Early Endosomal Membranes Research Articles

Rab11-FIP2 Functions in Transferrin Recycling and Associates with Endosomal Membranes via Its COOH-terminal Domain

Rab11-FIP2 is a recently described member of the Rip11/Rab11-FIP/Rab coupling protein family of Rab11 interacting proteins. Rab11-FIP2 interacts with both Rab11 and myosin Vb and co-localizes with Rab11 in both HeLa and Madin-Darby canine kidney cells (Hales, C. M., Griner, R., Hobdy-Henderson, K. C., Dorn, M. C., Hardy, D., Kumar, R., Navarre, J., Chan, E. K., Lapierre, L. A., and Goldenring, J. R. (2001) J. Biol. Chem. 276, 39067-390751). Here, we characterized the specificity of the interaction between Rab11-FIP2 and Rab11 and report that it does not interact with Rab4, Rab3, Rab5, Rab6, or Rab7. We demonstrate that the COOH-terminal region of Rab11-FIP2, which contains the Rab11 binding domain (RBD), is necessary and sufficient for its early endosomal membrane association. In contrast, the amino-terminal region, which contains a phospholipid binding C2-domain, by itself was insufficient for membrane binding. Expression of a deletion mutant of Rab11-FIP2, containing the RBD, caused tubulation of a transferrin receptor-positive early endosomal compartment in HeLa cells. Endogenous Rab11 was also associated with this compartment. This phenotype cannot be reversed by excess wild-type Rab11, or dominant-positive Rab11 (Rab11Q70L), suggesting that Rab11-FIP2 functions downstream of Rab11 in endosomal trafficking.

Cell-surface attachment of pedestal-forming enteropathogenic E. coli induces a clustering of raft components and a recruitment of annexin 2.

Annexin 2 is a Ca2+-regulated membrane- and F-actin-binding protein implicated in the stabilization or regulation of membrane/cytoskeleton contacts, or both, at the plasma membrane and at early endosomal membranes. To analyze the dynamic nature of such action we investigated whether annexin 2 could be found at sites of localized actin rearrangements occurring at the plasma membrane of HeLa cells infected with noninvading enteropathogenic Escherichia coli (EPEC). We show that adherent EPEC microcolonies, which are known to induce the formation of actin-rich pedestals beneath them, specifically recruit annexin 2 to the sites of their attachment. Mutant EPEC (EPECtir), which lack a functional receptor for intimate attachment (Tir, translocated intimin receptor) and which fail to produce full pedestal formation, are still capable of recruiting annexin 2 to the bacterial contact sites. Accumulation of annexin 2 at sites of EPEC or EPECtir attachment is accompanied by a recruitment of the annexin 2 protein ligand S100A10. EPEC and EPECtir attachment also induces a concentration of cholesterol and glycosyl phosphatidylinositol-anchored proteins at sites of bacterial contact. This indicates that membrane components present in rafts or raft-like microdomains are clustered upon EPEC adherence and that annexin 2 is recruited to the cytoplasmic membrane surface of such clusters, possibly stabilizing raft patches and their linkage to the actin cytoskeleton beneath adhering EPEC.

Pathogenic Mycobacterium avium remodels the phagosome membrane in macrophages within days after infection

As part of their strategy for intracellular survival, mycobacteria prevent maturation of the phagosomes in which they reside inside macrophages. The molecular basis for this inhibition is only now beginning to emerge, by way of the molecular characterisation of the phagosome membrane when it encloses virulent mycobacteria. Our own work has shown that at 15 days after the phagocytic uptake of Mycobacterium avium by mouse bone marrow-derived macrophages, the phagosome membrane is depleted about 4-fold for cell surface-derived membrane glycoconjugates, labelled by exogalactosylation, in comparison to the membrane of early endosomes with which it continues to interact. Here we asked whether this depletion occurred at early or late stages after infection. We found that only about half of the depletion had occurred at about 5 hours after the beginning of phagocytic uptake, with the remainder becoming established thereafter, with a half-time of about 2.5 days. Phagosomes became depleted in relation to early endosomes with which they continued to exchange membrane constituents. Early endosomes themselves became gradually depleted by about 30% during the 15-day post-infection period. In contrast, late endosomes/lysosomes remained unchanged, with a concentration of surface-derived glycoconjugates between that of early endosomes and of phagosomes at day 15 post infection. In view of the slowness of the post-infection change of phagosome membrane composition, we proposed that this change did not play a role in preventing maturation immediately after phagosome formation, but rather correlated with the process of maintaining the phagosomes in an immature state.

