An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes

In this paper, we show that beta COP is present on endosomes and is required for the formation of vesicles which mediate transport from early to late endosomes. Both the association of beta COP to endosomal membranes as well as transport vesicle formation depend on the lumenal pH. We find that epsilon COP, but not gamma COP, is also associated to endosomes, and that this association is also lumenal pH dependent. Our data, thus, indicate that a subset of COPs is part of the mechanism regulating endosomal membrane transport, and that membrane association of these COPs is controlled by the acidic properties of early endosomes, presumably via a trans-membrane pH sensor.     

Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments

The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.     

Functional Dissection of COP-I Subunits in the Biogenesis of Multivesicular Endosomes

In the present paper, we show that transport from early to late endosomes is inhibited at the restrictive temperature in a mutant CHO cell line (ldlF) with a ts-defect in ε coatomer protein (εCOP), although internalization and recycling continue. Early endosomes then appear like clusters of thin tubules devoid of the typical multivesicular regions, which are normally destined to become vesicular intermediates during transport to late endosomes. We also find that the in vitro formation of these vesicles from BHK donor endosomes is inhibited in cytosol prepared from ldlF cells incubated at the restrictive temperature. Although εCOP is rapidly degraded in ldlF cells at the restrictive temperature, cellular amounts of the other COP-I subunits are not affected. Despite the absence of εCOP, we find that a subcomplex of β, β′, and ζCOP is still recruited onto BHK endosomes in vitro, and this binding exhibits the characteristic properties of endosomal COPs with respect to stimulation by GTPγS and sensitivity to the endosomal pH. Previous studies showed that γ and δCOP are not found on endosomes. However, αCOP, which is normally present on endosomes, is no longer recruited when εCOP is missing. In contrast, all COP subunits, except obviously εCOP itself, still bind BHK biosynthetic membranes in a pH-independent manner in vitro. Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins. Our data also show that membrane association and function of endosomal COPs can be dissected: whereas β, β′, and ζCOP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of α and/or εCOP.     

Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis

Ubiquitin functions as a signal for sorting cargo at multiple steps of the endocytic pathway and controls the activity of trans-acting components of the endocytic machinery (reviewed in refs 1, and 2). By contrast to proteasome degradation, which generally requires a polyubiquitin chain that is at least four subunits long, internalization and sorting of endocytic cargo at the late endosome are mediated by mono-ubiquitination. Here, we demonstrate that ubiquitin-interacting motifs (UIMs) found in epsins and Vps27p (ref. 9) from Saccharomyces cerevisiae are required for ubiquitin binding and protein transport. Epsin UIMs are important for the internalization of receptors into vesicles at the plasma membrane. Vps27p UIMs are necessary to sort biosynthetic and endocytic cargo into vesicles that bud into the lumen of a late endosomal compartment, the multivesicular body. We propose that mono-ubiquitin regulates internalization and endosomal sorting by interacting with modular ubiquitin-binding domains in core components of the protein transport machinery. UIM domains are found in a broad spectrum of proteins, consistent with the idea that mono-ubiquitin can function as a regulatory signal to control diverse biological activities.     

ARF6 Targets Recycling Vesicles to the Plasma Membrane: Insights from an Ultrastructural Investigation

We have shown previously that the ADP- ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.     

Ncr1p, the yeast ortholog of mammalian Niemann Pick C1 protein, is dispensable for endocytic transport

The Niemann Pick C1 protein localizes to late endosomes and plays a key role in the intracellular transport of cholesterol in mammalian cells. Cholesterol and other lipids accumulate in a lysosomal or late endosomal compartment in cells lacking normal NPC1 function. Other than accumulation of lipids, defects in lysosomal retroendocytosis, sorting of a multifunctional receptor and endosomal movement have also been detected in NPC1 mutant cells. Ncr1p is an ortholog of NPC1 in the budding yeast Saccharomyces cerevisiae. In this study, we show that Ncr1p is a vacuolar membrane protein that transits through the biosynthetic vacuolar protein sorting pathway, and that it can be solubilized by Triton X-100 at 4 degrees C. Using well-established assays, we demonstrate that the absence of Ncr1p had no effect on fluid phase and receptor- mediated endocytosis, biosynthetic delivery to the vacuole, retrograde transport from endosome to Golgi and ubiquitin- and nonubiquitin-dependent multivesicular body sorting. We conclude that Ncr1p does not have an essential role in known endocytic transport pathways in yeast.     

