Osmotic swelling of endocytic compartments induced by internalized sucrose is restricted to mature lysosomes in cultured mammalian cells.

To determine which endocytic compartments are sensitive to sucrose-induced osmotic swelling, CHO and Vero cells were cultured for 1-3 days in media containing 0.03 to 0.05 M sucrose. (Sucrose is internalized but not digested by these cells.) To immunolocalize late endocytic compartments, cells were fixed with formaldehyde and labeled with antibodies against the 215-kDa mannose 6-phosphate receptor (prelysosomal compartment) and LAMP-1 and -2 (mature lysosomes). Early endosomes were labeled by a 2-min uptake of lucifer yellow, mature lysosomes by greater than or equal to 16-h uptake of lucifer yellow followed by a 2-h chase. The data showed that sucrose induced swelling of mature lysosomes only (mannose 6-phosphate receptor negative, LAMP-1 and LAMP-2 positive); early endosomes and the prelysosomal compartment were not affected by the presence of sucrose, i.e., osmotically swollen. Accumulation of lucifer yellow in the swollen compartment was insensitive to cycloheximide. These results suggest, by inference, that the complement of membrane transport proteins that regulate the osmotic properties of endocytic organelles must be discontinuously distributed along the endocytic pathway.     

Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts

Although endosomes and lysosomes are associated with different subcellular functions, we present evidence that a lysosomal enzyme, arylsulfatase-A, is present in prelysosomal vesicles which constitute part of the endosomal compartment. When human cultured fibroblasts were subfractionated with Percoll gradients, arylsulfatase-A activity was enriched in three subcellular fractions: dense lysosomes, light lysosomes, and light membranous vesicles. Pulsing the cells for 1 to 10 min with the fluid-phase endocytic marker, horseradish peroxidase, showed that endosomes enriched with the marker were distributed partly in the light lysosome fraction but mainly in the light membranous fraction. By pulsing the fibroblasts for 10 min with horseradish peroxidase conjugated to colloidal gold and then staining the light membranous and light lysosomal fractions for arylsulfatase-A activity with a specific cytochemical technique, the endocytic marker was detected under the electron microscope in the same vesicles as the lysosomal enzyme. The origin of the lysosomal enzyme in this endosomal compartment was shown not to be acquired through mannose 6-phosphate receptor-mediated endocytosis of enzymes previously secreted from the cell. Together with our recent finding that the light membranous fraction contains prelysosomes distinct from bona fide lysosomes and was highly enriched with newly synthesized arylsulfatase-A molecules, these results demonstrate that prelysosomes also constitute part of the endosomal compartment to which intracellular lysosomal enzymes are targeted.     

Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.     

Effects of temperature, pH elevators, and energy production inhibitors on horseradish peroxidase transport through endocytic vesicles

We investigated the effects of reduced temperature, the pH elevators NH4Cl, monensin, and HEPES (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) buffer, as well as the metabolic poisons NaF/KCN on transport of the fluid phase pinocytic marker, horseradish peroxidase (HRP), to lysosomes in Chinese hamster ovary (CHO) cells. In cell fractionation experiments, these agents appeared to block HRP transit at specific point(s) from "early" to "late" (i.e., low to high density) prelysosomal vesicles and lysosomes. Reduced temperature (17 degrees C) most strongly inhibited HRP transport from low density, early endosomes to lysosomes. In long-term HRP uptakes at 17 degrees C, marked peroxidase accumulation occurred both in early endosomes and in lysosomes. Loss (reversible pinocytosis) of HRP from "very early" endosomes occurred at 17 degrees C. All three pH elevators including the common media supplement HEPES buffer inhibited transit of internalized HRP into lysosomes. For all three pH elevators, inhibition was most pronounced at the "early" endosome stage. The respiratory inhibitors NaF/KCN also inhibited transport most strongly at the early endosome stage. Together these results suggest that "early" steps in the endocytic transport of HRP are the most sensitive and that the conditions tested may exert direct effects on the processing of endocytic vesicles.     

