNA/polyethylenimine transfection particles: influence of ligands, polymer size, and PEGylation on internalization and gene expression.

Receptor-binding ligands have been incorporated into DNA/polyethylenimine (PEI) complexes to enhance cell binding and cellular internalization. This study characterizes receptor-mediated uptake of DNA/PEI complexes on a cellular basis. A novel assay based on flow cytometry was applied, discriminating between total cell-associated and extracellularly bound DNA complexes. Receptor-mediated uptake of ligand-containing DNA/PEI (molecular weight, 800 kd) complexes was found to occur quickly (within 1 hour), whereas unspecific uptake through adsorptive endocytosis is less efficient or requires extended periods to reach the same degree of internalization. Rapid, receptor-mediated internalization requires a small complex size; however, large, aggregated complexes show higher gene expression. Using PEI 25 kd conjugated to large proteins such as transferrin or antibodies, improper condensation with DNA leads to suboptimal uptake and gene expression, whereas partial replacement of ligand-PEI with unconjugated PEI increases both uptake and transfection. In contrast, the 8 kd protein epidermal growth factor conjugated to PEI 25 kd properly condenses DNA and mediates specific uptake into human adenocarcinoma (KB) cells. Modification of the complex surface with appropriate amounts of poly(ethylene glycol) (PEG) does not block ligand-mediated internalization. A higher degree of PEGylation reduces the internalization of transferrin or antibody-containing complexes to a level similar to that of ligand-free complexes. In contrast, epidermal growth factor "mediated uptake is less effected by excessive PEGylation.

Clathrin-dependent and clathrin-independent endocytosis are differentially sensitive to insertion of poly (ethylene glycol)-derivatized cholesterol in the plasma membrane.

We examined the effect of a cholesterol derivative, poly (ethylene glycol) cholesteryl ether on the structure/function of clathrin-coated pits and caveolae. Addition of the compound to cultured cells induced progressive smoothening of the surface. Markedly, when the incorporated amount exceeded 10% equivalent of the surface area, fluid pinocytosis, but not endocytosis of transferrin, became inhibited in K562 cells. In A431 cells, both clathrin-independent fluid phase uptake and the internalization of fluorescent cholera-toxin B through caveolae were inhibited with concomitant flattening of caveolae. In contrast, clathrin-mediated internalization of transferrin was not affected until the incorporated poly (ethylene glycol) cholesteryl ether exceeded 20% equivalent of the plasma membrane surface area, at which point opened clathrin-coated pits accumulated. The cells were ruptured upon further addition of poly (ethylene glycol) cholesteryl ether. We propose that the primary reason for the differential effect of poly (ethylene glycol) cholesteryl ether is that the bulk membrane phase and caveolae are both more elastic than the rigid clathrin-coated pits. We analyzed the results with the current mechanical model (Rauch and Farge, Biophys J 2000;78:3036-3047) and suggest here that the functional clathrin-lattice is much stiffer than typical phospholipid bilayers.

Rabphilin dissociated from Rab3 promotes endocytosis through interaction with Rabaptin-5.

Rabphilin is a secretory vesicle protein that interacts with the GTP-bound form of the small GTPase Rab3. We investigated the involvement of Rabphilin in endocytosis using different point mutants of the protein. Overexpression of wild-type Rabphilin in the insulin-secreting cell line HIT-T15 did not affect receptor-mediated transferrin endocytosis. By contrast, Rabphilin V61A, a mutant that is unable to interact with Rab3, enhanced the rate of transferrin internalization. The effect of Rabphilin V61A was not mimicked by Rabphilin L83A, another mutant with impaired Rab3 binding. Careful analysis of the properties of the two mutants revealed that Rabphilin V61A and Rabphilin L83A are both targeted to secretory vesicles, have stimulatory activity on exocytosis, and bind equally well to alpha-actinin. However, Rabphilin L83A fails to interact with Rabaptin-5, an important component of the endocytotic machinery. These results indicate that Rabphilin promotes receptor-mediated endocytosis and that its action is negatively modulated by Rab3. We propose that the hydrolysis of GTP that is coupled to the exocytotic event disrupts the Rabphilin-Rab3 complex and permits the recruitment of Rabaptin-5 at the fusion site. Our data show that immediately after internalization the transferrin receptor and VAMP-2 colocalize on the same vesicular structures, suggesting that Rabphilin favors the rapid recycling of the components of the secretory vesicle.