ARF1 and ARF4 regulate recycling endosomal morphology and retrograde transport from endosomes to the Golgi apparatus

Small GTPases of the ADP-ribosylation factor (ARF) family, except for ARF6, mainly localize to the Golgi apparatus, where they trigger formation of coated carrier vesicles. We recently showed that class I ARFs (ARF1 and ARF3) localize to recycling endosomes, as well as to the Golgi, and are redundantly required for recycling of endocytosed transferrin. On the other hand, the roles of class II ARFs (ARF4 and ARF5) are not yet fully understood, and the complementary or overlapping functions of class I and class II ARFs have been poorly characterized. In this study, we find that simultaneous depletion of ARF1 and ARF4 induces extensive tubulation of recycling endosomes. Moreover, the depletion of ARF1 and ARF4 inhibits retrograde transport of TGN38 and mannose-6-phosphate receptor from early/recycling endosomes to the trans-Golgi network (TGN) but does not affect the endocytic/recycling pathway of transferrin receptor or inhibit retrograde transport of CD4-furin from late endosomes to the TGN. These observations indicate that the ARF1+ARF4 and ARF1+ARF3 pairs are both required for integrity of recycling endosomes but are involved in distinct transport pathways: the former pair regulates retrograde transport from endosomes to the TGN, whereas the latter is required for the transferrin recycling pathway from endosomes to the plasma membrane.     

Role of ARF4L in Recycling Between Endosomes and the Plasma Membrane

The human ADP-ribosylation factor-like protein, ARF4L is a member of the ARF family, which are small GTP-binding proteins that play significant roles in vesicle transport and protein secretion. However, little is known about the physiological roles of ARF4L. In this study, to understand the biological functions of ARF4L, we carried out immunocytochemical analysis of ARF4L molecules with mutations in the functional domains. ARF4L was shown to be distributed to the plasma membrane following binding to GTP (Q80L), and into endosomes following binding to GDP (T35N). Moreover, the inactive-form of ARF4L (T35N) causes localization of transferrin receptors to the endosomal compartment, while the active form (Q80L) causes transport to the plasma membrane. These findings indicate that ARF4L drive the transport of cargo protein and subsequent fusion of recycling vesicles with the plasma membrane for maintenance of the cell surface.     

Characterization of Coated Vesicles that Participate in Endocytic Recycling

While the recycling pathway of endocytosis has been shown to participate in many cellular functions, little is known regarding the transport carriers that mediate this pathway. In this study, we overexpressed a point mutant of ADP-ribosylation factor 6 (ARF6), that perturbs its GTPase cycle, to accumulate endosome-derived coated vesicles. Characterization by their purification revealed that, upon cell homogenization, these vesicles were mostly aggregated with larger noncoated membranes, and could be released with high-salt treatment. Equilibrium centrifugation revealed that these vesicles had buoyant density similar to the COP-coated vesicles. To purify the ARF6-regulated vesicles to homogeneity, enriched fractions from equilibrium centrifugation were subjected to immunoisolation through the hemagglutinin (HA) epitope of the mutant ARF6, by using a newly developed, high-affinity, anti-HA monoclonal antibody. Surface iodination of the purified vesicles revealed multiple prominent proteins. Immunoblotting with antibodies against subunits of the currently known coat proteins suggested that these vesicles have a novel coat complex. These vesicles are carriers for endocytic recycling, because they are enriched for transferrin receptor and also the v-SNARE cellubrevin that functions in transport from the recycling endosome to the plasma membrane. Thus, we have characterized transport vesicles that participate in endocytic recycling.     

