An effector domain mutant of Arf6 implicates phospholipase D in endosomal membrane recycling

In this study, we investigated the role of phospholipase D (PLD) in mediating Arf6 function in cells. Expression of Arf6 mutants that are defective in activating PLD, Arf6N48R and Arf6N48I, inhibited membrane recycling to the plasma membrane (PM), resulting in an accumulation of tubular endosomal membranes. Additionally, unlike wild-type Arf6, neither Arf6 mutant could generate protrusions or recruit the Arf6 GTPase activating protein (GAP) ACAP1 onto the endosome in the presence of aluminum fluoride. Remarkably, all of these phenotypes, including accumulated tubular endosomes, blocked recycling, and failure to make protrusions and recruit ACAP effectively, could be recreated in either untransfected cells or cells expressing wild-type Arf6 by treatment with 1-butanol to inhibit the formation of phosphatidic acid (PA), the product of PLD. Moreover, most of the defects present in cells expressing Arf6N48R or N48I could be reversed by treatment with agents expected to elevate PA levels in cells. Together, these observations provide compelling evidence that Arf6 stimulation of PLD is required for endosomal membrane recycling and GAP recruitment.     

H-ras resides on clathrin-independent ARF6 vesicles that harbor little RAF-1, but not on clathrin-dependent endosomes

Internalization of H-Ras from the cell surface onto endomembranes through vesicular endocytic pathways may play a significant role(s) in regulating the outcome of Ras signaling. However, the identity of Ras-associated subcellular vesicles and the means by which Ras localize to these internal sites remain elusive. In this study, we show that H-Ras is absent from endosomes initially derived from a clathrin-dependent endocytic pathway. Instead, both oncogenic H-Ras-61L and wild type H-Ras (basal or EGF-stimulated) bind Arf6-associated clathrin-independent endosomes and vesicles of the endosomal-recycling center (ERC). K-Ras4B-12V can also be internalized via Arf6 endosomes, and the C-terminal tails of both H-Ras and K-Ras4B are sufficient to mediate localization of GFP chimeras to Arf6-associated vesicles. Interestingly, little Raf-1 was found on these Arf6-associated endosomes even when active H-Ras was present. Instead, endogenous Raf-1 distributed primarily on EEA1-containing vesicles, suggesting that this H-Ras effector, although accessible for H-Ras interaction on the plasma membrane, appears to separate from its regulator during early stages of endocytosis. The discrete and dynamic distribution of Ras pathway components with spatio-temporal complexity may contribute to the specificity of Ras:effector interaction.     

Convergence of non-clathrin- and clathrin-derived endosomes involves Arf6 inactivation and changes in phosphoinositides

The trafficking of two plasma membrane (PM) proteins that lack clathrin internalization sequences, major histocompatibility complex class I (MHCI), and interleukin 2 receptor alpha subunit (Tac) was compared with that of PM proteins internalized via clathrin. MHCI and Tac were internalized into endosomes that were distinct from those containing clathrin cargo. At later times, a fraction of these internalized membranes were observed in Arf6-associated, tubular recycling endosomes whereas another fraction acquired early endosomal autoantigen 1 (EEA1) before fusion with the "classical" early endosomes containing the clathrin-dependent cargo, LDL. After convergence, cargo molecules from both pathways eventually arrived, in a Rab7-dependent manner, at late endosomes and were degraded. Expression of a constitutively active mutant of Arf6, Q67L, caused MHCI and Tac to accumulate in enlarged PIP(2)-enriched vacuoles, devoid of EEA1 and inhibited their fusion with clathrin cargo-containing endosomes and hence blocked degradation. By contrast, trafficking and degradation of clathrin-cargo was not affected. A similar block in transport of MHCI and Tac was reversibly induced by a PI3-kinase inhibitor, implying that inactivation of Arf6 and acquisition of PI3P are required for convergence of endosomes arising from these two pathways.     

A unique platform for H-Ras signaling involving clathrin-independent endocytosis

Trafficking of H-Ras was examined to determine whether it can enter cells through clathrin-independent endocytosis (CIE). H-Ras colocalized with the CIE cargo protein, class I major histocompatibility complex, and it was sequestered in vacuoles that formed upon expression of an active mutant of Arf6, Q67L. Activation of Ras, either through epidermal growth factor stimulation or the expression of an active mutant of Ras, G12V, induced plasma membrane ruffling and macropinocytosis, a stimulated form of CIE. Live imaging of cells expressing H-RasG12V and fluorescent protein chimeras with pleckstrin homology domains that recognize specific phosphoinositides showed that incoming macropinosomes contained phosphatidylinositol 4,5-bisphosphate (PIP(2)) and phosphatiylinositol 3,4,5-trisphosphate (PIP(3)). PIP(2) loss from the macropinosome was followed by the recruitment of Rab5, a downstream target of Ras, and then PIP(3) loss. Our studies support a model whereby Ras can signal on macropinosomes that pass through three distinct stages: PIP(2)/PIP(3), PIP(3)/Rab5, and Rab5. Vacuoles that form in cells expressing Arf6Q67L trap Ras signaling in the first stage, recruiting the active form of the Ras effectors extracellular signal-regulated kinase and protein kinase B (Akt) but not Rab5. Arf6 stimulation of macropinocytosis also involves passage through the distinct lipid phases, but recruitment of Akt is not observed.     