Fusion accessibility of endocytic compartments along the recycling and lysosomal endocytic pathways in intact cells

A fluorescence assay developed for the quantitation of intracellular fusion of sequentially formed endocytic compartments (Salzman, N. H., and F. R. Maxfield. 1988 J. Cell Biol. 106:1083-1091) has been used to measure the time course of endosome fusion accessibility along the recycling and degradative endocytic pathways. Transferrin (Tf) was used to label the recycling pathway, and alpha2-macroglobulin (alpha 2 M) was used to label the lysosomal degradative pathway. Along the degradative pathway, accessibility of vesicles containing alpha 2M to fusion with subsequently formed endocytic vesicles decreased with apparent first order kinetics. The t12 for the loss of fusion accessibility was approximately 8 min. The behavior of Tf is more complex. Initially the fusion accessibility of Tf decayed rapidly (t1/2 less than 3 min), but a constant level of fusion accessibility was then observed for 10 min. This suggests that Tf moves through one fusion accessible endosome rapidly and then enters a second fusion accessible compartment on the recycling pathway. At 18 degrees C, fusion of antifluorescein antibodies (AFA) containing vesicles with F-alpha 2M was observed when the interval between additions was 10 min. However, if the interval was increased to 1 h, no fusion with incoming vesicles was observed. These results identify the site of F-alpha 2M accumulation at 18 degrees C as a prelysosomal late endosome that no longer fuses with newly formed endosomes since no delivery to lysosomes is observed at this temperature.     

Mannose-6-phosphate receptors for lysosomal enzymes cycle between the Golgi complex and endosomes

We have examined the distribution of mannose-6-phosphate (Man6P) receptors (215 kD) for lysosomal enzymes in cultured Clone 9 hepatocytes at various times after the addition or removal of lysosomotropic weak bases (chloroquine or NH4Cl). Our previous studies demonstrated that after treatment with these agents, Man6P receptors are depleted from their sorting site in the Golgi complex and accumulate in dilated vacuoles that could represent either endosomes or lysosomes (Brown, W. J., E. Constantinescu, and M. G. Farquhar, 1984, J. Cell Biol., 99:320-326). We have now investigated the nature of these vacuoles by labeling NH4Cl-treated cells simultaneously with anti-Man6P receptor IgG and lysosomal or endosomal markers. The structures in which the immunolabeled receptors are found were identified as endosomes based on the presence of endocytic tracers (lucifer yellow and cationized ferritin). The lysosomal membrane marker, lgp120, was associated with a separate population of swollen vacuoles that did not contain detectable Man6P receptors. When cells were allowed to recover from weak base treatment, the receptors reappeared in the Golgi cisternae of most cells (approximately 90%) within approximately 20 min, indicating that as the intra-endosomal pH drops and lysosomal enzymes dissociate, the entire population of receptors rapidly recycles to Golgi cisternae. When NH4Cl-treated cells were allowed to endocytose Man6P, a competitive inhibitor of lysosomal enzyme binding, the receptors also recycled to the Golgi cisternae, suggesting that lysosomal enzymes can dissociate from the receptors under these conditions (high pH + presence of competitive inhibitor). From these results it can be concluded that the intracellular itinerary of the 215-kD Man6P receptor involves its cycling via coated vesicles between the Golgi complex and endosomes, ligand dissociation is both necessary and sufficient to trigger the recycling of Man6P receptors to the Golgi complex, and endosomes rather than secondary lysosomes represent the junction where endocytosed material and primary lysosomes carrying receptor-bound lysosomal enzymes meet.(ABSTRACT TRUNCATED AT 400 WORDS)     

Penetration of Semliki Forest virus from acidic prelysosomal vacuoles

To identify and characterize the intracellular site from which the penetration of Semliki Forest virus (SFV) to the cytosolic compartment of the host cell occurs, we determined the time course and temperature dependence of nucleocapsid uncoating and infection in BHK-21 cells. At 37 degrees C the genome release to the cytosol was detected within 5-7 min after virus endocytosis, whereas delivery of the virus particles to secondary lysosomes occurred within 15-20 min. At temperatures of 15 degrees -20 degrees C virus particles were internalized by endocytosis, but they were not delivered to the secondary lysosomes. Nevertheless, at 20 degrees C nucleocapsid uncoating and infection occurred, indicating that secondary lysosomes are not required for SFV penetration. We conclude that the penetration reaction occurs in prelysosomal endocytic vacuoles (endosomes). As SFV penetration by membrane fusion requires a pH less than 6 and the presence of cholesterol in the target membrane, the data indicate that endosomes are acidic and contain cholesterol.     

Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes.

Brefeldin A (BFA) is a fungal metabolite that causes a redistribution of the stacked cisternae of the Golgi complex into the endoplasmic reticulum by inhibiting anterograde transport. We report that BFA also causes membrane tubules derived from the trans-Golgi network (TGN) to fuse with early endosomes. In the presence of BFA, a mannose-6-phosphate receptor (M6PR)-enriched tubular network rapidly forms from the TGN, not from the prelysosomal compartment, and can be labeled with endocytic tracers after only 5 min of uptake at either 20 degrees C or 37 degrees C, indicating that it is also functionally an early endosome. Formation of the TGN-early endosome network is microtubule dependent and may involve modification of membrane processes affected by microtubule-associated motor activity. Concomitant with the formation of the fused TGN-early endosome network, there is a greater than 5-fold increase in cell surface M6PRs. The data suggest that BFA has revealed a membrane transport cycle between the TGN and early endosomes, perhaps used for the secretion or delivery of molecules to the cell surface.     

Effects of inhibitors of the vacuolar proton pump on hepatic heterophagy and autophagy

Bafilomycin A(1) (BAF) and concanamycin A (ConcA) are selective inhibitors of the H(+)-ATPases of the vacuolar system. We have examined the effects of these inhibitors on different steps in endocytic pathways in rat hepatocytes, using [(125)I]tyramine-cellobiose-labeled asialoorosomucoid ([(125)I]TC-AOM) and [(125)I]tyramine-cellobiose-labeled bovine serum albumin ([(125)I]TC-BSA) as probes for respectively receptor-mediated endocytosis and pinocytosis (here defined as fluid phase endocytosis). The effects of BAF and ConcA were in principle identical, although ConcA was more effective than BAF. The main findings were as follows. (1) BAF/ConcA reduced the rate of uptake of both [(125)I]TC-AOM and [(125)I]TC-BSA. The reduced uptake of [(125)I]TC-AOM was partly due to a redistribution of the asialoglycoprotein receptors (ASGPR) such that the number of surface receptors was reduced approximately 40% without a change in the total number of receptors. (2) BAF/ConcA at the same time increased retroendocytosis (recycling) of both probes. The increased recycling of the ligand ([(125)I]TC-AOM) is partly a consequence of the enhanced pH in endosomes, which prevents dissociation of ligand. (3) It was furthermore found that the ligand remained bound to the receptor in presence of BAF/ConcA and that the total amount of ligand molecules internalized in BAF/ConcA-treated cells was only slightly in excess of the total number of receptors. These data indicate that reduced pH in endosomes is the prime cause of receptor inactivation and release of ligand in early endosomes. (4) Subcellular fractionation experiments showed that [(125)I]TC-AOM remained in early endosomes, well separated from lysosomes in sucrose gradients. The fluid phase marker, [(125)I]TC-BSA, on the other hand, seemed to reach a later endosome in the BAF/ConcA-treated cells. This organelle coincided with lysosomes in the gradient, but hypotonic medium was found to selectively release a lysosomal enzyme (beta-acetylglucosaminidase), indicating that even [(125)I]TC-BSA remained in a prelysosomal compartment in the BAF/ConcA-treated cells. (5) Electron microscopy using horseradish peroxidase (HRP) as a fluid phase marker verified that BAF/ConcA inhibited transfer of material from late endosomes ('multivesicular bodies'). (6) BAF/ConcA led to accumulation of lactate dehydrogenase (LDH) in autophagic vacuoles, but although the drugs partly inhibited fusion between autophagosomes and lysosomes a number of autolysosomes was formed in the presence of BAF/ConcA. This observation explains the reduced buoyant density of lysosomes (revealed in sucrose density gradients). In conclusion, BAF/ConcA inhibit transfer of endocytosed material from late endosomes to lysosomes, but do not at the same time prevent fusion between autophagosomes and lysosomes.     