Cooperation of phosphoinositides and BAR domain proteins in endosomal tubulation

Phosphorylated derivatives of phosphatidylinositol (PtdIns) regulate many intracellular events, including vesicular trafficking and actin remodeling, by recruiting proteins to their sites of function. PtdIns(4,5)-bisphosphate [PI(4,5)P2] and related phosphoinositides are mainly synthesized by type I PtdIns-4-phosphate 5-kinases (PIP5Ks). We found that PIP5K induces endosomal tubules in COS-7 cells. ADP-ribosylation factor (ARF) 6 has been shown to act upstream of PIP5K and regulate endocytic transport and tubulation. ARF GAP with coiled-coil, ankyrin repeat, and pleckstrin homology domains 1 (ACAP1) has guanosine triphosphatase-activating protein (GAP) activity for ARF6. While there were few tubules induced by the expression of ACAP1 alone, numerous endosomal tubules were induced by coexpression of PIP5K and ACAP1. ACAP1 has a pleckstrin homology (PH) domain known to bind phosphoinositide and a Bin/amphiphysin/Rvs (BAR) domain that has been reported to detect membrane curvature. Truncated and point mutations in the ACAP1 BAR and PH domains revealed that both BAR and PH domains are required for tubulation. These results suggest that two ARF6 downstream molecules, PIP5K and ACAP1, function together in endosomal tubulation and that phosphoinositide levels may regulate endosomal dynamics.     

Architecture of coatomer: molecular characterization of delta-COP and protein interactions within the complex

Coatomer is a cytosolic protein complex that forms the coat of COP I- coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.     

ATP binding regulates oligomerization and endosome association of RME-1 family proteins

Members of the RME-1/mRme-1/EHD1 protein family have recently been shown to function in the recycling of membrane proteins from recycling endosomes to the plasma membrane. RME-1 family proteins are normally found in close association with recycling endosomes and the vesicles and tubules emanating from these endosomes, consistent with the proposal that these proteins directly participate in endosomal transport. RME-1 family proteins contain a C-terminal EH (eps15 homology) domain thought to be involved in linking RME-1 to other endocytic proteins, a coiled-coil domain thought to be involved in homo-oligomerization and an N-terminal P-loop domain thought to mediate nucleotide binding. In the present study, we show that both Caenorhabditis elegans and mouse RME-1 proteins bind and hydrolyze ATP. No significant GTP binding or hydrolysis was detected. Mutation or deletion of the ATP-binding P-loop prevented RME-1 oligomerization and at the same time dissociated RME-1 from endosomes. In addition, ATP depletion caused RME-1 to lose its endosome association in the cell, resulting in cytosolic localization. Taken together, these results indicate that ATP binding is required for oligomerization of mRme-1/EHD1, which in turn is required for its association with endosomes.     

Sorting Out the Sorting Functions of Endosomes in Arabidopsis

In animals, sorting of membrane proteins following their internalization from the plasma membrane (PM) by endocytosis occurs through a series of different endosomal compartments. In plants, how and where these sorting events take place is still poorly understood and our current view of the endocytic pathway still largely relies on analogies made from the animal system. However, extensive differences seem to exist between animal and plant endosomal functions, as exemplified by the role of the trans-Golgi network (TGN) as an early endosomal compartment in plants or the functional diversification of conserved sorting complexes. By using the Arabidopsis root tip as a reference model, we and other have begun to shed light on the complexity of the plant endocytic pathways. Notably, we have recently characterized the functions of an endosomal compartment, the SNX1-endosomes, also referred to as the prevacuolar compartment (PVC) or multivesicular bodies (MVB), in the sorting of different cargo proteins, including two related auxin-efflux carriers, PIN1 and PIN2. We have shown that routing decisions take place at this endosomal level, such as the sorting of PIN2 toward the lytic vacuole for degradation or PIN1 toward the PM for recycling.Key Words: Arabidopsis, intracellular trafficking, endocytic recycling, endosomes, MVB, PVC, VPS29, SNX, PIN, cell polarity     

Regulation of the V-ATPase along the endocytic pathway occurs through reversible subunit association and membrane localization

The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptor or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase), a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The active V-ATPase is composed of two multi-subunit domains, the transmembrane V(0) and the cytoplasmic V(1). Here we found that the ratio of membrane associated V(1)/Vo varies along the endocytic pathway, the relative abundance of V(1) being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subunits were associated with detergent resistant membranes (DRM) isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cells with U18666A, a drug that leads to the accumulation of cholesterol in late endosomes, affected acidification of late endosome. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments.     

