In Vivo Study of Vitellogenin-Gold Transport in the Ovarian Follicle and Oocyte of Xenopus laevis

The transport of injected vitellogenin (VTG)-gold in the ovarian follicle and developing oocyte in Xenopus is described. The gold particles reached the extracellular spaces of the theca and interfollicular spaces within 1 and 2 hr, respectively, after a tracer injection at 20°C. The tracers moved through channels between the constitutive cells of both the capillary endothelium and the follicle cell layer.
Compartments in the peripheral cytoplasm of vitellogenic oocytes at stage IV, which relate to yolk formation, seemed to be segregated as follows: (a) internalization compartment consisting of coated pits and vesicles of the oolemma covering the oocyte "macrovilli", (b) transport compartment of endosomes and multivesicular endosomes in the oocyte cortex, and (c) crystallization compartment of primordial yolk platelets (PYP) in the sub-cortical region. The gold particles appeared in the internalization and transport compartments at 3–6 hr after the tracer injection and in the cystallization compartment at 12–18 hr. The VTG, internalized by receptor-mediated endocytosis, was transferred from coated vesicles to multivesicular endosomes by vesicle-to-vesicle fusion. VTG crystallization took place in globular-shaped PYPs of about 1 μm. At 24 hr after the tracer injection, the gold particles appeared in completely crystallized yolk platelets, most of them clustered in the superficial layer and some integrated into the crystals.     

Ferromagnetic isolation of endosomes involved in vitellogenin transfer into Xenopus oocytes

The transport pathway of the yolk precursor vitellogenin (VTG) has been followed using the techniques of ferrolabeling and ferromagnetic sorting, coupled with electron microscopic visualization. Vitellogenin conjugated to colloidal ferric particles of ca. 11 nm is selectively transported from the oolemma to the yolk platelets of vitellogenic Xenopus oocytes after gonadotropin stimulation of the female. Several cortical membrane compartments, labeled or unlabeled with ferric particles, are involved in the internalization and the transfer of vitellogenin to the yolk platelets. 1) Coated pits apparently fuse with coated vesicles, and coated vesicles fuse with each other in the outermost cortical cytoplasm. 2) Vesicles, depleted of their clathrin coat, fuse with cortical tubular endosomes and discharge their contents into yolk endosomes. 3) These endosomes are the direct precursors of the yolk organelles. 4) Endocytic vesicles fuse only with primordial yolk platelets of type I and not with type II or fully grown yolk platelets. After pulse-chase loading with ferric particles conjugated to vitellogenin and subsequent subcellular fractionation of the oocytes, ferromagnetic sorting of the various vesicle populations has been performed by using a "free-flow magnetic chamber". This novel method enables specification and characterization of purified endosomal compartments that accumulate protein yolk in Xenopus oocytes.     

Receptor-mediated endocytosis of transferrin and ferritin by guinea-pig reticulocytes. Uptake by a common endocytic pathway

Earlier studies have shown that transferrin binds to specific receptors on the reticulocyte surface, clusters in coated pits and is then internalized via endocytic vesicles. Guinea-pig reticulocytes also have specific receptors for ferritin. In this paper ferritin and transferrin endocytosis by guinea-pig reticulocytes was studied by electron microscopy using the natural electron density of ferritin and colloidal gold-transferrin (AuTf). At 4 degrees C both ligands bound to the cell surface. At 37 degrees C progressive uptake occurred by endocytosis. AuTf and ferritin clustered in the same coated pits and small intracellular vesicles. After 60 min incubations the ligands colocalized to large multivesicular endosomes (MVE), still membrane-bound. MVE subsequently fused with the plasma membrane and released AuTf, ferritin and inclusions by exocytosis. All endocytic structures labelled with AuTf contained ferritin, but 23 to 35% of ferritin-labelled endocytic structures contained no AuTf. These data suggest that ferritin and transferrin are internalized through the same pathway involving receptors, coated pits and vesicles, but that these proteins are recycled only partly in common.     