Intra-endosomal pH-sensitive recruitment of the Arf-nucleotide exchange factor ARNO and Arf6 from cytoplasm to proximal tubule endosomes

Kidney proximal tubule epithelial cells have an extensive apical endocytotic apparatus that is critical for the reabsorption and degradation of proteins that traverse the glomerular filtration barrier and that is also involved in the extensive recycling of functionally important apical plasma membrane transporters. We show here that an Arf-nucleotide exchange factor, ARNO (ADP-ribosylation factor nucleotide site opener) as well as Arf6 and Arf1 small GTPases are located in the kidney proximal tubule receptor-mediated endocytosis pathway, and that ARNO and Arf6 recruitment from cytosol to endosomes is pH-dependent. In proximal tubules in situ, ARNO and Arf6 partially co-localized with the V-ATPase in apical endosomes in proximal tubules. Arf1 was localized both at the apical pole of proximal tubule epithelial cells, but also in the Golgi. By Western blot analysis ARNO, Arf6, and Arf1 were detected both in purified endosomes and in proximal tubule cytosol. A translocation assay showed that ATP-driven endosomal acidification triggered the recruitment of ARNO and Arf6 from proximal tubule cytosol to endosomal membranes. The translocation of both ARNO and Arf6 was reversed by V-type ATPase inhibitors and by uncouplers of endosomal intralumenal pH, and was correlated with the magnitude of intra-endosomal acidification. Our data suggest that V-type ATPase-dependent acidification stimulates the selective recruitment of ARNO and Arf6 to proximal tubule early endosomes. This mechanism may play an important role in the pH-dependent regulation of receptor-mediated endocytosis in proximal tubules in situ.     

Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic

ADP-ribosylation factor (Arf) 6 regulates the movement of membrane between the plasma membrane (PM) and a nonclathrin-derived endosomal compartment and activates phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show that PIP2 visualized by expressing a fusion protein of the pleckstrin homology domain from PLCdelta and green fluorescent protein (PH-GFP), colocalized with Arf6 at the PM and on tubular endosomal structures. Activation of Arf6 by expression of its exchange factor EFA6 stimulated protrusion formation, the uptake of PM into macropinosomes enriched in PIP2, and recycling of this membrane back to the PM. By contrast, expression of Arf6 Q67L, a GTP hydrolysis-resistant mutant, induced the formation of PIP2-positive actin-coated vacuoles that were unable to recycle membrane back to the PM. PM proteins, such as beta1-integrin, plakoglobin, and major histocompatibility complex class I, that normally traffic through the Arf6 endosomal compartment became trapped in this vacuolar compartment. Overexpression of human PIP 5-kinase alpha mimicked the effects seen with Arf6 Q67L. These results demonstrate that PIP 5-kinase activity and PIP2 turnover controlled by activation and inactivation of Arf6 is critical for trafficking through the Arf6 PM-endosomal recycling pathway.     

Arf6 regulates AP-1B-dependent sorting in polarized epithelial cells

The epithelial cell-specific clathrin adaptor complex AP-1B facilitates the sorting of various transmembrane proteins from recycling endosomes (REs) to the basolateral plasma membrane. Despite AP-1B's clear importance in polarized epithelial cells, we still do not fully understand how AP-1B orchestrates basolateral targeting. Here we identify the ADP-ribosylation factor 6 (Arf6) as an important regulator of AP-1B. We show that activated Arf6 pulled down AP-1B in vitro. Furthermore, interfering with Arf6 function through overexpression of dominant-active Arf6Q67L or dominant-negative Arf6D125N, as well as depletion of Arf6 with short hairpin RNA (shRNA), led to apical missorting of AP-1B-dependent cargos. In agreement with these data, we found that Arf6 colocalized with AP-1B and transferrin receptor (TfnR) in REs. In addition, we observed specific recruitment of AP-1B into Arf6-induced membrane ruffles in nonpolarized cells. We conclude that activated Arf6 directs membrane recruitment of AP-1B, thus regulating AP-1B's functions in polarized epithelial cells.     

Arf6 and microtubules in adhesion-dependent trafficking of lipid rafts

Integrin-mediated adhesion regulates membrane binding sites for Rac1 within lipid rafts. Detachment of cells from the substratum triggers the clearance of rafts from the plasma membrane through caveolin-dependent internalization. The small GTPase Arf6 and microtubules also regulate Rac-dependent cell spreading and migration, but the mechanisms are poorly understood. Here we show that endocytosis of rafts after detachment requires F-actin, followed by microtubule-dependent trafficking to recycling endosomes. When cells are replated on fibronectin, rafts exit from recycling endosomes in an Arf6-dependent manner and return to the plasma membrane along microtubules. Both of these steps are required for the plasma membrane targeting of Rac1 and for its activation. These data therefore define a new membrane raft trafficking pathway that is crucial for anchorage-dependent signalling.     

