RICIN-BINDING PROTEINS ALONG THE ENDOCYTIC PATHWAY: THE MAJOR ENDOSOMAL RICIN-BINDING PROTEIN IS ENDOSOME-SPECIFIC

Ricin is internalized after binding at the cell surface via lectin activity of the B-chain recognizing terminal galactose residues. Ricin-A chain is then translocated to the cytosol from various endocytic structures. Cell death is the result of catalytic inactivation of protein synthesis. Using ¹²⁵I-ricin overlays, we examined the distribution of ricin binding-proteins within highly purified preparations of plasma membrane vesicles, endosomes and lysosomes from lymphocytes. All compartments of the endocytic pathway had distinct profiles; some ricin-binding proteins were present throughout the pathway; others were restricted to the plasma membrane and endosomes. The major endosomal protein recognized by ¹²⁵I-ricin, a 166kDa glycoprotein, was endosome-specific. When endosomal proteins were solubilized before chromatography onto ricin-agarose this protein was also by far the major specifically-bound glycoprotein. This 166 kDa glycoprotein might be involved in ricin translocation from this compartment.     

Genome-wide RNAi screens identify genes required for Ricin and PE intoxications

Protein toxins such as Ricin and Pseudomonas exotoxin (PE) pose major public health challenges. Both toxins depend on host cell machinery for internalization, retrograde trafficking from endosomes to?the ER, and translocation to cytosol. Although both toxins follow a similar intracellular route, it is unknown how much they rely on the same genes. Here we conducted two genome-wide RNAi screens identifying genes required for intoxication and demonstrating that requirements are strikingly different between PE and Ricin, with only 13% overlap. Yet factors required by both toxins are present from the endosomes to the ER, and, at the morphological level, the toxins colocalize in multiple structures. Interestingly, Ricin, but not PE, depends on Golgi complex integrity and colocalizes significantly with a medial Golgi marker. Our data are consistent with two intertwined pathways converging and diverging at multiple points and reveal the complexity of retrograde membrane trafficking in mammalian cells.     

Endocytic membrane traffic to the Golgi apparatus in a regulated secretory cell line

We have established a ricin-resistant glycosylation-defective PC12 pheochromocytoma cell line to study biochemically glycoprotein traffic from the cell surface to the Golgi apparatus in regulated secretory cells. The strategy employed in this study is a modification of that used previously (Duncan, J. R., and Kornfeld, S. (1988) J. Cell Biol. 106, 617-628) to demonstrate transport of the cation-independent and -dependent mannose 6-phosphate receptors from the cell surface to the trans-Golgi network in nonsecretory cell types. In ricin-resistant PC12 cells, radiolabeled galactose was incorporated enzymatically into surface glycoconjugates, primarily glycoproteins. Resistance to beta-galactosidase was acquired upon reculture at 37 degrees C due to further terminal glycosylation of the galactose residues. Treatment of N-linked oligosaccharides isolated from recultured cells with a variety of glycosidases in conjunction with beta-galactosidase demonstrated the addition of sialic acid N-acetylglucosamine and fucose residues to the galactose residues in recultured cells. Resistance to beta-galactosidase was not acquired in cells recultured at 19 degrees C, indicating that subsequent glycosylation of galactose residues did not occur at the cell surface or in endosomes. While glycosylation of galactose incorporated into asparagine oligosaccharides in Chinese hamster ovary clone 13 cells was not significant (less than 1%) after 6 h of reculture, approximately 10% of the galactose incorporated into surface oligosaccharides was further glycosylated in PC12 cells in this time. Analysis of total labeled versus beta-galactosidase-resistant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that endocytic traffic to the site of glycosylation activity in mutant PC12 cells was highly selective, but included a much greater number of proteins than were detected in Chinese hamster ovary clone 13 fibroblasts.     

