ABSTRACT Ordered lipid domains (rafts) in plasma membranes have been hypothesized to participate in endocytosis based on inhibition of endocytosis by removal or sequestration of cholesterol. To more carefully investigate the role of the sterol in endocytosis, we used a substitution strategy to replace cholesterol with sterols that show various raft-forming abilities and chemical structures. Both clathrin-mediated endocytosis of transferrin and clathrin-independent endocytosis of clustered placental alkaline phosphatase were measured. A subset of sterols reversibly inhibited both clathrin-dependent and clathrin-independent endocytosis. The ability of a sterol to support lipid raft formation was necessary for endocytosis. However, it was not sufficient, because a sterol lacking a 3β-OH group did not support endocytosis even though it had the ability to support ordered domain formation. Double bonds in the sterol rings and an aliphatic tail structure identical to that of cholesterol were neither necessary nor sufficient to support endocytosis. This study shows that substitution using a large number of sterols can define the role of sterol structure in cellular functions. Hypotheses for how sterol structure can similarly alter clathrin-dependent and clathrin-independent endocytosis are discussed.

The retrograde transport route links early endosomes and the TGN. Several endogenous and exogenous cargo proteins use this pathway, one of which is the well-explored bacterial Shiga toxin. ADP-ribosylation factors (Arfs) are ~20 kDa GTP-binding proteins that are required for protein traffic at the level of the Golgi complex and early endosomes. In this study, we expressed mutants and protein fragments that bind to Arf-GTP to show that Arf1, but not Arf6 is required for transport of Shiga toxin from early endosomes to the TGN. We depleted six Arf1-specific ARF-GTPase-activating proteins and identified AGAP2 as a crucial regulator of retrograde transport for Shiga toxin, cholera toxin and the endogenous proteins TGN46 and mannose 6-phosphate receptor. In AGAP2-depleted cells, Shiga toxin accumulates in transferrin-receptor-positive early endosomes, suggesting that AGAP2 functions in the very early steps of retrograde sorting. A number of other intracellular trafficking pathways are not affected under these conditions. These results establish that Arf1 and AGAP2 have key trafficking functions at the interface between early endosomes and the TGN.

Transferrin receptor 2 (TfR2) possesses a YQRV motif similar to the YTRF motif of transferrin receptor 1 (TfR1) responsible for the internalization and secretion through the endosomal pathway. Raft biochemical dissection showed that TfR2 is a component of the low-density Triton-insoluble (LDTI) plasma membrane domain, able to co-immunoprecipitate with caveolin-1 and CD81, two structural raft proteins. In addition, subcellular fractionation experiments showed that TfR1, which spontaneously undergoes endocytosis and recycling, largely distributed to intracellular organelles, whereas TfR2 was mainly associated with the plasma membrane. Given the TfR2 localization in lipid rafts, we tested its capability to activate cell signalling. Interaction with an anti-TfR2 antibody or with human or bovine holotransferrin showed that it activated ERK1/ERK2 and p38 MAP kinases. Integrity of lipid rafts was required for MAPK activation. Co-localization of TfR2 with CD81, a raft tetraspanin exported through exosomes, prompted us to investigate exosomes released by HepG2 and K562 cells into culture medium. TfR2, CD81 and to a lesser extent caveolin-1, were found to be part of the exosomal budding vesicles. In conclusion, the present study indicates that TfR2 localizes in LDTI microdomains, where it promotes cell signalling, and is exported out of the cells through the exosome pathway, where it acts as an intercellular messenger.

ADP ribosylation factors (Arf) regulate membrane trafficking at multiple intracellular sites by recruiting coat proteins to membranes. The site-specific regulation of Arf is thought to be mediated by regulatory proteins including the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Here, we test this hypothesis by comparing the site of action of the Arf GAP AGAP2 to the closely related AGAP1. AGAP1 has previously been found to associate with the adaptor protein complex AP-3 and regulate the function of AP-3 endosomes. We found that AGAP2 directly interacted with AP-1. AGAP2 colocalized with AP-1, transferrin receptor and Rab4 on endosomes. Overexpression of AGAP2 changed the intracellular distribution of AP-1 and promoted Rab4-dependent fast recycling of transferrin. Based on these results, we concluded that the closely related Arf GAPs, AGAP1 and AGAP2, distinguish between these related heterotetrameric adaptor protein complexes to specifically regulate AP-3 endosomes and AP-1 recycling endosomes.

