Cytoplasmic dynein-dependent vesicular transport from early to late endosomes [published erratum appears in J Cell Biol 1994 Feb;124(3):397]

We have used an in vitro fusion assay to study the mechanisms of transport from early to late endosomes. Our data show that the late endosomes share with the early endosomes a high capacity to undergo homotypic fusion in vitro. However, direct fusion of early with late endosomes does not occur. We have purified vesicles which are intermediates during transport from early to late endosomes in vivo, and analyzed their protein composition in two-dimensional gels. In contrast to either early or late endosomes, these vesicles do not appear to contain unique proteins. Moreover, these vesicles undergo fusion with late endosomes in vitro, but not with each other or back with early endosomes. In vitro, fusion of these endosomal vesicles with late endosomes is stimulated by polymerized microtubules, consistent with the known role of microtubules during early to late endosome transport in vivo. In contrast, homotypic fusion of early or late endosomes is microtubule-independent. Finally, this stimulation by microtubules depends on microtubule-associated proteins and requires the presence of the minus-end directed motor cytoplasmic dynein, but not the plus-end directed motor kinesin, in agreement with the microtubule organization in vivo. Our data strongly suggest that early and late endosomes are separate, highly dynamic organelles, which are connected by a microtubule-dependent vesicular transport step.     

A role for Rab5 activity in the biogenesis of endosomal and lysosomal compartments

Rab5 is a small GTPase that plays roles in the homotypic fusion of early endosomes and regulation of intracellular vesicle transport. We show here that expression of GFP-tagged GTPase-deficient form of Rab5b (Rab5bQ79L) in NRK cells results in the sequential formation of three morphologically and functionally distinct types of endosomes. Expression of GFP-Rab5bQ79L initially caused a homotypic fusion of early endosomes accompanying a redistribution of the TGN-resident cargo molecules, and subsequent fusion with late endosomes/lysosomes, leading to the formation of giant hybrid organelles with features of early endosomes and late endosomes/lysosomes. Surprisingly, the giant endosomes gradually fragmented and shrunk, leading to the accumulation of early endosome clusters and concurrent reformation of late endosomes/lysosomes, a process accelerated by treatment with a phosphatidylinositol-3-kinase (PI(3)K) inhibitor, wortmannin. We postulate that such sequential processes reflect the biogenesis and maintenance of late endosomes/lysosomes, presumably via direct fusion with early endosomes and subsequent fission from hybrid organelles. Thus, our findings suggest a regulatory role for Rab5 in not only the early endocytic pathway, but also the late endocytic pathway, of membrane trafficking in coordination with PI(3)K activity.     

Rab5-mediated endosome-endosome fusion regulates hemoglobin endocytosis in Leishmania donovani

To understand the trafficking of endocytosed hemoglobin (Hb) in Leishmania, we investigated the characteristics of in vitro fusion between endosomes containing biotinylated Hb (BHb) and avidin-horseradish peroxidase (AHRP). We showed that early endosome fusion in Leishmania is temperature and cytosol dependent and is inhibited by ATP depletion, ATPgammaS, GTPgammaS and N-ethylmaleimide treatment. The Rab5 homolog from Leishmania donovani, LdRab5, was cloned and expressed. Our results showed that homotypic fusion between the early endosomes in Leishmania is Rab5 dependent. Early endosomes containing BHb fused efficiently with late endosomes in a process regulated by Rab7, whereas no fusion between early and late endosomes was detected using fluid phase markers. Pre-treatment of early endosomes containing BHb with monoclonal antibody specific for the C-terminus of the Hb receptor (HbR) or the addition of the C-terminal cytoplasmic fragment of the HbR specifically inhibited the fusion with late endosomes, suggesting that signal(s) mediated through the HbR cytoplasmic tail promotes the fusion of early endosomes containing Hb with late endosomes.     