A PtdIns(3)P-specific probe cycles on and off host cell membranes during Salmonella invasion of mammalian cells

Salmonella invade nonphagocytic cells by eliciting their own internalization; upon contact with the host cell, the bacteria induce membrane ruffles highly localized to the point of contact between the invading bacterium and the host cell [1, 2]. The bacterium is then internalized into an unusual cytosolic organelle, the Salmonella-containing vacuole (SCV). Early endosomal markers (including EEA1) have recently been shown to be associated with the SCV shortly after invasion [3]. EEA1, a protein involved in early endosome fusion, is recruited to early endosomal membranes in part by the interaction between its FYVE finger and phosphatidylinositol 3-phosphate [PtdIns(3)P] [4], a characteristic lipid of early endosomes. This suggests a possible role for PtdIns(3)P during Salmonella infection. To investigate this, we generated a highly specific probe for PtdIns(3)P that was used to follow invasion of Salmonella in nonphagocytic cells. Here, we show that PtdIns(3)P is present on the membranes of SCVs shortly after invasion and also that it is present on the membrane ruffles produced immediately prior to invasion. We also show that this specific probe cycles on and off the membranes of nascent SCVs even when PtdIns 3-kinase activity is inhibited, demonstrating that invading Salmonella influence the composition of the membranes that envelop them during invasion.

Natural-resistance-associated macrophage protein 1 is an H+/bivalent cation antiporter

In mammals, natural-resistance-associated macrophage protein 1 (Nramp1) regulates macrophage activation and is associated with infectious and autoimmune diseases. Nramp2 is associated with anaemia. Both belong to a highly conserved eukaryote/prokaryote protein family. We used Xenopus oocytes to demonstrate that, like Nramp2, Nramp1 is a bivalent cation (Fe2+, Zn2+ and Mn2+) transporter. Strikingly, however, where Nramp2 is a symporter of H+ and metal ions, Nramp1 is a highly pH-dependent antiporter that fluxes metal ions in either direction against a proton gradient. At pH9.0, oocytes injected with cRNA from wild-type murine Nramp1 with a glycine residue at position 169 (Nramp1G169; P = 3.22×10-6) and human NRAMP1 (P = 3.87×10-5) showed significantly enhanced uptake of radiolabelled Zn2+ compared with water-injected controls. At pH5.5, Nramp1G169 (P = 1.34×10-13) and NRAMP1 (P = 1.09×10-6) oocytes showed significant efflux of Zn2+. Zn2+ transport was abolished when the proton gradient was dissipated using carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Using pre-acidified oocytes, currents of 130±57 nA were evoked by 100µM Zn2+ at pH7.5, and 139±47 nA by 100µM Fe2+ at pH7.0, in Nramp1G169 oocytes; currents of 254±49 nA and 242±26 nA were evoked, respectively, in NRAMP1 oocytes. Steady-state currents evoked by increasing concentrations of Zn2+ were saturable, with apparent affinity constants of approx. 614nM for Nramp1G169 and approx. 562nM for NRAMP1 oocytes, and a curvilinear voltage dependence of transporter activity (i.e. the data points approximate to a curve that approaches a linear asymptote). In the present study we propose a new model for metal ion homoeostasis in macrophages. Under normal physiological conditions, Nramp2, localized to early endosomal membranes, delivers extracellularly acquired bivalent cations into the cytosol. Nramp1, localized to late endosomal/lysosomal membranes, delivers bivalent cations from the cytosol into this acidic compartment where they may directly affect antimicrobial activity.

Natural-resistance-associated macrophage protein 1 is an H+/bivalent cation antiporter.