Properties and action mechanism of the toxic lectin modeccin: Interaction with cell lines resistant to modeccin,abrin, and ricin

The toxic lectin modeccin, which inhibits protein synthesis in eukaryotic cells, is cleaved upon treatment with 2-mercaptoethanol into two peptide chains which move in polyacrylamide gels at rates corresponding to molecular weights 28,000 and 38,000. After reduction, the toxin loses its effect on cells, while its ability to inhibit cell-free protein synthesis increases. Like abrin and ricin it inhibits protein synthesis by inactivating the 60S ribosomal subunits. Modeccin binds to surface receptors containing terminal galactose residues. Competition experiments with various glycoproteins indicate that the modeccin receptors are different from the abrin receptors. In addition, they were present on HeLa cells in much smaller numbers. Moreover, mutant lines resistant to abrin and ricin were not resistant to modeccin and vice-versa. The toxin resistance of various mutant cell lines could not be accounted for by a reduced number of binding sites on cells. The data are consistent with the view that the cells possesss different populations of binding sites with differences in ability to facilitate the uptake of the toxins and that in the resistant lines the most active receptors have been reduced or eliminated.     

Toxin entry: how bacterial proteins get into mammalian cells

Certain bacteria secrete protein toxins that catalytically modify and disrupt essential processes in mammalian cells, often leading to cell death. As the substrates modified by these toxins are located in the mammalian cell cytosol, a catalytically active toxin polypeptide must reach this compartment in order to act. The toxins bind to receptors on the surface of susceptible cells and enter them by endocytic uptake. Endocytosed toxins initially accumulate in endosomes, where some of these proteins take advantage of the acidic environment within these organelles to form, or contribute to the formation of, protein-conducting channels through which the catalytic polypeptide is able to translocate into the cytosol. Other toxins are unable to respond to low pH in this way and must undergo intracellular vesicular transport to reach a compartment where pre-existing protein-conducting channels occur and can be exploited for membrane translocation — the endoplasmic reticulum. In this way, cell entry by this second group of toxins demonstrates that the secretory pathway of mammalian cells is completely reversible.     

Pathways followed by ricin and Shiga toxin into cells

The plant toxin ricin and the bacterial toxin Shiga toxin belong to a group of protein toxins that inhibit protein synthesis in cells enzymatically after entry into the cytosol. Ricin and Shiga toxin, which both have an enzymatically active moiety that inactivates ribosomes and a moiety that binds to cell surface receptors, enter the cytosol after binding to the cell surface, endocytosis by different mechanisms, and retrograde transport to the Golgi apparatus and the endoplasmic reticulum (ER). The toxins can be used to investigate the various transport steps involved, both the endocytic mechanisms as well as pathways for retrograde transport to the ER. Recent studies show that not only do several endocytic mechanisms exist in the same cell, but they are not equally sensitive to removal of cholesterol. New data have revealed that there is also more than one pathway leading from endosomes to the Golgi apparatus and retrogradely from the Golgi to the ER. Trafficking of protein toxins along these pathways will be discussed in the present article.     

Differential effects of nitrated ricin and nitrated and dithionite-reduced ricin on protein-synthesis inhibition and transmembrane tramsport in eukaryotic cells.

Ricin, was nitrated with tetranitromethane and reduced with sodium dithionite. Of the 8.0 nitro groups incorporated, 3.2 were on the A chain and 5.1 were on the B chain. Nitrated ricin1 was somewhat less active than nitrated and reduced ricin1 in inhibiting protein synthesis in vitro, but both were highly inhibitory. However, the modified toxins were less than 1% as active as ricin in inhibiting protein synthesis in cultured cells. Indirect immunofluorescence assays demonstrated tha both modified toxins were specifically bound to the cell surface and could be displaced by galactose.     