Ferromagnetic isolation of endosomes: a novel method for subcellular fractionation of Xenopus oocytes

A novel method has been developed using ferric particles to label endosomes, and to achieve magnetic sorting of the various endocytic compartments involved in lipoprotein uptake into cells. Ferric particles conjugated to a receptor-recognized ligand are bound to coated membrane pits and become internalized into the cytoplasm inside coated vesicles. After apparent fusion of the vesicles to tubular endosomes, the conjugates accumulate and finally discharge into multivesicular endosomes. Pulse-chase experiments elucidate the pathway of internalized conjugates and allow both early compartments (pinosomes and tubular endosomes) and late compartments (multivesicular endosomes and storage organelles) to be selectively labelled. After ferroloading of the various transport compartments, the cells are homogenized and subcellularly fractionated. Sorting of labelled endosomes is performed by a specially designed "free-flow" magnetic chamber. Prophase I-arrested oocytes of the toad Xenopus laevis are used as a model system for studying the transport pathway and the conversion of the yolk precursor vitellogenin. It is possible to follow the route of internalization of vitellogenin-iron conjugates via coated pits, coated vesicles, uncoated vesicles, tubular endosomes, multivesicular endosomes, and light primordial yolk platelets. These endosomes shuttle the ferric particles together with the vitellogenin from oolemma to performed heavy yolk organelles which are still growing. In addition, these various compartments can be isolated according to their function and subjected to electron microscopy and to gel electrophoresis for detailed characterization of their limiting membranes as well as their contents.     

Multivesicular bodies play a key role in vitellogenin endocytosis by Xenopus oocytes

A combination of electron microscopic tracers and subcellular fractionation has been used to examine the endocytic pathway of the yolk protein precursor, vitellogenin (VG), in Xenopus oocytes. VG was adsorbed to colloidal gold, and the organelles traversed by newly internalized ligand were examined at various time intervals after endocytosis. VG-Au enters oocytes via coated pits and vesicles and then appears rapidly in tubular endosomes and multivesicular bodies (MVBs). MVBs play a central role in VG processing for storage; the large majority of newly internalized VG enters this compartment, remaining there for up to several hours. Condensation of VG into crystalline bodies begins in MVBs, and continues with growth of the crystals until typical platelets are formed. When oocytes are exposed to high [VG], MVBs containing large amounts of internalized VG are morphologically indistinguishable from the primordial yolk platelets described earlier (Dumont, 1978). The use of VG-Au particles of two sizes demonstrates that gold particles in early MVBs were generally associated with the limiting membrane of these organelles, while older MVB compartments have gold particles well separated from the limiting membranes, suggesting that dissociation of VG from its receptor occurs in this compartment. Newly internalized ligand preferentially forms a new MVB, rather than fusing and mixing with previously formed MVBs. Progressive yolk protein condensation gradually transforms MVBs into yolk platelets over a period of several hours. Analysis of 125I-VG-Au behavior after sucrose gradient fractionation of oocytes allowed correlation of biochemical compartments with those observed in the electron microscope. MVBs containing yolk in progressive stages of condensation were found at densities from 1.16 up to 1.21 g/cc. The final, rate-limiting step in VG transport is a shift of ligand from light (1.21 g/cc) to heavy (1.23 g/cc) platelet compartments (Wall and Meleka, 1985). The morphological correlate of this process is movement of VG-Au from small (less than 3-4 microns diameter) to large (greater than 4 microns diameter) platelets.     