Structural and Functional Stability of the Mature Transferrin Receptor from Human Placenta

The transferrin receptor (TfR) is a N- and O-glycosylated transmembrane protein mediating the cellular iron uptake by binding and internalization of diferric transferrin. In this study, rate constants and dissociation constants of 125I-ferri-transferrin binding to the human TfR were examined dependent on receptor glycan composition, pH, bivalent cations, and temperature. To do so, purified human placental TfR was noncovalently immobilized to polystyrene surfaces and subjected to alterations in various parameters. We found that transferrin binding was clearly dependent on a receptor pretreatment with buffers of various pH in that most of the TfR molecules irreversibly lost transferrin binding activity below pH 6.5. However, the dissociation constant of the remaining active binding sites was not affected. Similarly, we were able to define the thermal stability of the receptor as a function of transferrin binding ability. Binding of transferrin was completely lost provided that the receptor was pretreated at temperatures of at least 65°C. Treatment with EDTA also caused an irreversible loss of transferrin binding activity, indicating that the functionally active conformation of the mature TfR depends on bivalent cations. In order to examine the role of the receptor glycans, we enzymatically removed the sialic acid residues, the hybrid and oligomannosidic N-glycans, or all types of N-glycans. In contrast to the parameters described above, all desialylated and N-deglycosylated TfR variants had exactly the same transferrin binding properties as the native TfR. To assess changes in the secondary structure of the receptor, circular dichroic spectra were recorded from TfR at pH 5.0, from heat pretreated receptor and from deglycosylated TfR. Since the receptor did not exhibit detectable changes in the CD spectrum of the deglycosylated receptor, it can be concluded that the N-linked carbohydrates of the mature, fully processed TfR are not essential for transferrin binding and conformational stability.

Receptor mediated endocytosis and cytotoxicity of transferrin-mitomycin C conjugate in the HepG2 cell and primary cultured rat hepatocyte.

Intracellular disposition and cytotoxicity of macromolecular conjugate of mitomycin C (MMC) with transferrin (TF) were examined in the human hepatoma cell line HepG2 cell and normal cultured rat hepatocyte. The conjugate (TF-MMC) was specifically bound to the HepG2 cell as well as TF. The number of the binding site and the association constant of TF-MMC in the HepG2 cell were 396000+/-31000 molecules/cell and 3.24 x 10(7)+/-0.58 x 10(7) M(-1), respectively. No difference in the binding parameters of TF-MMC and TF can be detected in the HepG2 cell. The association constant for the TF receptor was almost identical between HepG2 cell and hepatocyte, however, the numbers of the binding site of TF-MMC and TF in the HepG2 cell were from 40-times to 50-times greater than those in the hepatocyte. Furthermore, TF-MMC was internalized into the HepG2 cell and the hepatocyte as well as TF. The rates of internalization of TF-MMC and TF into the HepG2 cell were nearly identical to those into the hepatocyte. However, the levels of the internalization into the HepG2 cell were remarkably higher than those into the hepatocyte because the number of receptors in the HepG2 cell was larger than that in the hepatocyte, and the rate of release from the HepG2 cell was slower than that from the hepatocyte. TF-MMC inhibited the growth of the HepG2 cells. The 50% growth inhibition (GI50) of TF-MMC against the HepG2 cell was 0.9 microg MMC/ml, which was a little higher than that of MMC (GI50=0.5 microg/ml). These results indicated that the TF-MMC might be useful for delivery of MMC to the HepG2 cell.

Dual interaction of synaptotagmin with mu2- and alpha-adaptin facilitates clathrin-coated pit nucleation.

The synaptic vesicle protein synaptotagmin was proposed to act as a major docking site for the recruitment of clathrin coats implicated in endocytosis, including the recycling of synaptic vesicles. We show here that the C2B domain of synaptotagmin binds mu2- and alpha-adaptin, two of the four subunits of the endocytic adaptor complex AP-2. mu2 represents the major interacting subunit of AP-2 within this complex. Its binding to synaptotagmin is mediated by a site in subdomain B that is distinct from the binding site for tyrosine-based sorting motifs located in subdomain A. The presence of the C2B domain of synaptotagmin at the surface of liposomes enhances the recruitment of AP-2 and clathrin. Conversely, perturbation of the interaction between synaptotagmin and AP-2 by synprint, the cytoplasmic synaptotagmin-binding domain of N-type calcium channels, inhibits transferrin internalization in living cells. We conclude that a dual interaction of synaptotagmin with the clathrin adaptor AP-2 plays a key physiological role in the nucleation of endocytic clathrin-coated pits.