Annexin A2 binding to endosomes and functions in endosomal transport are regulated by tyrosine 23 phosphorylation

The phospholipid-binding annexin A2 (AnxA2) is known to play a role in the regulation of membrane and actin dynamics, in particular in the endocytic pathway. The protein is present on early endosomes, where it regulates membrane traffic, including the biogenesis of multivesicular transport intermediates destined for late endosomes. AnxA2 membrane association depends on the protein N terminus and membrane cholesterol but does not involve the AnxA2 ligand p11/S100A10. However, the precise mechanisms that control AnxA2 membrane association and function are not clear. In the present study, we have investigated the role of AnxA2 N-terminal phosphorylation in controlling association to endosomal membranes and functions. We found that endosomal AnxA2 was partially tyrosine-phosphorylated and that mutation of Tyr-23 to Ala (AnxA2Y23A), but not of Ser-25 to Ala, impaired AnxA2 endosome association. We then found that the AnxA2Y23A mutant was unable to bind endosomes in vivo, whereas a phospho-mimicking AnxA2 mutant (Y23D) showed efficient endosome binding capacity. Similarly, we found that AnxA2Y23D interacted more efficiently with liposomes in vitro when compared with AnxA2Y23A. To investigate the role of Tyr-23 in vivo, AnxA2 was knocked down with small interfering RNAs, and then cells were recomplemented with RNA interference-resistant forms of the protein. Using this strategy, we could show that AnxA2Y23D, but not AnxA2Y23A, could restore early-to-late endosome transport after AnxA2 depletion. We conclude that phosphorylation of Tyr-23 is essential for proper endosomal association and function of AnxA2, perhaps because it stabilizes membrane-associated protein via a conformational change.     

The chondroitin sulfate form of invariant chain trimerizes with conventional invariant chain and these complexes are rapidly transported from the trans-Golgi network to the cell surface

Targeting of MHCII-invariant chain complexes from the trans-Golgi network to endosomes is mediated by two di-leucine-based signals present in the cytosolic domain of invariant chain. Generation of this endosomal targeting signal is also dependent on multimerization of the invariant chain cytosolic domain sequences, mediated through assembly of invariant chain into homotrimers. A small subset of invariant chain is modified by the addition of chondroitin sulfate and is expressed on the cell surface in association with MHCII. In the present study, we have followed the biosynthetic pathway and route of intracellular transport of this proteoglycan form of invariant chain. We found that the efficiency of chondroitin sulfate modification can be increased by altering the invariant chain amino acid sequence around Ser-201 to the xylosylation consensus sequence. Our results also indicate that, following sulfation, the proteoglycan form is transported rapidly from the trans-Golgi network to the cell surface and is degraded following internalization into an endocytic compartment. Invariant chain-chondroitin sulfate is present in invariant chain trimers that also include conventional non-proteoglycan forms of invariant chain. These data indicate that invariant chain-chondroitin sulfate-containing complexes are transported rapidly from the trans-Golgi network to the cell surface in spite of the presence of an intact endosomal localization signal. Furthermore, these results suggest that invariant chain-chondroitin sulfate may play an important role in the generation of cell-surface pools of invariant chain that can serve as receptors for CD44 and macrophage migration inhibitory factor.     

Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes

BACKGROUND: In contrast to the intense attention devoted to research on intracellular sterol trafficking in animal cells, knowledge about sterol transport in plant cells remains limited, and virtually nothing is known about plant endocytic sterol trafficking. Similar to animals, biosynthetic sterol transport occurs from the endoplasmic reticulum (ER) via the Golgi apparatus to the plasma membrane. The vesicle trafficking inhibitor brefeldin A (BFA) has been suggested to disrupt biosynthetic sterol transport at the Golgi level. RESULTS: Here, we report on early endocytic sterol trafficking in Arabidopsis root epidermal cells by introducing filipin as a tool for fluorescent sterol detection. Sterols can be internalized from the plasma membrane and localize to endosomes positive for the early endosomal Rab5 GTPase homolog ARA6 fused to green fluorescent protein (GFP) (ARA6-GFP). Early endocytic sterol transport is actin dependent and highly BFA sensitive. BFA causes coaccumulation of sterols, endocytic markers like ARA6-GFP, and PIN2, a polarly localized presumptive auxin transport protein, in early endosome agglomerations that can be distinguished from ER and Golgi. Sterol accumulation in such aggregates is enhanced in actin2 mutants, and the actin-depolymerizing drug cytochalasin D inhibits sterol redistribution from endosome aggregations. CONCLUSIONS: Early endocytic sterol trafficking involves transport via ARA6-positive early endosomes that, in contrast to animal cells, is actin dependent. Our results reveal sterol-enriched early endosomes as targets for BFA interference in plants. Early endocytic sterol trafficking and recycling of polar PIN2 protein share a common pathway, suggesting a connection between plant endocytic sterol transport and polar sorting events.     

