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.     

Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3'-kinase-dependent machinery

In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3'-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes.     

Re-localization of activated EGF receptor and its signal transducers to multivesicular compartments downstream of early endosomes in response to EGF

The rapid internalization of receptor tyrosine kinases after ligand binding has been assumed to be a negative modulation of signal transduction. However, accumulating data indicate that signal transduction from internalized cell surface receptors also occurs from endosomes. We show that a substantial fraction of tyrosine-phosphorylated epidermal growth factor receptor (EGFR) and Shc, Grb2 and Cbl after internalization relocates from early endosomes to compartments which are negative for the early endosomes, recycling vesicle markers EEA1 and transferrin in EGF-stimulated cells. These compartments contained the multivesicular body and late endosome marker CD63, and the late endosome and lysosome marker LAMP-1, and showed a multivesicular morphology. Subcellular fractionation revealed that activated EGFR, adaptor proteins and activated ERK 1 and 2 were located in EEA1-negative and LAMP-1-positive fractions. Co-immunoprecipitations showed EGFR in complex with both Shc, Grb2 and Cbl. Treatment with the weak base chloroquine or inhibitors of lysosomal enzymes after EGF stimulation induced an accumulation of tyrosine-phosphorylated EGFR and Shc in EEA1-negative and CD63-positive vesicles after a 120-min chase period. This was accompanied by a sustained activation of ERK 1 and 2. These results suggest that EGFR signaling is not spatially restricted to the plasma membrane, primary vesicles and early endosomes, but is continuing from late endocytic trafficking organelles maturing from early endosomes.     

APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

Endocytosis allows cargo to enter a series of specialized endosomal compartments, beginning with early endosomes harboring Rab5 and its effector EEA1. There are, however, additional structures labeled by the Rab5 effector APPL1 whose role in endocytic transport remains unclear. It has been proposed that APPL1 vesicles are transport intermediates that convert into EEA1 endosomes. Here, we tested this model by analyzing the ultrastructural morphology, kinetics of cargo transport, and stability of the APPL1 compartment over time. We found that APPL1 resides on a tubulo-vesicular compartment that is capable of sorting cargo for recycling or degradation and that displays long lifetimes, all features typical of early endosomes. Fitting mathematical models to experimental data rules out maturation of APPL1 vesicles into EEA1 endosomes as a primary mechanism for cargo transport. Our data suggest instead that APPL1 endosomes represent a distinct population of Rab5-positive sorting endosomes, thus providing important insights into the compartmental organization of the early endocytic pathway.     

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.     

EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and fibroblasts

EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of "basolateral-type" endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.     

Selective membrane recruitment of EEA1 suggests a role in directional transport of clathrin-coated vesicles to early endosomes

The molecular mechanisms ensuring directionality of endocytic membrane trafficking between transport vesicles and target organelles still remain poorly characterized. We have been investigating the function of the small GTPase Rab5 in early endocytic transport. In vitro studies have demonstrated a role of Rab5 in two membrane fusion events: the heterotypic fusion between plasma membrane-derived clathrin-coated vesicles (CCVs) and early endosomes and in the homotypic fusion between early endosomes. Several Rab5 effectors are required in homotypic endosome fusion, including EEA1, which mediates endosome membrane docking, as well as Rabaptin-5 x Rabex-5 complex and phosphatidylinositol 3-kinase hVPS34. In this study we have examined the localization and function of Rab5 and its effectors in heterotypic fusion in vitro. We report that the presence of active Rab5 is necessary on both CCVs and early endosomes for a heterotypic fusion event to occur. This process requires EEA1 in addition to the Rabaptin-5 complex. However, whereas Rab5 and Rabaptin-5 are symmetrically distributed between CCVs and early endosomes, EEA1 is recruited selectively onto the membrane of early endosomes. Our results suggest that EEA1 is a tethering molecule that provides directionality to vesicular transport from the plasma membrane to the early endosomes.     