In mammals, natural-resistance-associated macrophage protein 1 (Nramp1) regulates macrophage activation and is associated with infectious and autoimmune diseases. Nramp2 is associated with anaemia. Both belong to a highly conserved eukaryote/prokaryote protein family. We used Xenopus oocytes to demonstrate that, like Nramp2, Nramp1 is a bivalent cation (Fe2+, Zn2+ and Mn2+) transporter. Strikingly, however, where Nramp2 is a symporter of H+ and metal ions, Nramp1 is a highly pH-dependent antiporter that fluxes metal ions in either direction against a proton gradient. At pH 9.0, oocytes injected with cRNA from wild-type murine Nramp1 with a glycine residue at position 169 (Nramp1(G169); P=3.22x10(-6)) and human NRAMP1 (P=3.87x10(-5)) showed significantly enhanced uptake of radiolabelled Zn2+ compared with water-injected controls. At pH 5.5, Nramp1(G169) (P=1.34x10(-13)) and NRAMP1 (P=1.09x10(-6)) oocytes showed significant efflux of Zn2+. Zn2+ transport was abolished when the proton gradient was dissipated using carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Using pre-acidified oocytes, currents of 130+/-57 nA were evoked by 100 microM Zn2+ at pH 7.5, and 139+/-47 nA by 100 microM Fe2+ at pH 7.0, in Nramp1(G169) oocytes; currents of 254+/-49 nA and 242+/-26 nA were evoked, respectively, in NRAMP1 oocytes. Steady-state currents evoked by increasing concentrations of Zn2+ were saturable, with apparent affinity constants of approx. 614 nM for Nramp1(G169) and approx. 562 nM for NRAMP1 oocytes, and a curvilinear voltage dependence of transporter activity (i.e. the data points approximate to a curve that approaches a linear asymptote). In the present study we propose a new model for metal ion homoeostasis in macrophages. Under normal physiological conditions, Nramp2, localized to early endosomal membranes, delivers extracellularly acquired bivalent cations into the cytosol. Nramp1, localized to late endosomal/lysosomal membranes, delivers bivalent cations from the cytosol into this acidic compartment where they may directly affect antimicrobial activity.

Modes of annexin-membrane interactions analyzed by employing chimeric annexin proteins

Annexin II is a member of the annexin family of Ca 2+- and phospholipid-binding proteins which is particularly enriched on early endosomal membranes and has been implicated in participating in endocytic events. In contrast to other endosomal annexins the association of annexin II with its target membrane can occur in the absence of Ca 2+ in a manner depending on the unique N-terminal domain of the protein. However, endosome binding of annexin II does not require formation of a protein complex with the intracellular ligand S100A10 (p11) as an annexin II mutant protein (PM AnxII) incapable of interacting with p11 is still present on endosomal membranes. Fusion of the N-terminal sequence of this PM AnxII (residues 1–27) to the conserved protein core of annexin I transfers the capability of Ca 2+-independent membrane binding to the otherwise Ca 2+-sensitive annexin I. These results underscore the importance of the N-terminal sequence of annexin II for the Ca 2+-independent endosome association and argue for a direct interaction of this sequence with an endosomal membrane receptor.

Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain.

Rab5 regulates endocytic membrane traffic by specifically recruiting cytosolic effector proteins to their site of action on early endosomal membranes. We have characterized a new Rab5 effector complex involved in endosomal fusion events. This complex includes a novel protein, Rabenosyn-5, which, like the previously characterized Rab5 effector early endosome antigen 1 (EEA1), contains an FYVE finger domain and is recruited in a phosphatidylinositol-3-kinase-dependent fashion to early endosomes. Rabenosyn-5 is complexed to the Sec1-like protein hVPS45. hVPS45 does not interact directly with Rab5, therefore Rabenosyn-5 serves as a molecular link between hVPS45 and the Rab5 GTPase. This property suggests that Rabenosyn-5 is a closer mammalian functional homologue of yeast Vac1p than EEA1. Furthermore, although both EEA1 and Rabenosyn-5 are required for early endosomal fusion, only overexpression of Rabenosyn-5 inhibits cathepsin D processing, suggesting that the two proteins play distinct roles in endosomal trafficking. We propose that Rab5-dependent formation of membrane domains enriched in phosphatidylinositol-3-phosphate has evolved as a mechanism for the recruitment of multiple effector proteins to mammalian early endosomes, and that these domains are multifunctional, depending on the differing activities of the effector proteins recruited.

The recycling endosome of Madin-Darby canine kidney cells is a mildly acidic compartment rich in raft components.

We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway.

Selective membrane permeabilization by the rotavirus VP5* protein is abrogated by mutations in an internal hydrophobic domain.