Meeting of the apical and basolateral endocytic pathways of the Madin- Darby canine kidney cell in late endosomes

Electron microscopic approaches have been used to study the endocytic pathways from the apical and basolateral surface domains of the polarized epithelial cell, MDCK strain I, grown on polycarbonate filters. The cells were incubated at 37 degrees C in the presence of two distinguishable markers administered separately to the apical or the basolateral domain. Initially each marker was visualized within distinct apical or basolateral peripheral endosomes. However, after 15 min at 37 degrees C, both markers were observed within common perinuclear structures. The compartment in which meeting first occurred was shown to be a late endosome (prelysosome) that labeled extensively with antibodies against the cation-independent mannose-6-phosphate receptor (MPR) on cryosections. With increasing incubation times, markers passed from these MPR-positive structures into a common set of MPR-negative lysosomes that were mainly located in the apical half of the cell. A detailed quantitative analysis of the endocytic pathways was carried out using stereological techniques in conjunction with horseradish peroxidase and acid phosphatase cytochemistry. This enabled us to estimate the absolute volumes and membrane surface areas of the endocytic organelles involved in apical and basolateral endocytosis.     

Evidence for the Involvement of annexin 6 in the trafficking between the endocytic compartment and lysosomes

Annexins are a family of calcium-dependent phospholipid-binding proteins, which have been implicated in a variety of biological processes including membrane trafficking. The annexin 6/lgp120 prelysosomal compartment of NRK cells was loaded with low-density lipoprotein (LDL) and then its transport from this endocytic compartment and its degradation in lysosomes were studied. NRK cells were microinjected with the mutated annexin 6 (anx6(1-175)), to assess the possible involvement of annexin 6 in the transport of LDL from the prelysosomal compartment. The results indicated that microinjection of mutated annexin 6, in NRK cells, showed the accumulation of LDL in larger endocytic structures, denoting retention of LDL in the prelysosomal compartment. To confirm the involvement of annexin 6 in the trafficking and the degradation of LDL we used CHO cells transfected with mutated annexin 6(1-175). Thus, in agreement with NRK cells the results obtained in CHO cells demonstrated a significant inhibition of LDL degradation in CHO cells expressing the mutated form of annexin 6 compared to controls overexpressing wild-type annexin 6. Therefore, we conclude that annexin 6 is involved in the trafficking events leading to LDL degradation.     

Immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages

We have used endocytic and phagocytic tracers in an EM immunocytochemical study to define the compartments of the phagocytic and endocytic pathways in mouse peritoneal macrophages. Endocytosed BSA-gold appeared successively in early endosomes, spherical endosomal vesicles, a late endosomal tubuloreticular compartment (TC), and terminal lysosomes. The TC appeared as an elaborate structure enriched for the lysosomal membrane glycoproteins Lamp 1 and Lamp 2, and expressing significant levels of rab7, a late endosome-specific GTP-binding protein. The cation-independent mannose-6-phosphate receptor was restricted to specialized regions of the TC that were predominantly adjacent to the Golgi complex. Both the early endosome and the TC had coated bud structures whose composition and function are presently unknown. Phagolysosomes containing latex beads expressed the same membrane antigens and received endocytic tracers simultaneously with the TC. Since the membrane surrounding both organelles was also in direct continuity, we assume that both structures form one functional compartment. Macrosialin, an antigen confined to macrophages and dendritic cells, was heavily expressed in TC and phagolysosomal membranes with low levels being detected in other endosomal compartments and on the cell surface. Treatment of cells with wheat germ agglutinin drastically altered the morphology of the TC, giving rise to sheets of tightly adherent membrane and greatly expanded vesicles, in which cell-associated wheat germ agglutinin was concentrated. The spherical endosomal carrier vesicles loaded with internalized gold tracers clustered nearby, often making contact without fusing. Since the delivery of endocytic tracer to the TC was significantly delayed these experiments suggest that the lectin is somehow preventing the endosome vesicles from fusing with the TC. Collectively, our data argue first that the PLC is equivalent to the "tubular lysosomes" commonly described in macrophages, and second that the meeting of the phagocytic and endocytic pathway occurs in this compartment.     