Cell vacuolization induced by Helicobacter pylori VacA cytotoxin does not depend on late endosomal SNAREs

Cellular vacuoles induced by the Helicobacter pylori vacuolating cytotoxin VacA originate from late endosomal compartments. Their biogenesis requires the activity of both rab7 GTPase and the ATPase proton pump. The toxin has been suggested to cause an increased luminal osmotic pressure via its anion-specific channel activity localized on late endosomal compartments after endocytosis. Here, we show that the extensive membrane fusion that takes place in the transition from the small late endosomal compartments to the large vacuoles does not depend on soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins. The process of vacuolization leads to disappearance of the large array of internal membranes of late endosomes. We suggest that most of the vacuole-limiting membrane derives from internal membranes.     

Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells

Using a microinjection approach to study apical plasma membrane protein trafficking in hepatic cells, we found that specific inhibition of Vps34p, a class III phosphoinositide 3 (PI-3) kinase, nearly perfectly recapitulated the defects we reported for wortmannin-treated cells (Tuma, P.L., C.M. Finnegan, J.-H Yi, and A.L. Hubbard. 1999. J. Cell Biol. 145:1089-1102). Both wortmannin and injection of inhibitory Vps34p antibodies led to the accumulation of resident apical proteins in enlarged prelysosomes, whereas transcytosing apical proteins and recycling basolateral receptors transiently accumulated in basolateral early endosomes. To understand how the Vps34p catalytic product, PI3P, was differentially regulating endocytosis from the two domains, we examined the PI3P binding protein early endosomal antigen 1 (EEA1). We determined that EEA1 distributed to two biochemically distinct endosomal populations: basolateral early endosomes and subapical endosomes. Both contained rab5, although the latter also contained late endosomal markers but was distinct from the transcytotic intermediate, the subapical compartment. When PI3P was depleted, EEA1 dissociated from basolateral endosomes, whereas it remained on subapical endosomes. From these results, we conclude that PI3P, via EEA1, regulates early steps in endocytosis from the basolateral surface in polarized WIF-B cells. However, PI3P must use different machinery in its regulation of the apical endocytic pathway, since later steps are affected by Vps34p inhibition.     

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.     

Gαs promotes EEA1 endosome maturation and shuts down proliferative signaling through interaction with GIV (Girdin)

The organization of the endocytic system into biochemically distinct subcompartments allows for spatial and temporal control of the strength and duration of signaling. Recent work has established that Akt cell survival signaling via the epidermal growth factor receptor (EGFR) occurs from APPL early endosomes that mature into early EEA1 endosomes. Less is known about receptor signaling from EEA1 endosomes. We show here that EGF-induced, proliferative signaling occurs from EEA1 endosomes and is regulated by the heterotrimeric G protein Gαs through interaction with the signal transducing protein GIV (also known as Girdin). When Gαs or GIV is depleted, activated EGFR and its adaptors accumulate in EEA1 endosomes, and EGFR signaling is prolonged, EGFR down-regulation is delayed, and cell proliferation is greatly enhanced. Our findings define EEA1 endosomes as major sites for proliferative signaling and establish that Gαs and GIV regulate EEA1 but not APPL endosome maturation and determine the duration and strength of proliferative signaling from this compartment.     

Endocytosed cation-independent mannose 6-phosphate receptor traffics via the endocytic recycling compartment en route to the trans-Golgi network and a subpopulation of late endosomes

Although the distribution of the cation-independent mannose 6-phosphate receptor (CI-MPR) has been well studied, its intracellular itinerary and trafficking kinetics remain uncertain. In this report, we describe the endocytic trafficking and steady-state localization of a chimeric form of the CI-MPR containing the ecto-domain of the bovine CI-MPR and the murine transmembrane and cytoplasmic domains expressed in a CHO cell line. Detailed confocal microscopy analysis revealed that internalized chimeric CI-MPR overlaps almost completely with the endogenous CI-MPR but only partially with individual markers for the trans-Golgi network or other endosomal compartments. After endocytosis, the chimeric receptor first enters sorting endosomes, and it then accumulates in the endocytic recycling compartment. A large fraction of the receptors return to the plasma membrane, but some are delivered to the trans-Golgi network and/or late endosomes. Over the course of an hour, the endocytosed receptors achieve their steady-state distribution. Importantly, the receptor does not start to colocalize with late endosomal markers until after it has passed through the endocytic recycling compartment. In CHO cells, only a small fraction of the receptor is ever detected in endosomes bearing substrates destined for lysosomes (kinetically defined late endosomes). These data demonstrate that CI-MPR takes a complex route that involves multiple sorting steps in both early and late endosomes.     