Different microtubule motors move early and late endocytic compartments

Important progress has been made during the past decade in the identification of molecular motors required in the distribution of early and late endosomes and the proper trafficking along the endocytic pathway. There is little direct evidence, however, that these motors drive movement of the endosomes. To evaluate the contributions of kinesin-1, dynein and kinesin-2 to the movement of early and late endosomes along microtubules, we made use of a cytosol-free motility assay using magnetically isolated early and late endosomes as well as biochemical analyses and live-cell imaging. By making use of specific antibodies, we confirmed that kinesin-1 and dynein move early endosomes and we found that kinesin-2 moves both early and late endosomes in the cell-free assay. Unexpectedly, dynein did not move late endosomes in the cell-free assay. We provide evidence from disruption of dynein function and latrunculin A treatment, suggesting that dynein regulates late endosome movement indirectly, possibly through a mechanism involving the actin cytoskeleton. These data provide new insights into the complex regulation of endosomes' motility and suggest that dynein is not the major motor required to move late endosomes toward the minus end of microtubules.     

A selective transport route from Golgi to late endosomes that requires the yeast GGA proteins

Pep12p is a yeast syntaxin located primarily in late endosomes. Using mutagenesis of a green fluorescent protein chimera we have identified a sorting signal FSDSPEF, which is required for transport of Pep12p from the exocytic pathway to late endosomes, from which it can, when overexpressed, reach the vacuole. When this signal is mutated, Pep12p instead passes to early endosomes, a step that is determined by its transmembrane domain. Surprisingly, Pep12p is then specifically retained in early endosomes and does not go on to late endosomes.By testing appropriate chimeras in mutant strains, we found that FSDSPEF-dependent sorting was abolished in strains lacking Gga1p and Gga2p, Golgi-associated coat proteins with homology to gamma adaptin. In the gga1 gga2 double mutant endogenous Pep12p cofractionated with the early endosome marker Tlg1p, and recycling of Snc1p through early endosomes was defective. Pep12p sorting was also defective in cells lacking the clathrin heavy or light chain. We suggest that specific and direct delivery of proteins to early and late endosomes is required to maintain the functional heterogeneity of the endocytic pathway and that the GGA proteins, probably in association with clathrin, help create vesicles destined for late endosomes.     

Syntaxin 7 mediates endocytic trafficking to late endosomes

The lysosome functions are ensured by accurate membrane trafficking in the cell. We found that mouse syntaxin 7 could complement yeast vam3 and pep12 mutants defective in docking/fusion to vacuolar and prevacuolar membranes, respectively. Immunohistochemical studies showed that syntaxin 7 is localized to late endosomes, but not to early endosomes. Induced expression of mutant syntaxin 7 blocked endocytic transport from early to late endosomes but did not block the transport of cathepsin D and lamp-2 from the trans-Golgi network to lysosomes. Thus, syntaxin 7 mediates the endocytic trafficking from early endosomes to late endosomes and lysosomes. These results also suggest that the biosynthetic pathway utilizes a different machinery from that of the endocytic pathway in the docking/fusion to late endosomes.     

Lumenal labeling of rat hepatocyte endocytic compartments. Distribution of several acid hydrolases and membrane receptors.

We used a combination of subcellular fractionation and lactoperoxidase-mediated iodination to examine the polypeptide compositions of three hepatocyte endocytic compartments: early endosomes, late endosomes, and lysosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase which binds specifically to asialoglycoprotein receptors was perfused through isolated rat livers at 37 degrees C. Subcellular fractions enriched in various endocytic compartments were then isolated by differential and isopycnic centrifugation, and the lactoperoxidase moiety of the internalized conjugate was used to catalyze the iodination of lumenal-facing proteins. The 125I profiles of early and late endosomes were strikingly similar after gel electrophoresis. Using immunoprecipitation, we directly identified and compared the relative amounts of the Na+,K(+)-ATPase and several different acid hydrolases and membrane receptors in all three fractions. The asialoglycoprotein receptor and the low density lipoprotein related protein were approximately nine times more abundant in early endosomes than late endosomes, suggesting that they recycle from early endosomes. In addition, cathepsin D, but not cathepsin L, beta-glucuronidase, and lgp 120, was detected in early endosomes; however, all of these molecules were detected in lysosomes. Our findings provide strong evidence that early endosomes mature into late endosomes and that there is either selective delivery or selective retention of hydrolases at discrete points in the endocytic pathway.     