Glycans as endocytosis signals: the cases of the asialoglycoprotein and hyaluronan/chondroitin sulfate receptors

Animal cells internalize specific extracellular macromolecules (ligands) by using specialized cell surface receptors that operate through a complex and highly regulated process known as receptor-mediated endocytosis, which involves the binding, internalization, and transfer of ligands through a series of distinct intracellular compartments. For the uptake of a variety of carbohydrate-containing macromolecules, such as glycoproteins, animal cells use specialized membrane-bound lectins as endocytic receptors that recognize different sugar residues or carbohydrate structures present on various ligands. The hepatic asialoglycoprotein receptor, which recognizes glycoconjugates containing terminal galactose or N-acetylgalactosamine residues, was the first membrane lectin discovered and has been a classical system for studying receptor-mediated endocytosis. Studies of how the asialoglycoprotein receptor functions have led to the discovery of two functionally distinct, parallel pathways of clathrin-mediated endocytosis (called the State 1 and State 2 pathways), which may also be utilized by all the other endocytic recycling receptor systems. Another endocytic membrane lectin, the hyaluronan/chondroitin sulfate receptor, which has recently been purified and cloned, is responsible for the turnover in mammals of these glycosaminoglycans, which are important components of extracellular matrices. We discuss the characteristics and physiological importance of these two proteins as examples of how lectins can function as endocytic receptors.     

Important relationships between Rab and MICAL proteins in endocytic trafficking

The internalization of essential nutrients, lipids and receptors is a crucial process for all eukaryotic cells. Accordingly, endocytosis is highly conserved across cell types and species. Once internalized, small cargo-containing vesicles fuse with early endosomes (also known as sorting endosomes), where they undergo segregation to distinct membrane regions and are sorted and transported on through the endocytic pathway. Although the mechanisms that regulate this sorting are still poorly understood, some receptors are directed to late endosomes and lysosomes for degradation, whereas other receptors are recycled back to the plasma membrane; either directly or through recycling endosomes. The Rab family of small GTP-binding proteins plays crucial roles in regulating these trafficking pathways. Rabs cycle from inactive GDP-bound cytoplasmic proteins to active GTP-bound membrane-associated proteins, as a consequence of the activity of multiple specific GTPase-activating proteins (GAPs) and GTP exchange factors (GEFs). Once bound to GTP, Rabs interact with a multitude of effector proteins that carry out Rab-specific functions. Recent studies have shown that some of these effectors are also interaction partners for the C-terminal Eps15 homology (EHD) proteins, which are also intimately involved in endocytic regulation. A particularly interesting example of common Rab-EHD interaction partners is the MICAL-like protein, MICAL-L1. MICAL-L1 and its homolog, MICAL-L2, belong to the larger MICAL family of proteins, and both have been directly implicated in regulating endocytic recycling of cell surface receptors and junctional proteins, as well as controlling cytoskeletal rearrangement and neurite outgrowth. In this review, we summarize the functional roles of MICAL and Rab proteins, and focus on the significance of their interactions and the implications for endocytic transport.     

The GTP/GDP cycling of rho GTPase TCL is an essential regulator of the early endocytic pathway

Rho GTPases are key regulators of actin dynamics. We report that the Rho GTPase TCL, which is closely related to Cdc42 and TC10, localizes to the plasma membrane and the early/sorting endosomes in HeLa cells, suggesting a role in the early endocytic pathway. Receptor-dependent internalization of transferrin (Tf) is unaffected by suppression of endogenous TCL by small interfering RNA treatment. However, Tf accumulates in Rab5-positive uncoated endocytic vesicles and fails to reach the early endosome antigen-1-positive early endosomal compartments and the pericentriolar recycling endosomes. Moreover, Tf release upon TCL knockdown is significantly slower. Conversely, in the presence of dominant active TCL, internalized Tf accumulates in early endosome antigen-1-positive early/sorting endosomes and not in perinuclear recycling endosomes. Tf recycles directly from the early/sorting endosomes and it is normally released by the cells. The same phenotype is generated by replacing the C terminus of dominant active Cdc42 and TC10 with that of TCL, indicating that all three proteins share downstream effector proteins. Thus, TCL is essential for clathrin-dependent endocytosed receptors to enter the early/sorting endosomes. Furthermore, the active GTPase favors direct recycling from early/sorting endosomes without accumulating in the perinuclear recycling endosomes.     

Cell surface and intracellular functions for ricin galactose binding.

The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.     