A role for EHD4 in the regulation of early endosomal transport

All four of the C-terminal Eps15 homology domain (EHD) proteins have been implicated in the regulation of endocytic trafficking. However, the high level of amino acid sequence identity among these proteins has made it challenging to elucidate the precise function of individual EHD proteins. We demonstrate here with specific peptide antibodies that endogenous EHD4 localizes to Rab5-, early embryonic antigen 1 (EEA1)- and Arf6-containing endosomes and colocalizes with internalized transferrin in the cell periphery. Knock-down of EHD4 expression by both small interfering RNA and short hairpin RNA leads to the generation of enlarged early endosomal structures that contain Rab5 and EEA1 as well as internalized transferrin or major histocompatibility complex class I molecules. In addition, cargo destined for degradation, such as internalized low-density lipoprotein, also accumulates in the enlarged early endosomes in EHD4-depleted cells. Moreover, we have demonstrated that these enlarged early endosomes are enriched in levels of the activated GTP-bound Rab5. Finally, we show that endogenous EHD4 and EHD1 interact in cells, suggesting coordinated involvement in the regulation of receptor transport along the early endosome to endocytic recycling compartment axis. The results presented herein provide evidence that EHD4 is involved in the control of trafficking at the early endosome and regulates exit of cargo toward both the recycling compartment and the late endocytic pathway.     

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.     

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.     

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.     

Mammalian CORVET Is Required for Fusion and Conversion of Distinct Early Endosome Subpopulations

Early endosomes are organized in a network of vesicles shaped by cycles of fusion, fission, and conversion to late endosomes. In yeast, endosome fusion and conversion are regulated, among others, by CORVET, a hexameric protein complex. In the mammalian endocytic system, distinct subpopulations of early endosomes labelled by the Rab5 effectors APPL1 and EEA1 are present. Here, the function of mammalian CORVET with respect to these endosomal subpopulations was investigated. Tgfbrap1 as CORVET‐specific subunit and functional ortholog of Vps3p was identified, demonstrating that it is differentially distributed between APPL1 and EEA1 endosomes. Surprisingly, depletion of CORVET‐specific subunits caused fragmentation of APPL1‐positive endosomes but not EEA1 endosomes in vivo. These and in vitro data suggest that CORVET plays a role in endosome fusion independently of EEA1. Depletion of CORVET subunits caused accumulation of large EEA1 endosomes indicative of another role in the conversion of EEA1 endosomes into late endosomes. In addition, depletion of CORVET‐specific subunits caused alterations in transport depending on both the type of cargo and the specific endosomal subpopulation. These results demonstrate that CORVET plays distinct roles at multiple stages in the mammalian endocytic pathway.      

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.     

The small GTP binding protein rab7 is essential for cellular vacuolation induced by Helicobacter pylori cytotoxin.

The VacA cytotoxin, produced by toxigenic strains of Helicobacter pylori, induces the formation of large vacuoles highly enriched in the small GTPase rab7. To probe the role of rab7 in vacuolization, HeLa cells were transfected with a series of rab mutants and exposed to VacA. Dominant-negative mutants of rab7 effectively prevented vacuolization, whereas homologous rab5 and rab9 mutants were only partially inhibitory or ineffective, respectively. Expression of wild-type or GTPase-deficient rab mutants synergized with VacA in inducing vacuolization. In vitro fusion of late endosomes was enhanced by active rab7 and inhibited by inactive rab7, consistent with vacuole formation by merging of late endosomes in a process that requires functional rab7. Taken together, the effects of overexpressed rab proteins described here indicate that continuous membrane flow along the endocytic pathway is necessary for vacuole growth.