The early endosome comprises morphologically distinct regions specialised in sorting cargo receptors. A central question is whether receptors move through a predetermined structural pathway, or whether cargo selection contributes to the generation of endosome morphology and membrane flux. Here, we show that depletion of tumour susceptibility gene 101 impairs the selection of epidermal growth factor receptor away from recycling receptors within the limiting membrane of the early endosome. Consequently, epidermal growth factor receptor sorting to internal vesicles of the multivesicular body and cargo recycling to the cell surface or Golgi complex are inhibited. These defects are accompanied by disruption of bulk flow transport to the lysosome and profound structural rearrangement of the early endosome. The pattern of tubular and vacuolar domains is replaced by enlarged vacuoles, many of which are folded into multicisternal structures resembling the `Class E' compartments that define several Saccharomyces cerevisiae vacuolar protein sorting mutants. The cisternae are interleaved by a fine matrix but lack other surface elaborations, most notably clathrin.

The insect low-density lipoprotein (LDL) receptor (LDLR) homologue LpR mediates endocytosis of an insect lipoprotein (lipophorin) that is structurally related to LDL. Despite these similarities, lipophorin and LDL follow distinct intracellular routes upon endocytosis by their receptors. Whereas LDL is degraded in lysosomes, lipophorin is recycled in a transferrin-like manner. We constructed several hybrid receptors composed of Locusta migratoria LpR and human LDLR regions to identify the domains implicated in LpR-mediated ligand recycling. Additionally, the triadic His562 residue of LDLR, which is putatively involved in ligand uncoupling, was mutated to Asn, corresponding to Asn643 in LpR, to analyse the role of the His triad in receptor functioning. The familial hypercholesterolaemia (FH) class 5 mutants LDLR H562Y and LDLR H190Y were also analysed in vitro. Fluorescence microscopic investigation and quantification suggest that LpR-mediated ligand recycling involves cooperation between the ligand-binding domain and epidermal growth factor (EGF) domain of LpR, whereas its cytosolic tail does not harbour motifs that affect this process. LDLR residue His562 appears to be essential for LDLR recycling after ligand endocytosis but not for constitutive receptor recycling. Like LDLR H562N , LDLR H562Y did not recycle bound ligand; moreover, the intracellular distribution of both mutant receptors after ligand incubation coincides with that of a lysosomal marker. The LDLR mutant characterization in vitro suggests that LDLR FH class 5 mutations might be divided into two subclasses.

Rab proteins comprise a family of monomeric GTPases that control cellular membrane traffic. Rab21 is a poorly characterised member with no known function. Human Rab21 cDNA from K562 cells was subcloned into GFP expression vectors to generate Rab21 and Rab21 mutants defective in either GTP hydrolysis (Rab21 Q78L) or binding (Rab21 T33N) for transfection studies in HeLa cells. Confocal fluorescence microscopy and ultrastructural studies revealed Rab21 to be predominantly localised to the early endocytic pathway, on vesicles containing earlyendosomal antigen 1 EEA1, transferrin receptor and internalised ligands. EEA1 was localised to enlarged endosomes in Rab21 wild-type expressing cells but the GTP hydrolysis and GDP binding mutants had unique phenotypes labelling tubular reticular structures and the trans-Golgi network, respectively. Early endosome localisation for Rab21 was confirmed in a hepatoma cell line that allowed analysis of the subcellular distribution of the endogenous protein. Comparison of the localisation of Rab21 with other Rabs revealed extensive colocalisation with early endocytic variants Rab4, Rab5, Rab17 and Rab22 but much less overlap with those associated with late endosomes, recycling endosomes and the early secretory pathway. Cells expressing Rab21 T33N had defects in endocytosis of transferrin and epidermal growth factor and failed to effectively deliver the latter ligand to late endosomes and lysosomes for degradation. Collectively, our data provide the first characterisation of Rab21 function in early endosome dynamics.