Two lysosomal membrane proteins,LGP85 and LGP107, are delivered to late endosomes/lysosomes through different intracellular routes after exiting from the trans-Golgi network

Lysosomal membrane proteins are delivered from their synthesis site, the endoplasmic reticulum (ER) to late endosomes/lysosomes through the Golgi complex. It has been proposed that after leaving the Golgi they are transported either directly or indirectly (via the cell surface) to late endosomes/lysosomes. In the present study, we examined the transport routes taken by two structurally different lysosomal membrane proteins, LGP85 and LGP107, in rat 3Y1-B cells. Here we show that newly synthesized LGP85 and LGP107 are delivered to late endosomes/lysosomes via a direct route without passing through the cell surface. Interestingly, although LGP107 is delivered from the Golgi to early endosomes containing internalized horseradish peroxidase-conjugated transferrin (HRP-Tfn) en route to lysosomes, LGP85 does not pass through the HRP-Tfn-positive early endosomes. These results suggest, therefore, that LGP85 and LGP107 are sorted into distinct transport vesicles at the post-Golgi, presumably the trans-Golgi network (TGN), after which LGP85 is delivered directly to late endosomes/lysosomes, but significant fractions of LGP107 are targeted to early endosomes before transport to late endosomes/lysosomes. This study provides the first evidence that after exiting from the Golgi, LGP85 and LGP107 are targeted to late endosomes/lysosomes via a different pathway.     

Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes

Hrs and the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are involved in the endosomal sorting of membrane proteins into multivesicular bodies and lysosomes or vacuoles. The ESCRT complexes are also required for formation of intraluminal endosomal vesicles and for budding of certain enveloped RNA viruses such as HIV. Here, we show that Hrs binds to the ESCRT-I subunit Tsg101 via a PSAP motif that is conserved in Tsg101-binding viral proteins. Depletion of Hrs causes a reduction in membrane-associated ESCRT-I subunits, a decreased number of multivesicular bodies and an increased size of late endosomes. Even though Hrs mainly localizes to early endosomes and Tsg101 to late endosomes, the two proteins colocalize on a subpopulation of endosomes that contain lyso-bisphosphatidic acid. Overexpression of Hrs causes accumulation of Tsg101 on early endosomes and prevents its localization to late endosomes. We conclude that Hrs mediates the initial recruitment of ESCRT-I to endosomes and, thereby, indirectly regulates multivesicular body formation.     

Effect of centrifugation on separation by aqueous two-phase partition of an early and late endosome model using inside-out plasma membrane vesicles from plants

Inside-out vesicles of plasma membranes prepared from a plant source were used as models to investigate effects of centrifugal forces on separations of early and late endosome populations by aqueous two-phase partition. Endosome subpopulations were resolved readily by preparative free-flow electrophoresis where acidification of the interiors of late endosomes occurred upon addition of ATP to activate a proton translocating ATPase. The resultant increased diffusion potential provided for a surface difference between late and early endosomes to permit electrophoretic separation. With the plant membranes, unincubated inside-out plasma membrane vesicles modeled early endosomes, whereas inside-out vesicles incubated with 1 mM ATP modeled late endosomes. A latent, 2,4-dichlorophenoxyacetic acid (2,4-D)-(auxin)-stimulated NADH:protein disulfide reductase measured spectrophotometrically was used as an enzymatic marker for both populations of inside-out vesicles. Phase partition behavior of each population was quantitated using total protein as the parameter.     

The essential role of early endosomes in autophagy is revealed by loss of COPI function

The role of early endosomes in the maturation of autophagosomes and autophagy has been inferred from morphological data accumulated in several cell models. Through the inhibition of early endosome function, by loss of COPI using siRNA depletion, we have found that early endosomes, and fusion of autophagosomes with functional early endosomes are essential for autophagy. Our results support the sequential, stepwise maturation of autophagosomes via fusion with the early endosomes, late endosomes and lysosomal compartments.     

The R-SNARE endobrevin/VAMP-8 mediates homotypic fusion of early endosomes and late endosomes

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of the trans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.     

Late endosomes derive from early endosomes by maturation.