Intersectin 2, a new multimodular protein involved in clathrin-mediated endocytosis

Intersectin 1 ( ITSN1) is a binding partner of dynamin that has been shown to participate in clathrin-mediated endocytosis. Here we report the characterization of a new human gene, ITSN2, highly similar to ITSN1. Alternative splicing of ITSN2 generates a short isoform with two EH domains, a coiled-coil region and five SH3 domains, and a longer isoform containing extra carboxy domains (DH, PH and C2 domains), suggesting that it could act as a guanine nucleotide exchange factor for Rho-like GTPases. ITSN2 expression analysis indicates that it is widely expressed in human tissues. Intersectin 2 isoforms show a subcellular distribution similar to other components of the endocytic machinery and co-localize with Eps15. Moreover, their overexpression, as well as the corresponding ITSN1 protein forms, inhibits transferrin internalization.

The p21 Rho-activating toxin cytotoxic necrotizing factor 1 is endocytosed by a clathrin-independent mechanism and enters the cytosol by an acidic-dependent membrane translocation step.

Cytotoxic necrotizing factor 1 (CNF1), a protein produced by pathogenic strains of Escherichia coli, activates the p21 Rho-GTP-binding protein, inducing a profound reorganization of the actin cytoskeleton. CNF1 binds to its cell surface receptor on HEp-2 cells with high affinity (K(d) = 20 pM). In HEp-2 cells the action of CNF1 is not blocked in the presence of filipin, a drug described to reduce cholera toxin internalization by the caveolae-like mechanism. Moreover, HEp-2 cells, which express a dominant negative form of proteins that impair the formation of clathrin coated-vesicles and internalization of transferrin (Eps15, dynamin or intersectin-Src homology 3), are still sensitive to CNF1. In this respect, the endocytosis of CNF1 is similar to the plant toxin ricin. However, unlike ricin toxin, CNF1 does not cross the Golgi apparatus and requires an acidic cell compartment to transfer its enzymatic activity into the cytosol in a manner similar to that required by diphtheria toxin. As shown for diphtheria toxin, the pH-dependent membrane translocation step of CNF1 could be mimicked at the level of the plasma membrane by a brief exposure to a pH of </=5.2. CNF1 is the first bacterial toxin described that uses both a clathrin-independent endocytic mechanism and an acidic-dependent membrane translocation step in its delivery of the catalytic domain to the cell cytosol.

Syndapin isoforms participate in receptor-mediated endocytosis and actin organization.

Syndapin I (SdpI) interacts with proteins involved in endocytosis and actin dynamics and was therefore proposed to be a molecular link between the machineries for synaptic vesicle recycling and cytoskeletal organization. We here report the identification and characterization of SdpII, a ubiquitously expressed isoform of the brain-specific SdpI. Certain splice variants of rat SdpII in other species were named FAP52 and PACSIN 2. SdpII binds dynamin I, synaptojanin, synapsin I, and the neural Wiskott-Aldrich syndrome protein (N-WASP), a stimulator of Arp2/3 induced actin filament nucleation. In neuroendocrine cells, SdpII colocalizes with dynamin, consistent with a role for syndapin in dynamin-mediated endocytic processes. The src homology 3 (SH3) domain of SdpI and -II inhibited receptor-mediated internalization of transferrin, demonstrating syndapin involvement in endocytosis in vivo. Overexpression of full-length syndapins, but not the NH(2)-terminal part or the SH3 domains alone, had a strong effect on cortical actin organization and induced filopodia. This syndapin overexpression phenotype appears to be mediated by the Arp2/3 complex at the cell periphery because it was completely suppressed by coexpression of a cytosolic COOH-terminal fragment of N-WASP. Consistent with a role in actin dynamics, syndapins localized to sites of high actin turnover, such as filopodia tips and lamellipodia. Our results strongly suggest that syndapins link endocytosis and actin dynamics.