Rotavirus infectivity is dependent on the proteolytic cleavage of the VP4 spike protein into VP8* and VP5* proteins. Proteolytically activated virus, as well as expressed VP5*, permeabilizes membranes, suggesting that cleavage exposes a membrane-interactive domain of VP5* which effects rapid viral entry. The VP5* protein contains a single long hydrophobic domain (VP5*-HD, residues 385 to 404) at an internal site. In order to address the role of the VP5*-HD in permeabilizing cellular membranes, we analyzed the entry of o-nitrophenyl-beta-D-galactopyranoside (ONPG) into cells induced to express VP5* or mutated VP5* polypeptides. Following IPTG (isopropyl-beta-D-thiogalactopyranoside) induction, VP5* and VP5* truncations containing the VP5*-HD permeabilized cells to the entry and cleavage of ONPG, while VP8* and control proteins had no effect on cellular permeability. Expression of VP5* deletions containing residues 265 to 474 or 265 to 404 permeabilized cells; however, C-terminal truncations which remove the conserved GGA (residues 399 to 401) within the HD abolished membrane permeability. Site-directed mutagenesis of the VP5-HD further demonstrated a requirement for residues within the HD for VP5*-induced membrane permeability. Functional analysis of mutant VP5*s indicate that conserved glycines within the HD are required and suggest that a random coiled structure rather than the strictly hydrophobic character of the domain is required for permeability. Expressed VP5* did not alter bacterial growth kinetics or lyse bacteria following induction. Instead, VP5*-mediated size-selective membrane permeability, releasing 376-Da carboxyfluorescein but not 4-kDa fluorescein isothiocyanate-dextran from preloaded liposomes. These findings suggest that the fundamental role for VP5* in the rotavirus entry process may be to expose triple-layered particles to low [Ca](i), which uncoats the virus, rather than to effect the detergent-like lysis of early endosomal membranes.

Rab15 differentially regulates early endocytic trafficking.

Rab GTPases play an important regulatory role in early endocytosis. We recently demonstrated that epitope-tagged Rab15 (HArab15) co-localizes with Rab4, -5, and -11 on early endosomal membranes in CHO cells (Zuk, P. A., and Elferink, L. A. (1999) J. Biol. Chem. 274, 22303-22312). To characterize the role of Rab15 in endocytosis, we prepared functional mutants of HArab15 and examined their effects on early endocytic trafficking. Wild-type HArab15 and its constitutively active, GTP-bound mutant (Q67L) reduce fluid phase and receptor-mediated endocytosis without affecting the rate of recycling from early endosomal compartments. Inhibition of early endocytosis appears to be due to a reduction in the rate of homotypic early endosome fusion. Conversely, mutations that constitutively inactivate HArab15 stimulate early endocytosis and the homotypic fusion of early endosomes in vitro. Unlike active forms of HArab15, constitutively inactive HArab15 mutants also affect recycling from early endosomal compartments. Moreover, the two constitutively inactive mutants, GDP-bound HArab15-T22N and the non-nucleotide binding mutant HArab15-N121I, differentially regulate the transit of fluid phase and receptor-mediated endocytic tracers through early/sorting endosomes. Together, these data suggest that HArab15 may counteract the reported stimulatory effect of Rab5 on early endocytosis. Consistent with this, overexpression of constitutively active HArab15-Q67L attenuates Rab5-stimulated endocytosis, whereas Rab5-stimulated endocytosis is augmented in cells overexpressing a constitutively inactive HArab15 mutant defective in guanine nucleotide binding (N121I). Our data indicate that HArab15 differentially regulates distinct steps in membrane trafficking through early/sorting and pericentriolar recycling endosomes.

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.

Direct interaction of EEA1 with Rab5b.

The early endosomal autoantigen EEA1 is essential for early endosomal membrane fusion. It binds to endosomes via a C-terminal domain (EEA1-CT). To identify proteins interacting with EEA1-CT, we screened a human brain library in the yeast two-hybrid system. Fourteen clones reacted strongly with EEA1-CT. Sequencing of these clones revealed that they all contained the ORF of the small GTPase, Rab5b. Further two-hybrid analysis suggested that Rab5b also interacts with the N-terminus of EEA1 (EEA1-NT). The interaction of both EEA1-CT and EEA1-NT with Rab5b was confirmed biochemically, and was found to be GTP dependent. Confocal immunofluorescence microscopy indicated that EEA1 colocalizes with Rab5b on early endosomes. Although EEA1-CT and EEA1-NT interacted strongly with wild-type Rab5b in the two-hybrid system, we detected no interaction with wild-type Rab5a, even though GTPase-deficient mutants of both Rab5a and Rab5b interacted equally well with EEA1. This difference could not be explained by differences in intrinsic GTPase activities, as these were found to be very similar. Instead, we speculate that yeast may contain a GTPase-activating protein (GAP) activity that stimulates Rab5a but not Rab5b. In contrast, pig brain cytosol was found to contain a GAP activity that stimulates the GTPase activity of Rab5b in preference to that of Rab5a. These data provide evidence that EEA1 interacts with both Rab5a and Rab5b, and that the GTPase activities of the two proteins are differentially regulated in vivo.