Regulation of endocytic recycling by C. elegans Rab35 and its regulator RME-4, a coated-pit protein

Using Caenorhabditis elegans genetic screens, we identified receptor-mediated endocytosis (RME)-4 and RME-5/RAB-35 as important regulators of yolk endocytosis in vivo. In rme-4 and rab-35 mutants, yolk receptors do not accumulate on the plasma membrane as would be expected in an internalization mutant, rather the receptors are lost from cortical endosomes and accumulate in dispersed small vesicles, suggesting a defect in receptor recycling. Consistent with this, genetic tests indicate the RME-4 and RAB-35 function downstream of clathrin, upstream of RAB-7, and act synergistically with recycling regulators RAB-11 and RME-1. We find that RME-4 is a conserved DENN domain protein that binds to RAB-35 in its GDP-loaded conformation. GFP-RME-4 also physically interacts with AP-2, is enriched on clathrin-coated pits, and requires clathrin but not RAB-5 for cortical association. GFP-RAB-35 localizes to the plasma membrane and early endocytic compartments but is lost from endosomes in rme-4 mutants. We propose that RME-4 functions on coated pits and/or vesicles to recruit RAB-35, which in turn functions in the endosome to promote receptor recycling.     

Specific proteolysis regulates fusion between endocytic compartments in Xenopus oocytes

We examined the role of proteolytic ligand modification in endosomal targeting using vitellogenin (VTG) uptake by Xenopus oocytes as a model system. Non-cleavable VTG is internalized, but does not appear in yolk platelets. We identified two inhibitors of VTG processing into the yolk proteins: the ionophore monensin and pepstatin A, a specific inhibitor of cathepsin D. Pepstatin neither affected ligand binding and internalization, nor inhibited the degradation of nonspecifically incorporated proteins, whereas monensin inhibited all of these processes. Inhibiting VTG processing prevented its deposition into yolk platelets by strongly interfering with endosome-yolk platelet fusion. Monensin treatment resulted in morphologically abnormal endosomes, while pepstatin only inhibited VTG cleavage and the subsequent fusion of endosomes with yolk platelets. Since VTG cleavage is initiated prior to its deposition in platelets, we postulate that ligand proteolysis could be necessary for normal endosomal targeting.     

Receptor-mediated endocytosis and cytoskeleton

Receptor-mediated endocytosis is the most specific pathway for macromolecules and macromolecular complexes generally designated as ligands to enter cells. Upon binding to their transmembrane receptors, the ligands enter endocytic vesicles that fuse with each other giving rise to the so-called early endosomes. The sorting of ligand-receptor complexes internalized in these endosomes depends on their nature: metabolic receptors are recycled back to the plasma membrane, while signaling receptors and their ligands (e.g. receptor tyrosine kinases or receptors associated with tyrosine kinase) are delivered to internal vesicles of the multivesicular late endosomes and finally are degraded after interaction with lysosomes. During these processes, endosomes undergo translocation from the cell periphery to the juxtanuclear region, which is accompanied by multiple fusion, invagination, tabulation, and membrane fission events. This review considers modern concepts of the sorting mechanisms of ligand-receptor complexes, the crosstalk between endosomes, microtubules, and actin, and the role of this crosstalk in endosome maturation.     

Molecules internalized by clathrin-independent endocytosis are delivered to endosomes containing transferrin receptors

We have previously demonstrated that the preendosomal compartment in addition to clathrin-coated vesicles, comprises distinct nonclathrin coated endocytic vesicles mediating clathrin-independent endocytosis (Hansen, S. H., K. Sandvig, and B. van Deurs. 1991. J. Cell Biol. 113:731-741). Using K+ depletion in HEp-2 cells to block clathrin- dependent but not clathrin-independent endocytosis, we have now traced the intracellular routing of these nonclathrin coated vesicles to see whether molecules internalized by clathrin-independent endocytosis are delivered to a unique compartment or whether they reach the same early and late endosomes as encountered by molecules internalized with high efficiency through clathrin-coated pits and vesicles. We find that Con A-gold internalized by clathrin-independent endocytosis is delivered to endosomes containing transferrin receptors. After incubation of K(+)- depleted cells with Con A-gold for 15 min, approximately 75% of Con A- gold in endosomes is colocalized with transferrin receptors. Endosomes containing only Con A-gold may be accounted for either by depletion of existing endosomes for transferrin receptors or by de novo generation of endosomes. Cationized gold and BSA-gold internalized in K(+)- depleted cells are also delivered to endosomes containing transferrin receptors. h-lamp-1-enriched compartments are only reached occasionally within 30 min in K(+)-depleted as well as in control cells. Thus, preendosomal vesicles generated by clathrin-independent endocytosis do not fuse to any marked degree with late endocytic compartments. These data show that in HEp-2 cells, molecules endocytosed without clathrin are delivered to the same endosomes as reached by transferrin receptors internalized through clathrin-coated pits.     