The transferrin receptor: role in health and disease

The transferrin receptor is a membrane glycoprotein whose only clearly defined function is to mediate cellular uptake of iron from a plasma glycoprotein, transferrin. Iron uptake from transferrin involves the binding of transferrin to the transferrin receptor, internalization of transferrin within an endocytic vesicle by receptor-mediated endocytosis and the release of iron from the protein by a decrease in endosomal pH. With the exception of highly differentiated cells, transferrin receptors are probably expressed on all cells but their levels vary greatly. Transferrin receptors are highly expressed on immature erythroid cells, placental tissue, and rapidly dividing cells, both normal and malignant. In proliferating nonerythroid cells the expression of transferrin receptors is negatively regulated post-transcriptionally by intracellular iron through iron responsive elements (IREs) in the 3′ untranslated region of transferrin receptor mRNA. IREs are recognized by specific cytoplasmic proteins (IRPs; iron regulatory proteins) that, in the absence of iron in the labile pool, bind to the IREs of transferrin receptor mRNA, preventing its degradation. On the other hand, the expansion of the labile iron pool leads to a rapid degradation of transferrin receptor mRNA that is not protected since IRPs are not bound to it. However, some cells and tissues with specific requirements for iron probably evolved mechanisms that can override the IRE/IRP-dependent control of transferrin receptor expression. Erythroid cells, which are the most avid consumers of iron in the organism, use a transcriptional mechanism to maintain very high transferrin receptor levels. Transcriptional regulation is also involved in the receptor expression during T and B lymphocyte activation. Macrophages are another example of a cell type that shows ‘unorthodox’ responses in terms of IRE/IRP paradigm since in these cells elevated iron levels increase (rather than decrease) transferrin receptor mRNA and protein levels. Erythroid cells contain the highest mass of the total organismal transferrin receptors which are released from reticulocytes during their maturation to erythrocytes. Hence, plasma contains small amounts of transferrin receptors which represent a soluble fragment of the extracellular receptor domain. Measurements of serum transferrin receptor concentrations are clinically useful since their levels correlate with the total mass of immature erythroid cells.

Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors.

The involvement of Ral and its downstream molecules in receptor-mediated endocytosis was examined. Expression of either RalG23V or RalS28N, which are known to be constitutively active and dominantnegative forms, respectively, in A431 cells blocked internalization of epidermal growth factor (EGF). Stable expression of RalG23V or RalS28N in CHO-IR cells also inhibited internalization of insulin. Internalization of EGF and insulin was not affected by full-length RalBP1 which is an effector protein of Ral, but was inhibited by its C-terminal region which binds directly to Ral and POB1. POB1 is a binding protein of RalBP1 and has the Eps15 homology (EH) domain. Deletion mutants of POB1 inhibited internalization of EGF and insulin. However, internalization of transferrin was unaffected by Ral, RalBP1, POB1 and their mutants. Epsin and Eps15 have been reported to be involved in the regulation of endocytosis of the receptors for EGF and transferrin. The EH domain of POB1 bound directly to Epsin and Eps15. Taken together with the observation that EGF and insulin activate Ral, these results suggest that Ral, RalBP1 and POB1 transmit the signal from the receptors to Epsin and Eps15, thereby regulating ligand-dependent receptor-mediated endocytosis.

3′-Azido-3′-deoxythymidine reduces the rate of transferrin receptor endocytosis in K562 cells

K562 cells, exposed for at least 24 h to 5 μM 3′-azido-3′-deoxythymidine (AZT), gave rise to an overall increase in the number of cell surface transferrin binding receptors (18–20%). This effect was ascertained either with binding experiments by using 125I-transferrin and with immunoprecipitation by using a specific monoclonal antibody against the transferrin receptor. At higher AZT concentrations (20 and 40 μM), a further increase was found, that is, up to 23% by binding experiments and up to 110% by immunoprecipitation. However, Scatchard analysis of the binding data indicated that although the number of cell surface transferrin receptors increased, the affinity of transferrin for its receptor did not change ( K a=4.0×10 8 M). Surprisingly, immunoprecipitation of total receptor molecules showed that the synthesis of receptor was not enhanced by the drug treatment. The effect of AZT on transferrin internalization and receptor recycling was also investigated. In this case, data indicated that the increase in the number of receptors at the cell surface was probably due to a slowing down of endocytosis rate rather than to an increased recycling rate of the receptor to cell surface. In fact, the time during which half the saturated amount of transferrin had been endocytosed ( t 1/2) was 2.15 min for control cells and 3.41, 3.04, and 3.74 min for 5, 20, and 40 μM AZT-treated cells, respectively. Conversely, recycling experiments did not show any significant differences between control and treated cells. A likely mechanism through which AZT could interfere with the transferrin receptor trafficking, together with the relevance of our findings, is extensively discussed.