Endocytosed proteins destined for degradation in lysosomes are targeted mainly to early endosomes following uptake. Late endosomes are the major site for entry of newly synthesized lysosomal hydrolases via the cation-independent mannose 6-phosphate receptor into the degradative pathway. No consensus exists as to the mechanism of transport from early to late endosomes. We used asialoorosomucoid and transferrin to label selected parts of the degradative and receptor-recycling pathways, respectively, in the human hepatoma cell line HepG2. Intracellular mixing of sequentially endocytosed 125I- and HRP-labeled ligands was monitored by using 3,3'-diaminobenzidine-mediated density perturbation. The entire endocytic pathway of asialoorosomucoid, except for the lysosomes, remained fully accessible to subsequently endocytosed transferrin conjugated to HRP with unchanged kinetics. These results together with immunoelectron microscopic data support a model in which early endosomes gradually mature into late endosomes.     

Infectious adenovirus type 2 transport through early but not late endosomes

Receptor-mediated endocytosis is a major gate for pathogens into cells. In this study, we analyzed the trafficking of human adenovirus type 2 and 5 (Ad2/5) and the escape-defective temperature-sensitive Ad2-ts1 mutant in epithelial cancer cells. Ad2/5 and Ad2-ts1 uptake into endosomes containing transferrin, major histocompatibility antigen 1 and the Rab5 effector early endosome antigen 1 (EEA1) involved dynamin, amphiphysin, clathrin and Eps15. Cointernalization experiments showed that most of the Ad2/5 and Ad2-ts1 visited the same EEA1-positive endosomes. In contrast to Ad2/5, Ad2-ts1 required functional Rab5 for endocytosis and lysosomal transport and was sensitive to the phosphatidyl-inositol-3 (PI3)-kinase inhibitor wortmannin or the ubiquitin-binding protein Hrs for sorting from early to late endosomes. Endosomal escape of Ad2 was not affected by incubation at 19 degrees C, which blocked membrane sorting in early endosomes and inhibited Ad2-ts1 transport to lysosomes. Unlike Semliki Forest Virus (SFV), sorting of Ad2-ts1 to late endosomes was independent of Rab7 and Ad2/5 infection independent of EEA1. The data indicate that Ad2/5 and Ad2-ts1 use an invariant machinery for clathrin-mediated uptake to early endosomes. We suggest that the infectious Ad2 particles are either directly released from early endosomes to the cytosol or sorted by a temperature-insensitive and PI3-kinase-independent mechanism to an escape compartment different from late endosomes or lysosomes.     

Wortmannin delays transfer of human rhinovirus serotype 2 to late endocytic compartments

Human rhinovirus 2 (HRV2) is internalized by members of the low-density lipoprotein receptor family into early endosomes (pH 6.2-6.0) where it dissociates from its receptors. After transfer into late endosomes, the virus undergoes a conformational change and RNA uncoating solely induced by pH < 5.6. Finally, virus capsids are degraded in lysosomes. To investigate the role of phosphatidylinositol 3-kinases (PI3K) in the HRV2 entry route, we used the inhibitor wortmannin. Although virus internalization was not altered by wortmannin, virus accumulated in enlarged early endosomes. Furthermore, the drug delayed HRV2 degradation and viral protein synthesis. Consequently, wortmannin-sensitive PI3K are involved in HRV2 transport from early to late compartments. However, wortmannin had no effect on the titer of infectious virus produced. Our data therefore suggest that virus retained in early endosomes for prolonged time periods can undergo the conformational change that otherwise occurs at pH < or = 5.6 in late endosomes.     

Quantitative correlative microscopy reveals the ultrastructural distribution of endogenous endosomal proteins

The key endosomal regulators Rab5, EEA1, and APPL1 are frequently applied in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as a marker for late endosomes and lysosomes. However, endogenous levels of these proteins localize poorly in immuno-EM, and systematic studies on their native ultrastructural distributions are lacking. To address this gap, we here present a quantitative, on-section correlative light and electron microscopy (CLEM) approach. Using the sensitivity of fluorescence microscopy, we label hundreds of organelles that are subsequently visualized by EM and classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 colocalizes with Rab5 on early endosomes, but unexpectedly also labels Rab5-negative late endosomes, which are positive for PI(3)P but lack Rab7. APPL1 is restricted to small Rab5-positive, tubulo-vesicular profiles. Rab7 primarily labels late endosomes and lysosomes. These data increase our understanding of the structural–functional organization of the endosomal system and introduce quantitative CLEM as a sensitive alternative for immuno-EM.     