Visualization of binding and receptor-mediated uptake of low density lipoproteins by human endothelial cells

Low density lipoproteins (LDL) were conjugated to colloidal gold for investigation of the ultrastructural aspects of binding and receptor-mediated internalization of LDL by cultured endothelial cells from the human umbilical artery and vein. The number of LDL receptors was increased by preincubation in lipoprotein-depleted serum. When the cells were incubated with LDL-gold particles for 2 h at 4 degrees C, the complexes were found in coated pits as well as in clusters attached to the plasma membrane. Small vesicles containing a few LDL-gold complexes appeared in the cytoplasm close to the plasma membrane when the cells were incubated with the conjugate for 5 min at 37 degrees C. After 15 min at 37 degrees C, larger vesicles with a pale matrix and membrane-orientated LDL-gold complexes were seen. After incubation for 30 min at 37 degrees C, colloidal gold particles were present in dense bodies. Quantification of the binding of LDL-gold complexes to the plasma membrane at 4 degrees C showed no differences between arterial and venous endothelial cells.     

Plasma membrane-derived vesicles containing receptor-ligand complexes are fusogenic with early endosomes in a cell-free system

Receptor-mediated endocytosis involves the transport of receptor-ligand complexes from the cell surface to an intracellular endocytic compartment. This study shows that plasma membrane-derived vesicles containing receptor-bound ligands (e.g. aggregated anti-dinitrophenol (DNP) IgG bound to Fc receptors) fuse with early endosomes containing DNP-beta-glucuronidase in a cell-free system. Plasma membrane vesicles were generated by homogenization of cells that had been allowed to bind ligands at 4 degrees C. Fusion between vesicles containing the two probes was assessed by (i) the formation of anti-DNP IgG-DNP-beta-glucuronidase complexes and (ii) the colocalization within closed vesicles of two different sizes of colloidal gold coated with ligands. Fusion required ATP, cytosol, and KCl. The requirements were similar to those described for endosome-endosome fusion in in vitro systems. Mild trypsinization of vesicles prior to their addition to the assay inhibited fusion. When DNP-beta-glucuronidase was chased into more mature endocytic compartments, fusion was not observed. The results indicate that cell surface regions involved in receptor-mediated endocytosis are capable of fusing to early endosomes. This fusion event may constitute the first step in the transport of ligands to an intracellular endocytic compartment.     

Intracellular transport of vitellogenin in Xenopus oocytes: An autoradiographic study

The role of primordial yolk platelets (PYPs) in the transport of the yolk precursor vitellogenin to the yolk platelets in Xenopus laevis oocytes has been demonstrated by electron microscopic autoradiography. Within 20 min after exposure of the oocyte to ³H-labeled-vitellogenin, silver grains are associated with small PYPs which are formed by the fusion of endosomes. At 40 min after incorporation of ³H-labeled vitellogenin, autoradiographic silver grains are associated with larger PYPs and with the superficial layer of yolk platelets. Thus, the results demonstrate that PYPs are an intermediate in the transport of vitellogenin from endosomes to yolk platelets. These observations are consonant with the general hypothesis that vitellogenin first associates (binds?) with the plasma membrane, then is incorporated by endocytosis into endosomes which fuse to form PYPs, and finally the contents of the PYPs are eventually deposited into yolk platelets.     