Cholesterol-dependent retention of GPI-anchored proteins in endosomes.

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the Cterminal end that serves as their sole means of membrane anchoring. Using fluorescently labeled ligands and digital fluorescence microscopy, we show that contrary to the potocytosis model, GPI-anchored proteins are internalized into endosomes that contain markers for both receptor-mediated uptake (e.g. transferrin) and fluid phase endocytosis (e.g. dextrans). This was confirmed by immunogold electron microscopy and the observation that a fluorescent folate derivative bound to the GPI-anchored folate receptor is internalized into the same compartment as co-internalized horseradish peroxidase-transferrin; the folate fluorescence was quenched when cells subsequently were incubated with diaminobenzidine and H2O2. Most of the GPI-anchored proteins are recycled back to the plasma membrane but at a rate that is at least 3-fold slower than C6-NBD-sphingomyelin or recycling receptors. This endocytic retention is regulated by the level of cholesterol in cell membranes; GPI-anchored proteins are recycled back to the cell surface at the same rate as recycling transferrin receptors and C6-NBD-sphingomyelin in cholesterol-depleted cells. Cholesterol-dependent endocytic sorting of GPI-anchored proteins is consistent with the involvement of specialized lipid domains or 'rafts' in endocytic sorting. These results provide an alternative explanation for GPI-requiring functions of some GPI-anchored proteins.     

The polymeric immunoglobulin receptor accumulates in specialized endosomes but not synaptic vesicles within the neurites of transfected neuroendocrine PC12 cells

We have expressed in neuroendocrine PC12 cells the polymeric immunoglobulin receptor (pIgR), which is normally targeted from the basolateral to the apical surface of epithelial cells. In the presence of nerve growth factor, PC12 cells extend neurites which contain synaptic vesicle-like structures and regulated secretory granules. By immunofluorescence microscopy, pIgR, like the synaptic vesicle protein synaptophysin, accumulates in both the cell body and the neurites. On the other hand, the transferrin receptor, which normally recycles at the basolateral surface in epithelial cells, and the cation-independent mannose 6-phosphate receptor, a marker of late endosomes, are largely restricted to the cell body. pIgR internalizes ligand into endosomes within the cell body and the neurites, while uptake of ligand by the low density lipoprotein receptor occurs primarily into endosomes within the cell body. We conclude that transport of membrane proteins to PC12 neurites as well as to specialized endosomes within these processes is selective and appears to be governed by similar mechanisms that dictate sorting in epithelial cells. Additionally, two types of endosomes can be identified in polarized PC12 cells by the differential uptake of ligand, a housekeeping type in the cell bodies and a specialized endosome in the neurites. Recent findings suggest that specialized axonal endosomes in neurons are likely to give rise to synaptic vesicles (Mundigl, O., M. Matteoli, L. Daniell, A. Thomas-Reetz, A. Metcalf, R. Jahn, and P. De Camilli. 1993. J. Cell Biol. 122:1207- 1221). Although pIgR reaches the specialized endosomes in the neurites of PC12 cells, we find by subcellular fractionation that under a variety of conditions it is efficiently excluded from synaptic vesicle- like structures as well as from secretory granules.     

Trafficking of green fluorescent protein tagged-vesicular acetylcholine transporter to varicosities in a cholinergic cell line

Synaptic vesicle proteins are suggested to travel from the trans-Golgi network to active zones via tubulovesicular organelles, but the participation of different populations of endosomes in trafficking remains a matter of debate. Therefore, we generated a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) and studied the localization of VAChT in organelles in the cell body and varicosities of living cholinergic cells. GFP-VAChT is distributed to both early and recycling endosomes in the cell body and is also observed to accumulate in endocytic organelles within varicosities of SN56 cells. GFP-VAChT positive organelles in varicosities are localized close to plasma membrane and are labeled with FM4-64 and GFP-Rab5, markers of endocytic vesicles and early endosomes, respectively. A GFP-VAChT mutant lacking a dileucine endocytosis motif (leucine residues 485 and 486 changed to alanine residues) accumulated at the plasma membrane in SN56 cells. This endocytosis-defective GFP-VAChT mutant is localized primarily at the somal plasma membrane and exhibits reduced neuritic targeting. Furthermore, the VAChT mutant did not accumulate in varicosities, as did VAChT. Our data suggest that clathrin-mediated internalization of VAChT to endosomes at the cell body might be involved in proper sorting and trafficking of VAChT to varicosities. We conclude that genesis of competent cholinergic secretory vesicles depends on multiple interactions of VAChT with endocytic proteins.     

