Invasion of host cells by JC virus identifies a novel role for caveolae in endosomal sorting of noncaveolar ligands

Invasion of glial cells by the human polyomavirus, JC virus (JCV), leads to a rapidly progressing and uniformly fatal demyelinating disease known as progressive multifocal leukoencephalopathy. The endocytic trafficking steps used by JCV to invade cells and initiate infection are not known. We demonstrated that JCV infection was inhibited by dominant defective and constitutively active Rab5-GTPase mutants that acted at distinct steps in endosomal sorting. We also found that labeled JCV colocalized with labeled cholera toxin B and with caveolin-1 (cav-1) on early endosomes following internalization by clathrin-dependent endocytosis. JCV entry and infection were both inhibited by dominant defective mutants of eps15 and Rab5-GTPase. Expression of a dominant-negative scaffolding mutant of cav-1 did not inhibit entry or infection by JCV. A single-cell knockdown experiment using cav-1 shRNA did not inhibit JCV entry but interfered with a downstream trafficking event important for infection. These data show that JCV enters cells by clathrin-dependent endocytosis, is transported immediately to early endosomes, and is then sorted to a caveolin-1-positive endosomal compartment. This latter step is dependent on Rab5-GTPase, cholesterol, caveolin-1, and pH. This is the first example of a ligand that enters cells by clathrin-dependent endocytosis and is then sorted from early endosomes to caveosomes, indicating that caveolae-derived vesicles play a more important role than previously realized in sorting cargo from early endosomes.     

Actin Dynamics Regulate Multiple Endosomal Steps during Kaposi's Sarcoma-Associated Herpesvirus Entry and Trafficking in Endothelial Cells

The role of actin dynamics in clathrin-mediated endocytosis in mammalian cells is unclear. In this study, we define the role of actin cytoskeleton in Kaposi''s sarcoma-associated herpesvirus (KSHV) entry and trafficking in endothelial cells using an immunofluorescence-based assay to visualize viral capsids and the associated cellular components. In contrast to infectivity or reporter assays, this method does not rely on the expression of any viral and reporter genes, but instead directly tracks the accumulation of individual viral particles at the nuclear membrane as an indicator of successful viral entry and trafficking in cells. Inhibitors of endosomal acidification reduced both the percentage of nuclei with viral particles and the total number of viral particles docking at the perinuclear region, indicating endocytosis, rather than plasma membrane fusion, as the primary route for KSHV entry into endothelial cells. Accordingly, a viral envelope protein was only detected on internalized KSHV particles at the early but not late stage of infection. Inhibitors of clathrin- but not caveolae/lipid raft-mediated endocytosis blocked KSHV entry, indicating that clathrin-mediated endocytosis is the major route of KSHV entry into endothelial cells. KSHV particles were colocalized not only with markers of early and recycling endosomes, and lysosomes, but also with actin filaments at the early time points of infection. Consistent with these observations, transferrin, which enters cells by clathrin-mediated endocytosis, was found to be associated with actin filaments together with early and recycling endosomes, and to a lesser degree, with late endosomes and lysosomes. KSHV infection induced dynamic actin cytoskeleton rearrangements. Disruption of the actin cytoskeleton and inhibition of regulators of actin nucleation such as Rho GTPases and Arp2/3 complex profoundly blocked KSHV entry and trafficking. Together, these results indicate an important role for actin dynamics in the internalization and endosomal sorting/trafficking of KSHV and clathrin-mediated endocytosis in endothelial cells.     