We have used fluorescence imaging of individual exocytic events in combination with immunogold electron microscopy and FM1-43 photoconversion to study the stimulus-dependent recycling of dense core vesicle content in isolated rat pituitary lactotrophs. Secretory stimulation with high external [K + ] resulted in 100 exocytic sites per cell that were labeled by extracellular antibodies against the peptide hormone prolactin. Morphological analysis demonstrated that the prolactin was retained and internalized in intact dense cores. Vesicles containing non-secreted, internalized prolactin did not colocalize with DiI-LDL that had been chased into lysosomes but did transiently colocalize with internalized transferrin. The recycling vesicles also trafficked through a syntaxin 6-positive compartment but not the TGN38-positive trans-Golgi. Recycling vesicles, which returned to the cell surface in a slow basal manner, could also be stimulated to undergo exocytosis with a high release probability during subsequent exocytic stimulation with external K + . These studies suggest a functional role for recycling vesicles that retain prolactin.

The pericentriolar endocytic recycling compartment (ERC) is involved in receptor and lipid recycling as well as in the delivery of internalized cargo from early endosomes to the trans Golgi network (TGN). We show that synaptotagmin (Syt) IX, a member of the Syt family of proteins, localizes to the ERC and is required for export from the ERC to the cell surface. We demonstrate that rat basophilic leukemia (RBL-2H3) mast cells endogenously express Syt IX mRNA and protein. Localization studies employing fractionation on linear sucrose gradients combined with confocal microscopy by indirect immunofluorescence or stable expression of a Syt IX-green fluorescent fusion protein demonstrate that Syt IX colocalizes with internalized transferrin (Tfn) and with Rab 11 at the perinuclear ERC. Syt IX also colocalizes with tubulin at the microtubules organizing center (MTOC) and remains associated with tubulin clusters formed in taxol-treated cells. Moreover, Syt IX coimmunoprecipitates with tubulin from intact RBL cells, and chimeric fusion proteins comprising either the C2A or the C2B domain of Syt IX are able to pull down tubulin from RBL cell lysates. To study the functional role of Syt IX, we have stably transfected RBL cells with Syt IX sense or antisense cDNA and monitored the routes of Tfn internalization and recycling in cells that overexpress (RBL-Syt IX + ) or display substantially reduced (<90%) levels of Syt IX (RBL-Syt IX – ). In these cells, Tfn binding and internalization into early endosomes and the ERC are unaltered. However, recycling from the ERC to the cell surface is significantly slowed down in the RBL-Syt IX – cells. These results therefore indicate that Syt IX is involved in regulating transport from the ERC to the cell surface, and suggest that it may play a role in linking vesicles that exit the ERC with the microtubules network.

Early endosomes and a perinuclear, Rab-11-positive compartment have been implicated in the recycling of internalized receptors. In this study, we show that synaptotagmin III (Syt III), a member of the Syt family of proteins, is required for the formation and delivery of cargo to the perinuclear endocytic recycling compartment (ERC). We demonstrate that rat basophilic leukemia(RBL-2H3) mast cells endogenously express Syt III, and >70% of this protein colocalizes with early endosomal markers, such as EEA1, annexin II and syntaxin 7, and the remaining protein colocalizes with secretory granule (SG)markers such as β-hexosaminidase, histamine and serotonin. To study the functional role of Syt III, we stably transfected RBL cells with Syt III antisense cDNA and monitored the route of transferrin (Tfn) internalization in cells that displayed substantially reduced (<90%) levels of Syt III(RBL-Syt III - ). In these cells, Tfn binding and internalization into early endosomes were unaltered. However, whereas in the mock-transfected cells Tfn was subsequently delivered to the ERC, in the RBL-Syt III - cells, Tfn remained associated with dispersed peripheral vesicles and Rab 11 remained cytosolic. Nevertheless, the rates of Tfn internalization and recycling were not affected. RBL-Syt III - cells also displayed enlarged SGs, reminiscent of the SGs present in Chediak-Higashi(beige) mice. However, morphometric analyses suggested that granule formation was unaltered and that the calculated unit granule volume is the same in both cell lines. Therefore, our results implicate Syt III as a critical factor for the generation and delivery of internalized cargo to the perinuclear endocytic recycling compartment and suggest a possible link between ERC and recycling from immature SGs during the process of SG maturation.