Chicken oocyte growth: receptor-mediated yolk deposition

During the rapid final stage of growth, chicken oocytes take up massive amounts of plasma components and convert them to yolk. The oocyte expresses a receptor that binds both major yolk lipoprotein precursors, vitellogenin (VTG) and very low density lipoprotein (VLDL). In the present study, in vivo transport tracing methodology, isolation of coated vesicles, ligand- and immuno-blotting, and ultrastructural immunocytochemistry were used for the analysis of receptor-mediated yolk formation. The VTG/VLDL receptor was identified in coated profiles in the oocyte periphery, in isolated coated vesicles, and within vesicular compartments both outside and inside membrane-bounded yolk storage organelles (yolk spheres). VLDL particles colocalized with the receptor, as demonstrated by ultrastructural visualization of VLDL-gold following intravenous administration, as well as by immunocytochemical analysis with antibodies to VLDL. Lipoprotein particles were shown to reach the oocyte surface by passage across the basement membrane, which possibly plays an active and selective role in yolk precursor accessibility to the oocyte surface, and through gaps between the follicular granulosa cells. Following delivery of ligands from the plasma membrane into yolk spheres, proteolytic processing of VTG and VLDL by cathepsin D appears to correlate with segregation of receptors and ligands which enter disparate sub-compartments within the yolk spheres. In small, quiescent oocytes, the VTG/VLDL receptor was localized to the central portion of the cell. At onset of the rapid growth phase, it appears that this pre-existing pool of receptors redistributes to the peripheral region, thereby initiating yolk formation. Such a redistribution mechanism would obliterate the need for de novo synthesis of receptors when the oocyte's energy expenditure is to be utilized for plasma membrane synthesis, establishment and maintenance of intracellular topography and yolk formation, and preparation for ovulation.     

Endocytosis and intracellular processing of transferrin and colloidal gold-transferrin in rat reticulocytes: demonstration of a pathway for receptor shedding

Endocytosis and intracellular processing of transferrin (Tf) and Tf receptors were examined in rat reticulocytes. Subcellular fractionation revealed that Tf enters a non-lysosomal endocytic compartment with a density between those of plasma membrane and lysosomes. After 20 min of uptake at (37 degrees C) 35 to 40% of cell-associated Tf was contained in this intermediate-density compartment. To test the fidelity of colloidal gold-Tf (AuTf) as a probe for Tf processing, reticulocytes were fractionated after uptake of 131I-Tf and 125I-AuTf. The subcellular distributions of the two ligands were indistinguishable by this method, a result suggesting that AuTf is processed similarly to Tf. Electron microscopy revealed that AuTf entered multivesicular endosomes (MVEs) as well as various small vesicles and tubular structures. In addition MVE exocytosis was observed with discharge of inclusion vesicles and associated AuTf. AuTf was bound to the outside of these vesicles both before and after exocytosis. These data suggest that Tf receptors are shed from developing reticulocytes by incorporation into the limiting membrane of inclusion vesicles, followed by discharge of these vesicles by MVE exocytosis. As further evidence of this process, we isolated inclusion vesicles after their discharge and found them to contain Tf receptors. Moreover, the rate of Tf receptor shedding by inclusion vesicle discharge matches Tf receptor loss rates closely enough to suggest that this is the primary path of receptor loss during reticulocyte development.     