Lysosomal membrane protein composition, acidic pH and sterol content are regulated via a light-dependent pathway in metazoan cells

In metazoans, lysosomes are characterized by a unique tubular morphology, acidic pH, and specific membrane protein (LAMP) and lipid (cholesterol) composition as well as a soluble protein (hydrolases) composition. Here we show that perturbation to the eye-color gene, light, results in impaired lysosomal acidification, sterol accumulation, altered endosomal morphology as well as compromised lysosomal degradation. We find that Drosophila homologue of Vps41, Light, regulates the fusion of a specific subset of biosynthetic carriers containing characteristic endolysosomal membrane proteins, LAMP1, V0-ATPase and the cholesterol transport protein, NPC1, with the endolysosomal system, and is then required for the morphological progression of the multivesicular endosome. Inhibition of Light results in accumulation of biosynthetic transport intermediates that contain these membrane cargoes, whereas under similar conditions, endosomal delivery of soluble hydrolases, previously shown to be mediated by Dor, the Drosophila homologue of Vps18, is not affected. Unlike Dor, Light is recruited to endosomes in a PI3P-sensitive fashion wherein it facilitates fusion of these biosynthetic cargoes with the endosomes. Depletion of the mammalian counterpart of Light, hVps41, in a human cell line also inhibits delivery of hLAMP to endosomes, suggesting an evolutionarily conserved pathway in metazoa.     

A Highlights from MBoC Selection: EHBP-1 Functions with RAB-10 during Endocytic Recycling in Caenorhabditis elegans

Caenorhabditis elegans RAB-10 functions in endocytic recycling in polarized cells, regulating basolateral cargo transport in the intestinal epithelia and postsynaptic cargo transport in interneurons. A similar role was found for mammalian Rab10 in MDCK cells, suggesting that a conserved mechanism regulates these related pathways in metazoans. In a yeast two-hybrid screen for binding partners of RAB-10 we identified EHBP-1, a calponin homology domain (CH) protein, whose mammalian homolog Ehbp1 was previously shown to function during endocytic transport of GLUT4 in adipocytes. In vivo we find that EHBP-1-GFP colocalizes with RFP-RAB-10 on endosomal structures of the intestine and interneurons and that ehbp-1 loss-of-function mutants share with rab-10 mutants specific endosome morphology and cargo localization defects. We also show that loss of EHBP-1 disrupts transport of membrane proteins to the plasma membrane of the nonpolarized germline cells, a defect that can be phenocopied by codepletion of RAB-10 and its closest paralog RAB-8. These results indicate that RAB-10 and EHBP-1 function together in many cell types and suggests that there are differences in the level of redundancy among Rab family members in polarized versus nonpolarized cells.     

GPI anchoring leads to sphingolipid-dependent retention of endocytosed proteins in the recycling endosomal compartment

Glycosylphosphatidylinositol (GPI) anchoring is important for the function of several proteins in the context of their membrane trafficking pathways. We have shown previously that endocytosed GPI-anchored proteins (GPI-APs) are recycled to the plasma membrane three times more slowly than other membrane components. Recently, we found that GPI-APs are delivered to endocytic organelles, devoid of markers of the clathrin-mediated pathway, prior to their delivery to a common recycling endosomal compartment (REC). Here we show that the rate-limiting step in the recycling of GPI-APs is their slow exit from the REC; replacement of the GPI anchor with a transmembrane protein sequence abolishes retention in this compartment. Depletion of endogenous sphingolipid levels using sphingolipid synthesis inhibitors or in a sphingolipid-synthesis mutant cell line specifically enhances the rate of endocytic recycling of GPI-APs to that of other membrane components. We have shown previously that endocytic retention of GPI-APs is also relieved by cholesterol depletion. These findings strongly suggest that functional retention of GPI-APs in the REC occurs via their association with sphingolipid and cholesterol-enriched sorting platforms or 'rafts'.