Lipid requirements for entry of protein toxins into cells

The plant toxin ricin and the bacterial toxin Shiga toxin both belong to a group of protein toxins having one moiety that binds to the cell surface, and another, enzymatically active moiety, that enters the cytosol and inhibits protein synthesis by inactivating ribosomes. Both toxins travel all the way from the cell surface to endosomes, the Golgi apparatus and the ER before the ribosome-inactivating moiety enters the cytosol. Shiga toxin binds to the neutral glycosphingolipid Gb3 at the cell surface and is therefore dependent on this lipid for transport into the cells, whereas ricin binds both glycoproteins and glycolipids with terminal galactose. The different steps of transport used by these toxins have specific requirements for lipid species, and with the recent developments in mass spectrometry analysis of lipids and microscopical and biochemical dissection of transport in cells, we are starting to see the complexity of endocytosis and intracellular transport. In this article we describe lipid requirements and the consequences of lipid changes for the entry and intoxication with ricin and Shiga toxin. These toxins can be a threat to human health, but can also be exploited for diagnosis and therapy, and have proven valuable as tools to study intracellular transport.     

A deubiquitinating enzyme UBPY regulates the level of protein ubiquitination on endosomes

Monoubiquitination of endocytosed cell surface receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. The sorting of ubiquitinated proteins is executed by concerted actions of class E vacuolar protein sorting (Vps) proteins. Some proteins in the sorting machinery undergo monoubiquitination, suggesting that their functions are also regulated by ubiquitination. The Hrs-STAM complex, a class E Vps protein complex essential for the initial step of the sorting pathway, binds two deubiquitinating enzymes, UBPY and AMSH. Here we examined the effects of inactivating UBPY on protein ubiquitination at endosomes. Overexpression of a catalytically inactive UBPY mutant or depletion of UBPY by RNA interference resulted in the accumulation of ubiquitinated proteins on morphologically aberrant endosomes. Electron microscopy showed that they are aggregates of multivesicular endosomes. Among the sorting machinery proteins that undergo ubiquitination, Eps15 was monoubiquitinated at an elevated level in UBPY-inactivated cells. UBPY also deubiquitinated Eps15 in vitro, suggesting that Eps15 is a cellular substrate for UBPY. Furthermore, inactivation of UBPY caused the accumulation of Eps15 on the endosomal aggregates. These results suggest that UBPY regulates the level of protein ubiquitination on endosomes, which is required for maintaining the morphology of the organelle.     

SweetBac: a new approach for the production of mammalianised glycoproteins in insect cells

Recombinant production of therapeutically active proteins has become a central focus of contemporary life science research. These proteins are often produced in mammalian cells, in order to obtain products with post-translational modifications similar to their natural counterparts. However, in cases where a fast and flexible system for recombinant production of proteins is needed, the use of mammalian cells is limited. The baculoviral insect cell system has proven to be a powerful alternative for the expression of a wide range of recombinant proteins in short time frames. The major drawback of baculoviral systems lies in the inability to perform mammalian-like glycosylation required for the production of therapeutic glycoproteins. In this study we integrated sequences encoding Caenorhabditis elegans N-acetylglucosaminyltransferase II and bovine β1,4-galactosyltransferase I into the backbone of a baculovirus genome. The thereby generated SweetBac virus was subsequently used for the production of the human HIV anti-gp41 antibody 3D6 by integrating heavy and light chain open reading frames into the SweetBac genome. The parallel expression of target genes and glycosyltransferases reduced the yield of secreted antibody. However, the overall expression rate, especially in the recently established Tnao38 cell line, was comparable to that of transient expression in mammalian cells. In order to evaluate the ability of SweetBac to generate mammalian-like N-glycan structures on 3D6 antibody, we performed SDS-PAGE and tested for the presence of terminal galactose using Riccinus communis agglutinin I. The mammalianised variants of 3D6 showed highly specific binding to the lectin, indicating proper functionality. To confirm these results, PNGase A released N-glycans were analyzed by MALDI-TOF-MS and shown to contain structures with mainly one or two terminal galactose residues. Since the presence of specific N-glycans has an impact on antibodies ability to exert different effector functions, we tested the binding to human Fc gamma receptor I present on U937 cells.     