Kinetic evidence that newly-synthesized endogenous lysosome-associated membrane protein-1 (LAMP-1) first transits early endosomes before it is delivered to lysosomes

After de novo synthesis of lysosome-associated membrane proteins (LAMPs), they are sorted in the trans-Golgi network (TGN) for delivery to lysosomes. Opposing views prevail on whether LAMPs are targeted to lysosomes directly, or indirectly via prelysosomal stages of the endocytic pathway, in particular early endosomes. Conflicting evidence is based on kinetic measurements with too limited quantitative data for sufficient temporal and organellar resolution. Using cells of the mouse macrophage cell line, P338D(1), this study presents detailed kinetic data that describe the extent of, and time course for, the appearance of newly-synthesized LAMP-1 in organelles of the endocytic pathway, which had been loaded selectively with horse-radish peroxidase (HRP) by appropriate periods of endocytosis. After a 5-min pulse of metabolic labelling, LAMP-1 was trapped in the respective organelles by HRP-catalyzed crosslinking with membrane-permeable diaminobenzidine (DAB). These kinetic observations provide sufficient quantitative evidence that in P338D(1) cells the bulk of newly-synthesized endogenous LAMP-1 first appeared in early endosomes, before it was delivered to late endosomes and lysosomes about 25 min later.     

Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis

Endosomes are prelysosomal organelles that serve as an intracellular site for the sorting, distribution, and processing of receptors, ligands, fluid phase components, and membrane proteins internalized by endocytosis. Whereas the overall functions of endosomes are increasingly understood, little is known about endosome structure, composition, or biogenesis. In this paper, we describe a rapid procedure that permits analytical and preparative isolation of endosomes from a variety of tissue culture cells. The procedure relies on a combination of density gradient centrifugation and free flow electrophoresis. It yields a fraction of highly purified, functionally intact organelles. As markers for endosomes in Chinese hamster ovary cells, we used endocytosed horseradish peroxidase, FITC-conjugated dextran, and [35S]methionine-labeled Semliki Forest virus. Total postnuclear supernatants, crude microsomal pellets, or partially purified Golgi fractions were subjected to free flow electrophoresis. Endosomes and lysosomes migrated together as a single anodally deflected peak separated from most other organelles (plasma membrane, mitochondria, endoplasmic reticulum, and Golgi). The endosomes and lysosomes were then resolved by centrifugation in Percoll density gradients. Endosomes prepared in this way were enriched up to 70-fold relative to the initial homogenate and were still capable of ATP-dependent acidification. By electron microscopy, the isolated organelles were found to consist of electron lucent vacuoles and tubules, many of which could be shown to contain an endocytic tracer (e.g., horseradish peroxidase). SDS PAGE analysis of integral and peripheral membrane proteins (separated from each other by condensation in Triton X-114) revealed a unique and restricted subset of proteins when compared with lysosomes, the unshifted free flow electrophoresis peak, and total cell protein. Altogether, the purification procedure takes 5-6 h and yields amounts of endosomes (150-200 micrograms protein) sufficient for biochemical, immunological, and functional analysis.     

The mannose 6-phosphate receptor and the biogenesis of lysosomes

Localization of the 215 kd mannose 6-phosphate receptor (MPR) was studied in normal rat kidney cells. Low levels of receptor were detected in the trans Golgi network, Golgi stack, plasma membrane, and peripheral endosomes. The bulk of the receptor was localized to an acidic, reticular-vesicular structure adjacent to the Golgi complex. The structure also labeled with antibodies to lysosomal enzymes and a lysosomal membrane glycoprotein (lgp120). While lysosome-like, this structure is not a typical lysosome that is devoid of MPRs. The endocytic marker alpha 2 macroglobulin-gold entered the structure at 37 degrees C, but not at 20 degrees C. With prolonged chase, most of the marker was transported from the structure into lysosomes. We propose that the MPR/lgp-enriched structure is a specialized endosome (prelysosome) that serves as an intermediate compartment into which endocytic vesicles discharge their contents, and where lysosomal enzymes are released from the MPR and packaged along with newly synthesized lysosomal glycoproteins into lysosomes.     