Analysis of foot-and-mouth disease virus internalization events in cultured cells

It has been demonstrated that foot-and-mouth disease virus (FMDV) can utilize at least four members of the alpha(V) subgroup of the integrin family of receptors in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the betaG-betaH loop of VP1. While there have been extensive studies of virus-receptor interactions at the cell surface, our understanding of the events during viral entry into the infected cell is still not clear. We have utilized confocal microscopy to analyze the entry of two FMDV serotypes (types A and O) after interaction with integrin receptors at the cell surface. In cell cultures expressing both the alphaVbeta3 and alphaVbeta6 integrins, virus adsorbed to the cells at 4 degrees C appears to colocalize almost exclusively with the alphaVbeta6 integrin. Upon shifting the infected cells to 37 degrees C, FMDV capsid proteins were detected within 15 min after the temperature shift, in association with the integrin in vesicular structures that were positive for a marker of clathrin-mediated endocytosis. In contrast, virus did not colocalize with a marker for caveola-mediated endocytosis. Virus remained associated with the integrin until about 1 h after the temperature shift, when viral proteins appeared around the perinuclear region of the cell. By 15 min after the temperature shift, viral proteins were seen colocalizing with a marker for early endosomes, while no colocalization with late endosomal markers was observed. In the presence of monensin, which raises the pH of endocytic vesicles and has been shown to inhibit FMDV replication, viral proteins were not released from the recycling endosome structures. Viral proteins were not observed associated with the endoplasmic reticulum or the Golgi. These data indicate that FMDV utilizes the clathrin-mediated endocytosis pathway to infect the cells and that viral replication begins due to acidification of endocytic vesicles, causing the breakdown of the viral capsid structure and release of the genome by an as-yet-unidentified mechanism.     

Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor.

Many of the actions of the neuropeptide substance P (SP) that are mediated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We delineated this endocytic pathway in transfected cells by confocal microscopy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were internalized into the same clathrin immunoreactive vesicles, and then sorted into different compartments. The NK1-R was colocalized with a marker of early endosomes, but not with markers of late endosomes or lysosomes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, there was a loss and subsequent recovery of surface NK1-R. The loss was prevented by hypertonic sucrose and potassium depletion, inhibitors of clathrin-mediated endocytosis. Recovery was independent of new protein synthesis because it was unaffected by cycloheximide. Recovery required endosomal acidification because it was prevented by an H(+)-ATPase inhibitor. The fate of internalized 125I-SP was examined by chromatography. SP was intact at the cell surface and in early endosomes, but slowly degraded in perinuclear vesicles. We conclude that SP induces clathrin-dependent internalization of the NK1-R. The SP/NK1-R complex dissociates in acidified endosomes. SP is degraded, whereas the NK1-R recycles to the cell surface.     

MARCH ubiquitin ligases alter the itinerary of clathrin-independent cargo from recycling to degradation

Following endocytosis, internalized plasma membrane proteins can be recycled back to the cell surface or trafficked to late endosomes/lysosomes for degradation. Here we report on the trafficking of multiple proteins that enter cells by clathrin-independent endocytosis (CIE) and determine that a set of proteins (CD44, CD98, and CD147) found primarily in recycling tubules largely failed to reach late endosomes in HeLa cells, whereas other CIE cargo proteins, including major histocompatibility complex class I protein (MHCI), trafficked to both early endosome antigen 1 (EEA1) and late endosomal compartments in addition to recycling tubules. Expression of the membrane-associated RING-CH 8 (MARCH8) E3 ubiquitin ligase completely shifted the trafficking of CD44 and CD98 proteins away from recycling tubules to EEA1 compartments and late endosomes, resulting in reduced surface levels. Cargo affected by MARCH expression, including CD44, CD98, and MHCI, still entered cells by CIE, suggesting that the routing of ubiquitinated cargo occurs after endocytosis. MARCH8 expression led to direct ubiquitination of CD98 and routing of CD98 to late endosomes/lysosomes.     