Domains within the GARP subunit Vps54 confer separate functions in complex assembly and early endosome recognition

Tethering complexes contribute to the specificity of membrane fusion by recognizing organelle features on both donor and acceptor membranes. The Golgi-associated retrograde protein (GARP) complex is required for retrograde traffic from both early and late endosomes to the trans-Golgi network (TGN), presenting a paradox as to how a single complex can interact specifically with vesicles from multiple upstream compartments. We have found that a subunit of the GARP complex, Vps54, can be separated into N- and C-terminal regions that have different functions. Whereas the N-terminus of Vps54 is important for GARP complex assembly and stability, a conserved C-terminal domain mediates localization to an early endocytic compartment. Mutation of this C-terminal domain has no effect on retrograde transport from late endosomes. However, a specific defect in retrieval of Snc1 from early endosomes is observed when recycling from late endosomes to the Golgi is blocked. These data suggest that separate domains recruit tethering complexes to different upstream compartments to regulate individual trafficking pathways.     

Distribution and trafficking of MPR300 is normal in cells with cholesterol accumulated in late endocytic compartments: evidence for early endosome-to-TGN trafficking of MPR300

It has been reported that an accumulation of cholesterol within late endosomes/lysosomes in Niemann-Pick type C (NPC) fibroblasts and U18666A-treated cells causes impairment of retrograde trafficking of the cation-independent mannose 6-phosphate/IGF-II receptor (MPR300) from late endosomes to the trans-Golgi network (TGN). In apparent conflict with these results, here we show that as in normal fibroblasts, MPR300 localizes exclusively to the TGN in NPC fibroblasts as well as in normal fibroblasts treated with U18666A. This localization can explain why several lysosomal properties and functions, such as intracellular lysosomal enzyme activity and localization, the biosynthesis of cathepsin D, and protein degradation, are all normal in NPC fibroblasts. These results, therefore, suggest that the accumulation of cholesterol in late endosomes/lysosomes does not affect the retrieval of MPR300 from endosomes to the TGN. Furthermore, treatment of normal and NPC fibroblasts with chloroquine, which inhibits membrane traffic from early endosomes to the TGN, resulted in a redistribution of MPR300 to EEA1 and internalized transferrin-positive, but LAMP-2-negative, early-recycling endosomes. We propose that in normal and NPC fibroblasts, MPR300 is exclusively targeted from the TGN to early endosomes, from where it rapidly recycles back to the TGN without being delivered to late endosomes. This notion provides important insights into the definition of late endosomes, as well as the biogenesis of lysosomes.     

The ERM proteins interact with the HOPS complex to regulate the maturation of endosomes

In the degradative pathway, the progression of cargos through endosomal compartments involves a series of fusion and maturation events. The HOPS (homotypic fusion and protein sorting) complex is part of the machinery that promotes the progression from early to late endosomes and lysosomes by regulating the exchange of small GTPases. We report that an interaction between subunits of the HOPS complex and the ERM (ezrin, radixin, moesin) proteins is required for the delivery of EGF receptor (EGFR) to lysosomes. Inhibiting either ERM proteins or the HOPS complex leads to the accumulation of the EGFR into early endosomes, delaying its degradation. This impairment in EGFR trafficking observed in cells depleted of ERM proteins is due to a delay in the recruitment of Rab7 on endosomes. As a consequence, the maturation of endosomes is perturbed as reflected by an accumulation of hybrid compartments positive for both early and late endosomal markers. Thus, ERM proteins represent novel regulators of the HOPS complex in the early to late endosomal maturation.     

Retrograde Transport: Two (or More) Roads Diverged in an Endosomal Tree?

Some proteins and lipids traffic from the plasma membrane to the trans Golgi network (TGN)/Golgi apparatus and the endoplasmic reticulum, via the retrograde transport route. Endosomes are an obligatory through station. Whether early, recycling and late endosomes all hand off material to the TGN have remained a matter of debate. In this review, we give a short historical overview on how retrograde transport was discovered and explored. We then summarize and critically discuss data that have been put forward in favour of the existence of trafficking interfaces between each of the different endocytic localizations and the TGN. We finally point out some conceptual and technological challenges that will have to be met to establish definite conclusions for each of these scenarios.