Bacterial Lipopolysaccharide Can Enter Monocytes Via Two CD14-Dependent Pathways

Host recognition and disposal of LPS, an important Gram-negative bacterial signal molecule, may involve intracellular processes. We have therefore analyzed the initial pathways by which LPS, a natural ligand of glycosylphosphatidylinositol (GPI)-anchored CD14 (CD14-GPI), enters CD14-expressing THP-1 cells and normal human monocytes. Exposure of the cells to hypertonic medium obliterated coated pits and blocked 125I-labeled transferrin internalization, but failed to inhibit CD14-mediated internalization of [3H]LPS monomers or aggregates. Immunogold electron microscope analysis found that CD14-bound LPS moved principally into noncoated structures (mostly tubular invaginations, intracellular tubules, and vacuoles), whereas relatively little moved into coated pits and vesicles. When studied using two-color laser confocal microscopy, internalized Texas Red-LPS and BODIPY-transferrin were found in different locations and failed to overlap completely even after extended incubation. In contrast, in THP-1 cells that expressed CD14 fused to the transmembrane and cytosolic domains of the low-density lipoprotein receptor, a much larger fraction of the cell-associated LPS moved into coated pits and colocalized with intracellular transferrin. These results suggest that CD14 (GPI)-dependent internalization of LPS occurs predominantly via noncoated plasma membrane invaginations that direct LPS into vesicles that are distinct from transferrin-containing early endosomes. A smaller fraction of the LPS enters via coated pits. Aggregation, which greatly increases LPS internalization, accelerates its entry into the nonclathrin-mediated pathway.

Biogenesis In Vitro of Vesicles From Endosomes

41 The endocytosis and recycling of plasma membrane proteins allows the composition of the cell surface to be modulated. Transport of membrane proteins from endosomes to the surface or to other membranous compartments has been proposed to occur via vesicular intermediates. However, such intermediates have yet to be identified. We set out to determine whether endosomes can give rise to vesicular intermediates and if so, which cytosolic proteins are involved. We set up a system using CHO cells stably transfected with a myc-tagged version of the facilitative transporter, GLUT4. Endosomes are labeled by internalization of transferrin or anti-myc monoclonal antibodies. After disruption of the cells, labeled membranes are incubated with cytosol and an ATP regenerating system and warmed. We found that endosomal membranes incubated with cytosol from a variety of tissues as well as from yeast gave rise to small vesicles containing either transferrin or GLUT4. Vesicle formation was insensitive to the fungal metabolite brefeldin A but was completely inhibited by non-hydrolyzable forms of GTP (GTPγ-S). Cytosol from yeast strains carrying a disruption in clathrin heavy chain showed a reduced level of vesicle formation suggesting that clathrin is involved in vesicle biogenesis. When vesicles were incubated with cytosol and GTPγ-S, their density increased and clathrin was recruited. Our data suggests that endosomes can give to vesicular intermediates and that clathrin is one of the proteins involved in vesicle biogenesis from endosomes. We propose that such intermediates may represent those involved in transport of recycling membrane proteins from endosomes either directly to the plasma membrane or to other membranous compartments.

The clathrin endocytic pathway in viral infection.

How important is the clathrin-dependent endocytic pathway for entry of viruses into host cells? While it is widely accepted that Semliki Forest virus (SFV), an enveloped virus, requires this pathway there are conflicting data concerning the closely related Sindbis virus, as well as varying results with picornaviruses such as human rhinovirus 14 (HRV 14) and poliovirus. We have examined the entry mode of SFV, Sindbis virus, HRV 14 and poliovirus using a method that identifies single infected cells. This assay takes advantage of the observation that the clathrin-dependent endocytic pathway is specifically and potently arrested by overexpression of dynamin mutants that prevent clathrin-coated pit budding. Using HeLa cells and conditions of low multiplicity of infection to favor use of the most avid pathway of cell entry, it was found that SFV, Sindbis virus and HRV 14 require an active clathrin-dependent endocytic pathway for successful infection. In marked contrast, infection of HeLa cells by poliovirus did not appear to require the clathrin pathway.