Entry of Rice dwarf virus into cultured cells of its insect vector involves clathrin-mediated endocytosis

Electron microscopy revealed that the entry of Rice dwarf virus (RDV) into insect vector cells involved endocytosis via coated pits. The treatment of cells with drugs that block receptor-mediated or clathrin-mediated endocytosis significantly reduced RDV infectivity. However, the drug that blocks caveola-mediated endocytosis had a negligible effect on such infection. Infection was also inhibited when cells had been pretreated with bafilomycin A1, which interferes with acidification of endosomes. Moreover, immunofluorescence staining indicated that the virus is internalized into early endosomes. Together, our data indicate that RDV enters insect vector cells through receptor-mediated, clathrin-dependent endocytosis and is sequestered in early endosomes.     

Transfer of M2 muscarinic acetylcholine receptors to clathrin-derived early endosomes following clathrin-independent endocytosis

Upon agonist stimulation, many G protein-coupled receptors such as beta(2)-adrenergic receptors are internalized via beta-arrestin- and clathrin-dependent mechanisms, whereas others, like M(2) muscarinic acetylcholine receptors (mAChRs), are internalized by clathrin- and arrestin-independent mechanisms. To gain further insight into the mechanisms that regulate M(2) mAChR endocytosis, we investigated the post-endocytic trafficking of M(2) mAChRs in HeLa cells and the role of the ADP-ribosylation factor 6 (Arf6) GTPase in regulating M(2) mAChR internalization. Here, we report that M(2) mAChRs are rapidly internalized by a clathrin-independent pathway that is inhibited up to 50% by expression of either GTPase-defective Arf6 Q67L or an upstream Arf6 activator, Galpha(q) Q209L. In contrast, M(2) mAChR internalization was not affected by expression of dominant-negative dynamin 2 K44A, which is a known inhibitor of clathrin-dependent endocytosis. Nevertheless, M(2) mAChRs, which are initially internalized in structures that lack clathrin-dependent endosomal markers, quickly localize to endosomes that contain the clathrin-dependent, early endosomal markers early endosome autoantigen-1, transferrin receptor, and GTPase-defective Rab5 Q79L, which is known to swell early endosomal compartments. These results suggest that M(2) mAChRs initially internalize via an Arf6-associated, clathrin-independent pathway but then quickly merge with the clathrin endocytic pathway at the level of early endosomes.     

Integrin alphavbeta8 functions as a receptor for foot-and-mouth disease virus: role of the beta-chain cytodomain in integrin-mediated infection

Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use three alphav integrins, alphavbeta1, alphavbeta3, and alphavbeta6, as cellular receptors. Binding to the integrin is mediated by a highly conserved RGD motif located on a surface-exposed loop of VP1. The RGD tripeptide is recognized by several other members of the integrin family, which therefore have the potential to act as receptors for FMDV. Here we show that SW480 cells are made susceptible to FMDV following transfection with human beta8 cDNA and expression of alphavbeta8 at the cell surface. The involvement of alphavbeta8 in infection was confirmed by showing that virus binding and infection of the transfected cells are inhibited by RGD-containing peptides and by function-blocking monoclonal antibodies specific for either the alphavbeta8 heterodimer or the alphav chain. Similar results were obtained with a chimeric alphavbeta8 including the beta6 cytodomain (alphavbeta8/6), showing that the beta6 cytodomain can substitute efficiently for the corresponding region of beta8. In contrast, virus binding to alphavbeta6 including the beta8 cytodomain (alphavbeta6/8) was lower than that of the wild-type integrin, and this binding did not lead to infection. Further, the alphavbeta6 chimera was recognized poorly by antibodies specific for the ectodomain of alphavbeta6 and displayed a relaxed sequence-binding specificity relative to that of wild-type integrin. These data suggest that the beta6 cytodomain is important for maintaining alphavbeta6 in a conformation required for productive infection by FMDV.     