Anthrax toxin fusion proteins for intracellular delivery of macromolecules

The dominant role played by the anthrax toxin in Bacillus anthracis pathogenesis shows that the toxin has evolved to be an efficient system for delivering its two catalytic protein components, oedema factor and lethal factor (LF), into the cytosol of host cells. This system involves binding of the protective antigen (PA) toxin component to a ubiquitous (and still unidentified) receptor, proteolytic activation at the cell surface, internalization by endocytosis and translocation through an early endosome membrane to the cytosol (Leppla 1995). We and colleagues showed that the system can be exploited to deliver heterologous polypeptides to the cytosol (Arora et al. 1992; Milne et al. 1995). This work used the catalytic domains of other toxins which are normally translocated across membranes (Arora & Leppla 1994). Immunity to intracellular pathogens depends on the cytosolic processing of antigens to produce peptides that are presented on the cell surface bound to MHC Class I molecules. The anthrax toxin delivery system provides a way to mimic this process. We made a fusion protein containing the (non-catalytic) amino terminal domain of LF and the gp120 envelope glycoprotein of HIV-1. Administration of this recombinant protein along with PA to antigen-presenting cells sensitized them to cytolysis by cytotoxic T-cells specific to gp120 peptides (Goletz et al. 1997). Further exploitation of the anthrax toxin system as a cell-targeting reagent would be facilitated by achieving cell type specificity. The recent determination of the PA structure (Petosa et al. 1997) allows rational engineering to modify or replace the receptor-binding domain with specific ligand structures. A model system was produced by fusing a c-Myc peptide to the carboxyl terminus of PA so as to target hybridoma cells expressing cell surface antibodies to this peptide. Killing of the hybridoma cells was shown to be specific by competition with the peptide and with non-toxic mutants of PA (Varughese et al. 1998).

Rab15 Mediates an Early Endocytic Event in Chinese Hamster Ovary Cells

Rab GTPases comprise a large family of monomeric proteins that regulate a diverse number of membrane trafficking events, including endocytosis. In this paper, we examine the subcellular distribution and function of the GTPase Rab15. Our biochemical and confocal immunofluorescence studies demonstrate that Rab15 associates with the transferrin receptor, a marker for the early endocytic pathway, but not with Rab7 or the cation-independent mannose 6-phosphate receptor, markers for late endosomal membranes. Furthermore, Rab15 colocalizes with Rab4 and -5 on early/sorting endosomes, as well as Rab11 on pericentriolar recycling endosomes. Consistent with its localization to early endosomal membranes, overexpression of the constitutively active mutant HArab15Q67L reduces receptor-mediated and fluid phase endocytosis. Therefore, our functional studies suggest that Rab15 may function as an inhibitory GTPase in early endocytic trafficking.

Chemical Chaperones Enhance Superantigen and Conventional Antigen Presentation by HLA-DM-Deficient as well as HLA-DM-Sufficient Antigen-Presenting Cells and Enhance IgG2a Production In Vivo

Chemical chaperones, first defined in studies of mutant cystic fibrosis transmembrane conductance regulator proteins, are small molecules that act as stabilizers of proteins in their native state and have the ability in some cases to rescue protein-folding mutants within cells. HLA-DM is an MHC II-specific molecular chaperone that facilitates peptide loading onto MHC II proteins and also stabilizes empty MHC II molecules prior to their acquisition of antigenic peptides. APC that lack HLA-DM exhibit quantitative defects in protein Ag as well as superantigen presentation. Here we show that both the superantigen and protein presentation defect in MHC II-transfected, HLA-DM-deficient T2 cells can be partially overcome by treating the APC with the chemical chaperones glycerol, DMSO, or trimethylamine oxide. These chemical chaperones also enhance superantigen and conventional Ag presentation by wild-type APC. In vivo, glycerol was found to act as an adjuvant and resulted in enhanced IgG2a production to trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH). In vitro, the enhancement of Ag presentation by chemical chaperones was found to take place at the level of the APC and took several hours to develop. Subcellular fractionation experiments show that HLA-DM enhances presentation of peptides by dense endosome fractions whereas chemical chaperones enhance presentation by light membrane fractions (early endosome or plasma membrane). The mechanism by which these chemical chaperones augment Ag presentation is not defined, but flow cytometric analysis suggests that the enhancement may be due to a subtle effect on the stability of several different proteins at the cell surface.