Rab5, a member of the small GTPase family of proteins, is primarily localized on early endosomes and has been proposed to participate in the regulation of early endosome trafficking. It has been reported that phosphatidylinositol 3-kinases and FYVE domain proteins, such as EEA1, can be recruited onto early endosomes and act as Rab5 effectors. SARA (Smad anchor for receptor activation), also a FYVE domain protein, was initially isolated as a participant in signal transduction from the transforming growth factorβ receptor. Overexpressed SARA has been found on EEA1-positive early endosomes. In this report, we show that endogenous SARA is present on early endosomes and overexpression of SARA causes endosomal enlargement. Functionally, SARA overexpression significantly delays the recycling of transferrin. The transferrin receptor distributed on the cell surfaces was also greatly reduced in cells overexpressing SARA. However, the internalization rate of transferrin is not affected by SARA overexpression. The morphological and functional alterations caused by SARA overexpression resemble those caused by overexpression of Rab5:GTP mutant Rab5Q79L. Finally,all SARA-mediated phenotypic changes can be counteracted by overexpression Rab5:GDP mutant Rab5S34N. These results collectively suggested that SARA plays an important functional role downstream of Rab5-regulated endosomal trafficking.

The lipoprotein of insects, high-density lipophorin (HDLp), is homologous to that of mammalian low-density lipoprotein (LDL) with respect to its apolipoprotein structure. Moreover, an endocytic receptor for HDLp has been identified (insect lipophorin receptor, iLR) that is homologus to the LDL receptor. We transfected LDL-receptor-expressing CHO cells with iLR cDNA to study the endocytic uptake and intracellular pathways of LDL and HDLp simultaneously. Our studies provide evidence that these mammalian and insect lipoproteins follow distinct intracellular routes after receptor-mediated endocytosis. Multicolour imaging and immunofluorescence was used to visualize the intracellular trafficking of fluorescently labeled ligands in these cells. Upon internalization, which can be completely inhibited by human receptor-associated protein (RAP), mammalian and insect lipoproteins share endocytic vesicles. Subsequently, however, HDLp evacuates the LDL-containing endosomes. In contrast to LDL, which is completely degraded in lysosomes after dissociating from its receptor, both HDLp and iLR converge in a nonlysosomal juxtanuclear compartment. Colocalization studies with transferrin identified this organelle as the endocytic recycling compartment via which iron-depleted transferrin exits the cell. Fluorescently labeled RAP is also transported to this recycling organelle upon receptor-mediated endocytosis by iLR. Internalized HDLp eventually exits the cell via the recycling compartment, a process that can be blocked by monensin, and is re-secreted with a t½of ∼13 minutes. From these observations, we conclude that HDLp is the first non-exchangeable apolipoprotein-containing lipoprotein that follows a transferrin-like recycling pathway despite the similarities between mammalian and insect lipoproteins and their receptors.

Recent studies showing thorough intermixing of apical and basolateral endosomes have demonstrated that endocytic sorting is critical to maintaining the plasma membrane polarity of epithelial cells. Our studies of living, polarized cells show that disrupting endocytosis with brefeldin-A rapidly destroys the polarity of transferrin receptors in MDCK cells while having no effect on tight junctions. Brefeldin-A treatment induces tubulation of endosomes, but the sequential compartments and transport steps of the transcytotic pathway remain intact. Transferrin is sorted from LDL, but is then missorted from common endosomes to the apical recycling endosome, as identified by its nearly neutral pH, and association with GFP chimeras of Rabs 11a and 25. From the apical recycling endosome, transferrin is then directed to the apical plasma membrane. These data are consistent with a model in which polarized sorting of basolateral membrane proteins occurs via a brefeldin-A-sensitive process of segregation into basolateral recycling vesicles. Although disruption of polar sorting correlates with dissociation of γ-adaptin from endosomes, γ-adaptin does not appear to be specifically involved in sorting into recycling vesicles, as we find it associated with the transcytotic pathway, and particularly to the post-sorting transcytotic apical recycling endosome. Movies available on-line