Galactose-rich glycoproteins are on the cell surface of herpes virus-infected cells. 1. Surface labeling and serial lectin binding studies of Asn-linked oligosaccharides of glycoprotein gC

Cell-surface glycoproteins of mock-infected and herpes simplex virus type 1 (HSV-1)-infected BHK-21 and HEp-2 cells were radiolabeled by incubation with galactose oxidase followed by reduction with NaB3H4. The incorporation of radiolabel into glycoconjugates in both BHK-21 and HEp-2 cells was increased several fold following infection with HSV, showing an increase in surface-exposed Gal residues in the infected cells. This was further confirmed by an increase in binding of cell-surface-labeled glycoproteins gC and gB from HSV-infected BHK-21 cells to Ricinus communis agglutinin I, which is specific for beta-D-Gal residues. Prior treatment of cells with Clostridium perfringens neuraminidase enhanced the surface radiolabeling by the galactose oxidase/NaB3H4 method: HEp-2 cells exhibited over sixfold enhancement in labeling, while BHK-21 cells showed only a slight increase. HSV glycoprotein gC was the predominant cell-surface glycoprotein radiolabeled by the galactose oxidase/NaB3H4 method in virus-infected BHK-21 cells. The glycoprotein gC was purified by immunoaffinity column chromatography on monoclonal anti-gC-antibody-Sepharose. The radiolabel in the glycopeptides of gC was resistant to beta elimination, showing that it was associated only with Asn-linked oligosaccharides. A serial lectin affinity chromatography of glycopeptides on columns of concanavalin A-Sepharose, lentil (Lens culinaris) lectin-Sepharose, and Ricin I-agarose allowed the assignment of minimal oligosaccharide structures bearing terminal Gal residues in gC.     

Differential use of two AP-3-mediated pathways by lysosomal membrane proteins

The adaptor protein complex AP-3 is involved in the sorting of lysosomal membrane proteins to late endosomes/lysosomes. It is unclear whether AP-3-containing vesicles form at the trans-Golgi network (TGN) or early endosomes. We have compared the trafficking routes of endolyn/CD164 and 'typical' lysosomal membrane glycoproteins (lgp120/lamp-1 and CD63/lamp-3) containing cytosolic YXXPhi-targeting motifs preceded by asparagine and glycine, respectively. Endolyn, which has a NYHTL-motif, is concentrated in lysosomes, but also occurs in endosomes and at the cell surface. We observed predominant interaction of the NYHTL-motif with the mu-subunits of AP-3 in the yeast two-hybrid system. Endolyn was mislocalized to the cell surface in AP-3-deficient pearl cells, confirming a major role of AP-3 in endolyn traffic. However, lysosomal delivery of endolyn (or a NYHTL-reporter), but not GYXXPhi-containing proteins, was practically abolished when AP-2-mediated endocytosis or traffic from early to late endosomes was inhibited in NRK and 3T3 cells. This indicates that endolyn is mostly transported along the indirect lysosomal pathway (via the cell surface), rather than directly from the TGN to late endosomes/lysosomes. Our results suggest that AP-3 mediates lysosomal sorting of some membrane proteins in early endosomes in addition to sorting of proteins with intrinsically strong AP-3-interacting lysosomal targeting motifs at the TGN.