Role of protein kinase C betaII in influenza virus entry via late endosomes

Many viruses take advantage of receptor-mediated endocytosis in order to enter target cells. We have utilized influenza virus and Semliki Forest virus (SFV) to define a role for protein kinase C betaII (PKCbetaII) in endocytic trafficking. We show that specific PKC inhibitors prevent influenza virus infection, suggesting a role for classical isoforms of PKC. We also examined virus entry in cells overexpressing dominant-negative forms of PKCalpha and -beta. Cells expressing a phosphorylation-deficient form of PKCbetaII (T500V), but not an equivalent mutant form of PKCalpha, inhibited successful influenza virus entry-with the virus accumulating in late endosomes. SFV, however, believed to enter cells from the early endosome, was unaffected by PKCbetaII T500V expression. We also examined the trafficking of two cellular ligands, transferrin and epidermal growth factor (EGF). PKCbetaII T500V expression specifically blocked EGF receptor trafficking and degradation, without affecting transferrin receptor recycling. As with influenza virus, in PKCbetaII kinase-dead cells, EGF receptor was trapped in a late endosome compartment. Our findings suggest that PKCbetaII is an important regulator of a late endosomal sorting event needed for influenza virus entry and infection.     

Rab11a and its binding partners regulate the recycling of the ß1-adrenergic receptor

ß1-adrenergic receptors (ß1-AR) are internalized in response to agonists and then recycle back for another round of signaling. The serine 312 to alanine mutant of the ß1-AR (S312A) is internalized but does not recycle. We determined that WT ß1-AR and S312A were internalized initially to an early sorting compartment because they colocalized by > 70% with the early endosomal markers rab5a and early endosomal antigen-1 (EEA1). Subsequently, the WT ß1-AR trafficked via rab4a-expressing sorting endosomes to recycling endosomes. In recycling endosomes WT ß1-AR were colocalized by > 70% with the rab11 GTPase. S312A did not colocalize with either rab4a or rab11, instead they exited from early endosomes to late endosomes/lysosomes in which they were degraded. Rab11a played a prominent role in recycling of the WT ß1-AR because dominant negative rab11a inhibited, while constitutively active rab11a accelerated the recycling of the ß1-AR. Next, we determined the effect of each of the rab11-interacting proteins on trafficking of the WT ß1-AR. The recycling of the ß1-AR was markedly inhibited when myosin Vb, FIP2, FIP3 and rabphillin were knocked down. These data indicate that rab11a and a select group of its binding partners play a prominent role in recycling of the human ß1-AR.     

Bovine Ephemeral Fever Virus Uses a Clathrin-Mediated and Dynamin 2-Dependent Endocytosis Pathway That Requires Rab5 and Rab7 as Well as Microtubules

The specific cell pathways involved in bovine ephemeral fever virus (BEFV) cell entry have not been determined. In this work, colocalization of the M protein of BEFV with clathrin or dynamin 2 was observed under a fluorescence microscope. To better understand BEFV entry, we carried out internalization studies with a fluorescently labeled BEFV by using a lipophilic dye, 3,30-dilinoleyloxacarbocyanine perchlorate (DiO), further suggesting that BEFV uses a clathrin-mediated endocytosis pathway. Our results suggest that clathrin-mediated and dynamin 2-dependent endocytosis is an important avenue of BEFV entry. Suppression of Rab5 or Rab7a through the use of a Rab5 dominant negative mutant and Rab7a short hairpin RNA (shRNA) demonstrated that BEFV requires both early and late endosomes for endocytosis and subsequent infection in MDBK and Vero cells. Treatment of BEFV-infected cells with nocodazole significantly decreased the M protein synthesis and viral yield, indicating that microtubules play an important role in BEFV productive infection, likely by mediating trafficking of BEFV-containing endosomes. Furthermore, BEFV infection was strongly blocked by different inhibitors of endosomal acidification, suggesting that virus enters host cells by clathrin-mediated and dynamin 2-dependent endocytosis in a pH-dependent manner.     