Mutant Rab7 Causes the Accumulation of Cathepsin D and Cation-independent Mannose 6–Phosphate Receptor in an Early Endocytic Compartment

Stable BHK cell lines inducibly expressing wild-type or dominant negative mutant forms of the rab7 GTPase were isolated and used to analyze the role of a rab7-regulated pathway in lysosome biogenesis. Expression of mutant rab7N125I protein induced a dramatic redistribution of cation-independent mannose 6–phosphate receptor (CI-MPR) from its normal perinuclear localization to large peripheral endosomes. Under these circumstances ~50% of the total receptor and several lysosomal hydrolases cofractionated with light membranes containing early endosome and Golgi markers. Late endosomes and lysosomes were contained exclusively in well-separated, denser gradient fractions. Newly synthesized CI-MPR and cathepsin D were shown to traverse through an early endocytic compartment, and functional rab7 was crucial for delivery to later compartments. This observation was evidenced by the fact that 2 h after synthesis, both markers were more prevalent in fractions containing light membranes. In addition, both were sensitive to HRP-DAB– mediated cross-linking of early endosomal proteins, and the late endosomal processing of cathepsin D was impaired. Using similar criteria, the lysosomal membrane glycoprotein 120 was not found accumulated in an early endocytic compartment. The data are indicative of a post-Golgi divergence in the routes followed by different lysosome-directed molecules.     

Kinetics of endosome acidification detected by mutant and wild-type Semliki Forest virus.

The fusogenic properties of Semliki Forest virus (SFV) and its mutants were used to follow the kinetics of acidification during the endocytic uptake of virus by BHK-21 cells. It has previously been shown that the low pH of endocytic vacuoles triggers a conformational change in the SFV spike glycoprotein, activating membrane fusion and initiating virus infection. This conformational alteration was here shown to occur in endosomes and to follow the same time course as the intracellular fusion reaction, demonstrating that fusion occurs rapidly after virus exposure to endosome acidity. The kinetics of endosome acidification were monitored using wild type (wt) SFV and fus-1, an SFV mutant with a lower fusion pH threshold. The results presented here demonstrated that wt and mutant virus were internalized with a t1/2 of 10 min, and that endosomes were acidified to the wt threshold of pH 6.2 with a t1/2 of 15 min. In contrast, endosome pH reached the fus-1 threshold of 5.3 with a much longer t1/2 of 45 min. The subsequent degradation of SFV in lysosomes had a t1/2 of 90 min. It was found that after the initial uptake of virus from the plasma membrane, its transit through the endocytic pathway, exposure to endosome acidity and eventual delivery to lysosomes were markedly asynchronous.     

Evidence for a sorting endosome in Arabidopsis root cells

In eukaryotic cells, the endocytic and secretory pathways are key players in several physiological processes. These pathways are largely inter-connected in animal and yeast cells through organelles named sorting endosomes. Sorting endosomes are multi-vesicular compartments that redirect proteins towards various destinations, such as the lysosomes or vacuoles for degradation, the trans-Golgi network for retrograde transport and the plasma membrane for recycling. In contrast, cross-talk between the endocytic and secretory pathways has not been clearly established in plants, especially in terms of cargo protein trafficking. Here we show by co-localization analyses that endosomes labelled with the AtSORTING NEXIN1 (AtSNX1) protein overlap with the pre-vacuolar compartment in Arabidopsis root cells. In addition, alteration of the routing functions of AtSNX1 endosomes by drug treatments leads to mis-routing of endocytic and secretory cargo proteins. Based on these results, we propose that the AtSNX1 endosomal compartment represents a sorting endosome in root cells, and that this specialized organelle is conserved throughout eukaryotes.     

The biogenesis of lysosomes: Is it a kiss and run,continuous fusion and fission process?

Molecules are transferred to lysosomes, the major, acid pH, digestive compartment in eukaryotic cells, by a complex series of pathways that converge at a late endosome/prelysosomal compartment. Here, we discuss the relationship between this compartment and the lysosome. We propose that lysosomes are maintained within cells by a repeated series of kiss and run, transient fusion and fission processes with the late endosome/prelysosome compartment. Directionality to these processes may be conferred by pH gradients and retrieval mechanisms. The future challenge in testing this and any other proposed hypothesis for lysosomal biogenesis will be the establishment of molecular mechanisms.     

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.