A novel class of clathrin-coated vesicles budding from endosomes

Clathrin-coated vesicles transport selective integral membrane proteins from the plasma membrane to endosomes and from the TGN to endosomes. Recycling of proteins from endosomes to the plasma membrane occurs via unidentified vesicles. To study this pathway, we used a novel technique that allows for the immunoelectron microscopic examination of transferrin receptor-containing endosomes in nonsectioned cells. Endosomes were identified as separate discontinuous tubular-vesicular entities. Each endosome was decorated, mainly on the tubules, with many clathrin-coated buds. Endosome-associated clathrin-coated buds were discerned from plasma membrane-derived clathrin-coated vesicles by three criteria: size (60 nm and 100 nm, respectively), continuity with endosomes, and the lack of labeling for alpha-adaptin. They were also distinguished from TGN-derived clathrin-coated vesicles by their location at the periphery of the cell, size, and the lack of labeling for gamma-adaptin. In the presence of brefeldin A, a large continuous endosomal network was formed. Transferrin receptor recycling as well as the formation of clathrin-coated pits at endosomes was inhibited in the presence of brefeldin A. Together with the localization of transferrin receptors at endosome-associated buds, this indicates that a novel class of clathrin-coated vesicles serves an exit pathway from endosomes. The target organelles for endosome-derived clathrin-coated vesicles remain, however, to be identified.     

The relationship between lumenal and limiting membranes in swollen late endocytic compartments formed after wortmannin treatment or sucrose accumulation

Immunofluorescence and electron microscopy were used to evaluate the formation of swollen endosomes in NRK cells after treatment with wortmannin or sucrose and to study the relationship between lumenal and limiting membrane. Both treatments resulted in the formation of two populations of swollen late endocytic vacuoles, positive for lysosomal glycoproteins or cation-independent mannose 6-phosphate receptors, but those induced by wortmannin were characterised by time-dependent accumulation of lumenal vesicles, whereas those induced by sucrose uptake did not accumulate lumenal vesicles. In both cases, the distribution of the late endosomal marker, lysobisphosphatidic acid, remained unchanged and was present within the lumen of the swollen vacuoles. Consumption of plasma membrane and peripheral early endosomes, and the appearance of transferrin receptors in swollen late endosomes, indicated that continued membrane influx from early endocytic compartments, together with inhibition of membrane traffic out of the swollen compartments, is sufficient to account for the observed phenotype of cells treated with wortmannin. The accumulation of organelles with the characteristic morphology of endocytic carrier vesicles in cells that have taken up sucrose offers an explanation for the paucity of lumenal vesicles in swollen sucrosomes. Our data suggest that in fibroblast cells the swollen endosome phenotype induced by wortmannin is a consequence of endocytic membrane influx, coupled with the failure to recycle membrane to other cellular destinations, and not the inhibition of multivesicular body biogenesis.     

Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomes

Cells rely on the correct sorting of endocytic ligands and receptors for proper function. Early endosomes have been considered as the initial sorting station where cargos for degradation separate from those for recycling. Using live-cell imaging to monitor individual endosomes and ligand particles in real time, we have discovered a sorting mechanism that takes place prior to early endosome entry. We show that early endosomes are in fact comprised of two distinct populations: a dynamic population that is highly mobile on microtubules and matures rapidly toward late endosomes and a static population that matures much more slowly. Several cargos destined for degradation are preferentially targeted to the dynamic endosomes, whereas the recycling ligand transferrin is nonselectively delivered to all early endosomes and effectively enriched in the larger, static population. This pre-early endosome sorting process begins at clathrin-coated vesicles, depends on microtubule-dependent motility, and appears to involve endocytic adaptors.     

Intracellular pathways of endocytosed transferrin and non-specific tracers in epithelial cells lining the rete testis of the rat