Integrin αvβ1 Is a Receptor for Foot-and-Mouth Disease Virus

Infection by field strains of Foot-and-mouth disease virus (FMDV) is initiated by binding to certain species of arginine-glycine-aspartic acid (RGD)-dependent integrin including alphavbeta3 and the epithelial integrin alphavbeta6. In this report we show that the integrin alphavbeta1, when expressed as a human/hamster heterodimer on transfected CHOB2 cells, is a receptor for FMDV. Virus binding and infection mediated by alphavbeta1 was inefficient in the presence of physiological concentrations of calcium and magnesium but were significantly enhanced by reagents that activate the integrin and promote ligand binding. The ability of chimeric alpha5/alphav integrin subunits, in association with the beta1 chain, to bind FMDV and mediate infection matched the ligand binding specificity of alphavbeta1, not alpha5beta1, thus providing further evidence for the receptor role of alphavbeta1. In addition, data are presented suggesting that amino acid residues near the RGD motif may be important for differentiating between the binding specificities of alphavbeta1 and alphavbeta6.     

Specificity of the VP1 GH loop of Foot-and-Mouth Disease virus for alphav integrins

Foot-and-mouth disease virus (FMDV) can use a number of integrins as receptors to initiate infection. Attachment to the integrin is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) tripeptide located on the GH loop of VP1. Other residues of this loop are also conserved and may contribute to integrin binding. In this study we have used a 17-mer peptide, whose sequence corresponds to the GH loop of VP1 of type O FMDV, as a competitor of integrin-mediated virus binding and infection. Alanine substitution through this peptide identified the leucines at the first and fourth positions following RGD (RGD+1 and RGD+4 sites) as key for inhibition of virus binding and infection mediated by alphavbeta6 or alphavbeta8 but not for inhibition of virus binding to alphavbeta3. We also show that FMDV peptides containing either methionine or arginine at the RGD+1 site, which reflects the natural sequence variation seen across the FMDV serotypes, are effective inhibitors for alphavbeta6. In contrast, although RGDM-containing peptides were effective for alphavbeta8, RGDR-containing peptides were not. These observations were confirmed by showing that a virus containing an RGDR motif uses alphavbeta8 less efficiently than alphavbeta6 as a receptor for infection. Finally, evidence is presented that shows alphavbeta3 to be a poor receptor for infection by type O FMDV. Taken together, our data suggest that the integrin binding loop of FMDV has most likely evolved for binding to alphavbeta6 with a higher affinity than to alphavbeta3 and alphavbeta8.     

Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants

During endocytosis in Chinese hamster ovary (CHO) cells, Semliki Forest virus (SFV) passes through two distinct subpopulations of endosomes before reaching lysosomes. One subpopulation, defined by cell fractionation using free flow electrophoresis as "early endosomes," constitutes the major site of membrane and receptor recycling; while "late endosomes," an electrophoretically distinct endosome subpopulation, are involved in the delivery of endosomal content to lysosomes. In this paper, the pH-sensitive conformational changes of the SFV E1 spike glycoprotein were used to study the acidification of these defined endosome subpopulations in intact wild-type and acidification-defective CHO cells. Different virus strains were used to measure the kinetics at which internalized SFV was delivered to endosomes of pH less than or equal to 6.2 (the pH at which wild-type E1 becomes resistant to trypsin digestion) vs. endosomes of pH less than or equal to 5.3 (the threshold pH for E1 of the SFV mutant fus-1). By correlating the kinetics of acquisition of E1 trypsin resistance with the transfer of SFV among distinct endosome subpopulations defined by cell fractionation, we found that after a brief residence in vesicles of relatively neutral pH, internalized virus encountered pH less than or equal to 6.2 in early endosomes with a t1/2 of 5 min. Although a fraction of the virus reached a pH of less than or equal to 5.3 in early endosomes, most fus-1 SFV did not exhibit the acid-induced conformational change until arrival in late endosomes (t1/2 = 8-10 min). Thus, acidification of both endosome subpopulations was heterogeneous. However, passage of SFV through a less acidic early endosome subpopulation always preceded arrival in the more acidic late endosome subpopulation. In mutant CHO cells with temperature-sensitive defects in endosome acidification in vitro, acidification of both early and late endosomes was found to be impaired at the restrictive temperature (41 degrees C). The acidification defect was also found to be partially penetrant at the permissive temperature, resulting in the inability of any early endosomes in these cells to attain pH less than or equal to 5.3. In vitro studies of endosomes isolated from mutant cells suggested that the acidification defect is most likely in the proton pump itself. In one mutant, this defect resulted in increased sensitivity of the electrogenic H+ pump to fluctuations in the endosomal membrane potential.     