NSF is required for transport from early to late endosomes.

Protein transport between early and late endosomes is a major membrane trafficking pathway in the cell followed by many proteins, including all down-regulated receptors. Yet, little is known at the molecular level about the mechanisms regulating membrane interactions in the endocytic pathway beyond early endosomes. In this study, we have used an in vitro transport assay to study the biochemical properties of endosome docking/fusion events. Our data demonstrate that N-ethylmaleimide (NEM) sensitive factor (NSF) and its soluble associated proteins (SNAPs) are required for transport from early to late endosomes, as well as at all other steps of endosomal membrane transport. We also find that these proteins are enriched on endosomal membranes. In addition, our studies suggest that besides NSF/SNAPs, another NEM-sensitive component may also be involved in docking/fusion at this late stage of the pathway. Finally, we find that, in contrast to Golgi membranes, NSF association to both early and late endosomal membranes occurs via an ATP-independent mechanism, indicating that the binding properties of endosomal and biosynthetic NSF are different. Our data thus show that NSF/SNAPs, perhaps together with another NEM-sensitive factor, are part of the basic molecular machinery which controls docking/fusion events during transport from early to late endosomes, along the lysosomal degradation pathway.

Early endosome membrane dynamics characterized by flow cytometry.

Early endosomes are very dynamic intracellular membrane organelles that undergo multiple fusion and fission events. In this study, we developed a novel assay based on multiparametric flow cytometric analyses and early endosome sorting to characterize better the mechanisms of early endosome membrane dynamics in vitro. In particular, we have investigated the role of rab4 and rab5, two small GTPases known to regulate distinct steps of membrane traffic in the endocytic pathway. We show that early endosomes undergo homotypic fusion reactions, which lead to the formation of fusion intermediates with increased size. Fusion is efficiently stimulated by recombinant rab5 but not by recombinant rab4. Subsequently, membrane fission consumes this larger fusion compartment. This fission process is stimulated by rab4 and by the GTP hydrolysis-defective mutant rab4Q67L.

Circulating collagen is catabolized by endocytosis mainly by endothelial cells of endocardium in cod (Gadus morhua)

The cells responsible for the clearance of collagen were studied in cod. Cod collagen labelled with the lysosomal trap-label 125I-tyramine cellobiose was cleared from the circulation with a t1/2 of 15 min. 1 h After injection 75%, 17% and 8% of the label were recovered in the heart, liver and blood, respectively. 24 h After administration of collagen labelled conventionally with 125I to allow escape of labelled degradation product from the site of uptake, 80% of the label had left the heart, signifying degradation. When collagen was tagged with 125I-tyramine cellobiose, heart-associated radioactivity did not decrease after 24 h, indicating intralysosomal degradation. Fluorescence microscopy revealed that i.v. injected fluorescently-labelled collagen accumulated in discrete vesicles of cells lining the endocardial blood space of both atrium and ventricle. Conventional and immuno-electron microscopy showed that these cells contained numerous coated pits and vesicles reflecting active endocytosis, and that ligand lined the limiting membrane of early endosomes. Intravenously injected 2 μm latex accumulated mainly in kidney. We conclude that the population of non-macrophagic endocardial cells are important for the turnover of collagen in cod. These cells therefore resemble sinusoidal endothelial cells of salmon kidney and mammalian liver.

Invariant Chain Induces a Delayed Transport from Early to Late Endosomes

Invariant chain associated with class II molecules is proteolytically processed in several distinct intermediates during its transport through the endocytic pathway. Using subcellular fractionation, early and late endosomal compartments were separated in human fibroblasts transfected with HLA-DR (4N5 cells) and supertransfected with invariant chain (4N5Ii cells) or invariant chain lacking most of the cytoplasmic tail (4N5 delta 20Ii cells). Early and late endosome membrane fractions were characterized by morphology and by analyzing the presence of the Rab5 and Rab7 GTPases as markers of early and late endosomes, respectively. The transfer of endocytosed horseradish peroxidase from early to late endosomes proceeded relatively rapid both in 4N5 and 4N5 delta 20Ii cells (t1/2 = 25 min), whereas this transfer was significantly delayed (t1/2 = 2 h) in 4N5Ii cells. Pulse-chase experiments showed that invariant chain and its degradation products were first observed in early endosomes and thereafter in late endosomes. Our results strongly suggest that invariant chain induces a retention mechanism in the endocytic pathway.