Summary The uptake and pathway of different markers and ligands for fluid-phase, adsorptive and receptor mediated endocytosis were analyzed in the epithelial cells lining the rete testis after their infusion into the lumen of these anastomotic channels. At 2 min after injection, diferric transferrin bound to colloidal gold was seen attached to the apical plasma membrane and to the membrane of endocytic coated and uncoated pits and vesicles. The injection of transferrin-gold in the presence of a 100-fold excess of unconjugated diferric transferrin revealed no binding or internalization of transferrin-gold. Similarly, apotransferrin-gold was neither bound to the apical plasma membrane nor internalized by these cells. These results thus indicate the presence of specific binding sites for diferric transferrin. At 5 min, internalized diferric transferrin-gold reached endosomes. At 15 and 30 min, the endosomes were still labeled but at these time intervals the transferrin-gold also appeared in tubular elements connected to or associated with these bodies or seen in close proximity to the apical plasma membrane. At 60 and 90 min, most of the transferrin-gold was no longer present in these organelles and was seen only exceptionally in secondary lysosomes. These results thus suggest that the tubular elements may be involved in the recycling of transferrin back to the lumen of the rete testis. The coinjection of transferrin-gold and the fluid-phase marker native ferritin revealed that both proteins were often internalized in the same endocytic pit and vesicle and shared the same endosome. However, unlike transferrin, native ferritin at the late time intervals appeared in dense multivesicular bodies and secondary lysosomes. When the adsorptive marker cationic ferritin and the fluid-phase marker albumin-gold were coinjected, again both proteins often shared the same endocytic pit and vesicle, endosome, pale and dense multivesicular body and secondary lysosomes. However, several endocytic vesicles labeled only with cationic ferritin appeared to bypass the endosomal and lysosomal compartments and to reach the lateral intercellular space and areas of the basement membrane. The rete epithelial cells, therefore, appear to be internalizing proteins and ligands by receptor-mediated and non-specific endocytosis which, after having shared the same endocytic vesicle and endosome, appear to be capable of being segregated and routed to different destinations.     

The Coronavirus Transmissible Gastroenteritis Virus Causes Infection after Receptor-Mediated Endocytosis and Acid-Dependent Fusion with an Intracellular Compartment

Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive infection, or whether they fuse directly with the plasma membrane. We have studied the interaction between TGEV and a cell line (MDCK) stably expressing recombinant pig aminopeptidase N (pAPN). By electron microscopy and flow cytometry, TGEV was found to be associated with the plasma membrane after adsorption to the pAPN-MDCK cells. TGEV was also observed in endocytic pits and apical vesicles after 3 to 10 min of incubation at 38°C. The number of pits and apical vesicles was increased by the TGEV incubation, indicating an increase in endocytosis. After 10 min of incubation, a distinct TGEV-pAPN-containing population of large intracellular vesicles, morphologically compatible with endosomes, was found. A higher density of pAPN receptors was observed in the pits beneath the virus particles than in the surrounding plasma membrane, indicating that TGEV recruits pAPN receptors before endocytosis. Ammonium chloride and bafilomycin A₁ markedly inhibited the TGEV infection as judged from virus production and protein biosynthesis analyses but did so only when added early in the course of the infection, i.e., about 1 h after the start of endocytosis. Together our results point to an acid intracellular compartment as the site of fusion for TGEV.     

Subpopulations of endosomes generated at sequential stages in the endocytic pathway of asialoganglioside-containing ferrite ligands in rat liver

Subpopulations of endosomes generated at different stages of the endocytic pathway were isolated by a high-gradient magnetic separation followed by a Percoll density gradient centrifugation. Rat livers were perfused for 5 min with asialoganglioside (ASG)-containing ferrite particles and chased at 37 degrees C. At various times after the internalization, the endocytic vesicles containing ferrite particles were isolated by the magnetic separation. Isolated fractions contained endosomes until 15-min perfusion, after which most of the particles were transported to lysosomes. The endosomal fractions isolated after the 5- or 15-min perfusions were further analyzed by 30% Percoll density gradient centrifugation. The endosomes after 5-min perfusion showed peaks around the density of 1.05 g/ml (peak I) and 1.07 g/ml (peak Is), both of which contained asialoglycoprotein receptors. In the 15-min perfusion, another peak of endosomes (peak II) was observed at the higher density of 1.09 g/ml without the receptors, in addition to peak I. These endosomes had their own characteristic proteins. Some proteins were common in the subgroups of endosomes. These results suggest that the endosome I containing the ligands and the receptors was first produced after endocytosis and, through the endosome is, was scissioned into the endosome II containing the ligands. The endosome II was then fused with primary lysosomes for proteolytic cleavage of ligands.