Cholesterol is required for endocytosis and endosomal escape of adenovirus type 2

The species C adenovirus type 2 (Ad2) and Ad5 bind the coxsackievirus B Ad receptor and alphav integrin coreceptors and enter epithelial cells by clathrin-mediated endocytosis. This pathway is rapid and efficient. It leads to cell activation and the cholesterol-dependent formation of macropinosomes. Macropinosomes are triggered to release their contents when incoming Ad2 escapes from endosomes. Here, we show that cholesterol extraction of epithelial cells by methyl-beta-cyclodextrin (mbetaCD) treatment reduced Ad5-mediated luciferase expression approximately 4-fold. The addition of cholesterol to normal cells increased gene expression in a dose-dependent manner up to threefold, but it did not restore gene expression in mbetaCD-treated cells. mbetaCD had no effect in the presence of excess cholesterol, indicating that the inhibition of gene expression was due specifically to cholesterol depletion. Cholesterol depletion inhibited rapid Ad2 endocytosis, endosomal escape, and nuclear targeting, consistent with the notion that clathrin-dependent endocytosis of Ad2 is cholesterol dependent. In cholesterol-reduced cells, Ad2 internalized at a low rate, suggestive of an alternative, clathrin-independent, low-capacity entry pathway. While exogenous cholesterol completely restored rapid Ad2 endocytosis, macropinocytosis, and macropinosome disruption, it did not, surprisingly, restore viral escape from endosomes. Our results indicate that macropinosome disruption and endosomal escape of Ad2 are independent events in cells depleted of and then refilled with cholesterol, suggesting that viral escape from endosomes requires lipid-controlled membrane homeostasis, trafficking, or signaling.     

Extracellular signal-regulated kinase regulates clathrin-independent endosomal trafficking

Extracellular signal-regulated kinase (Erk) is widely recognized for its central role in cell proliferation and motility. Although previous work has shown that Erk is localized at endosomal compartments, no role for Erk in regulating endosomal trafficking has been demonstrated. Here, we report that Erk signaling regulates trafficking through the clathrin-independent, ADP-ribosylation factor 6 (Arf6) GTPase-regulated endosomal pathway. Inactivation of Erk induced by a variety of methods leads to a dramatic expansion of the Arf6 endosomal recycling compartment, and intracellular accumulation of cargo, such as class I major histocompatibility complex, within the expanded endosome. Treatment of cells with the mitogen-activated protein kinase kinase (MEK) inhibitor U0126 reduces surface expression of MHCI without affecting its rate of endocytosis, suggesting that inactivation of Erk perturbs recycling. Furthermore, under conditions where Erk activity is inhibited, a large cohort of Erk, MEK, and the Erk scaffold kinase suppressor of Ras 1 accumulates at the Arf6 recycling compartment. The requirement for Erk was highly specific for this endocytic pathway, because its inhibition had no effect on trafficking of cargo of the classical clathrin-dependent pathway. These studies reveal a previously unappreciated link of Erk signaling to organelle dynamics and endosomal trafficking.     

Heparan sulfate-binding foot-and-mouth disease virus enters cells via caveola-mediated endocytosis

Foot-and-mouth disease virus (FMDV) utilizes different cell surface macromolecules to facilitate infection of cultured cells. Virus, which is virulent for susceptible animals, infects cells via four members of the alpha(V) subclass of cellular integrins. In contrast, tissue culture adaptation of some FMDV serotypes results in the loss of viral virulence in the animal, accompanied by the loss of virus' ability to use integrins as receptors. These avirulent viral variants acquire positively charged amino acids on surface-exposed structural proteins, resulting in the utilization of cell surface heparan sulfate (HS) molecules as receptors. We have recently shown that FMDV serotypes utilizing integrin receptors enter cells via a clathrin-mediated mechanism into early endosomes. Acidification within the endosome results in a breakdown of the viral capsid, releasing the RNA, which enters the cytoplasm by a still undefined mechanism. Since there is evidence that HS internalizes bound ligands via a caveola-mediated mechanism, it was of interest to analyze the entry of FMDV by cell-surface HS. Using a genetically engineered variant of type O(1)Campos (O(1)C3056R) which can utilize both integrins and HS as receptors and a second variant (O(1)C3056R-KGE) which can utilize only HS as a receptor, we followed viral entry using confocal microscopy. After virus bound to cells at 4 degrees C, followed by a temperature shift to 37 degrees C, type O(1)C3056R-KGE colocalized with caveolin-1, while O(1)C3056R colocalized with both clathrin and caveolin-1. Compounds which either disrupt or inhibit the formation of lipid rafts inhibited the replication of O(1)C3056R-KGE. Furthermore, a caveolin-1 knockdown by RNA interference also considerably reduced the efficiency of O(1)C3056R-KGE infection. These results indicate that HS-binding FMDV enters the cells via the caveola-mediated endocytosis pathway and that caveolae can associate and traffic with endosomes. In addition, these results further suggest that the route of FMDV entry into cells is a function solely of the viral receptor.     

Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump

Endocytosed proteins are sorted in early endosomes to be recycled to the plasma membrane or transported further into the degradative pathway. We studied the role of endosomes acidification on the endocytic trafficking of the transferrin receptor (TfR) as a representative for the recycling pathway, the cation-dependent mannose 6-phosphate receptor (MPR) as a prototype for transport to late endosomes, and fluid-phase endocytosed HRP as a marker for transport to lysosomes. Toward this purpose, bafilomycin A1 (Baf), a specific inhibitor of the vacuolar proton pump, was used to inhibit acidification of the vacuolar system. Microspectrofluorometric measurement of the pH of fluorescein-rhodamine-conjugated transferrin (Tf)-containing endocytic compartments in living cells revealed elevated endosomal pH values (pH > 7.0) within 2 min after addition of Baf. Although recycling of endocytosed Tf to the plasma membrane continued in the presence of Baf, recycled Tf did not dissociate from its receptor, indicating failure of Fe3+ release due to a neutral endosomal pH. In the presence of Baf, the rates of internalization and recycling of Tf were reduced by a factor of 1.40 +/- 0.08 and 1.57 +/- 0.25, respectively. Consequently, little if any in TfR expression at the cell surface was measured during Baf treatment. Sorting between endocytosed TfR and MPR was analyzed by the HRP-catalyzed 3,3'- diaminobenzidine cross-linking technique, using transferrin conjugated to HRP to label the endocytic pathway of the TfR. In the absence of Baf, endocytosed surface 125I-labeled MPR was sorted from the TfR pathway starting at 10 min after uptake, reaching a plateau of 40% after 45 min. In the presence of Baf, sorting was initiated after 20 min of uptake, reaching approximately 40% after 60 min. Transport of fluid-phase endocytosed HRP to late endosomes and lysosomes was measured using cell fractionation and immunogold electron microscopy. Baf did not interfere with transport of HRP to MPR-labeled late endosomes, but nearly completely abrogated transport to cathepsin D- labeled lysosomes. From these results, we conclude that trafficking through early and late endosomes, but not to lysosomes, continued upon inactivation of the vacuolar proton pump.