HIV-1 requires Arf6-mediated membrane dynamics to efficiently enter and infect T lymphocytes

As the initial barrier to viral entry, the plasma membrane along with the membrane trafficking machinery and cytoskeleton are of fundamental importance in the viral cycle. However, little is known about the contribution of plasma membrane dynamics during early human immunodeficiency virus type 1 (HIV-1) infection. Considering that ADP ribosylation factor 6 (Arf6) regulates cellular invasion via several microorganisms by coordinating membrane trafficking, our aim was to study the function of Arf6-mediated membrane dynamics on HIV-1 entry and infection of T lymphocytes. We observed that an alteration of the Arf6-guanosine 5'-diphosphate/guanosine 5'-triphosphate (GTP/GDP) cycle, by GDP-bound or GTP-bound inactive mutants or by specific Arf6 silencing, inhibited HIV-1 envelope-induced membrane fusion, entry, and infection of T lymphocytes and permissive cells, regardless of viral tropism. Furthermore, cell-to-cell HIV-1 transmission of primary human CD4(+) T lymphocytes was inhibited by Arf6 knockdown. Total internal reflection fluorescence microscopy showed that Arf6 mutants provoked the accumulation of phosphatidylinositol-(4,5)-biphosphate-associated structures on the plasma membrane of permissive cells, without affecting CD4-viral attachment but impeding CD4-dependent HIV-1 entry. Arf6 silencing or its mutants did not affect fusion, entry, and infection of vesicular stomatitis virus G-pseudotyped viruses or ligand-induced CXCR4 or CCR5 endocytosis, both clathrin-dependent processes. Therefore we propose that efficient early HIV-1 infection of CD4(+) T lymphocytes requires Arf6-coordinated plasma membrane dynamics that promote viral fusion and entry.     

MICAL-L1 is a tubular endosomal membrane hub that connects Rab35 and Arf6 with Rab8a

Endocytosis is a conserved process across species in which cell surface receptors and lipids are internalized from the plasma membrane. Once internalized, receptors can either be degraded or be recycled back to the plasma membrane. A variety of small GTP-binding proteins regulate receptor recycling. Despite our familiarity with many of the key regulatory proteins involved in this process, our understanding of the mode by which these proteins co-operate and the sequential manner in which they function remains limited. In this study, we identify two GTP-binding proteins as interaction partners of the endocytic regulatory protein molecule interacting with casl-like protein 1 (MICAL)-L1. First, we demonstrate that Rab35 is a MICAL-L1-binding partner in vivo. Over-expression of active Rab35 impairs the recruitment of MICAL-L1 to tubular recycling endosomes, whereas Rab35 depletion promotes enhanced MICAL-L1 localization to these structures. Moreover, we demonstrate that Arf6 forms a complex with MICAL-L1 and plays a role in its recruitment to tubular endosomes. Overall, our data suggest a model in which Rab35 is a critical upstream regulator of MICAL-L1 and Arf6, while both MICAL-L1 and Arf6 regulate Rab8a function.     

EFA6 facilitates the assembly of the tight junction by coordinating an Arf6-dependent and -independent pathway

We have previously reported that EFA6, exchange factor for Arf6, is implicated upon E-cadherin engagement in the process of epithelial cell polarization. We had found that EFA6 acts through stabilization of the apical actin ring onto which the tight junction is anchored. Mutagenesis experiments showed that both the catalytic domain of EFA6 and its C-terminal domain were required for full EFA6 function. Here we address the contribution of the specific substrate of EFA6, the small G protein Arf6. Unexpectedly, depletion of Arf6 by RNA interference or expression of the constitutively active fast-cycling mutant (Arf6T157N) revealed that Arf6 plays an opposing role to EFA6 by destabilizing the apical actin cytoskeleton and the associated tight junction. However, in complementation experiments, when the C-terminal domain of EFA6 is co-expressed with Arf6T157N, it reverts the effects of Arf6T157N expressed alone to faithfully mimic the phenotypes induced by EFA6. In addition, we find that the two signaling pathways downstream of EFA6, i.e. the one originating from the activated Arf6GTP and the other one from the EFA6 C-terminal domain, need to be tightly balanced to promote the proper reorganization of the actin cytoskeleton. Altogether, our results indicate that to regulate the tight junction, EFA6 activates Arf6 through its Sec7 catalytic domain as it modulates this activity through its C-terminal domain.     

Role of the Arf6 GDP/GTP cycle and Arf6 GTPase-activating proteins in actin remodeling and intracellular transport

We have analyzed both biochemically and functionally a series of Arf6 mutants, providing new insights into the molecular mode of action of the small G protein Arf6. First, by comparing a fast-cycling mutant (Arf6(T157N)) and a GTPase-deficient mutant (Arf6(Q67L)), we established the necessity for completion of the Arf6 GDP/GTP cycle for recycling of major histocompatibility complex molecules to the plasma membrane. Second, we found that aluminum fluoride (AlF), known for inducing membrane protrusion in cells expressing exogenous wild-type Arf6, stabilized a functional wild-type Arf6.AlF(x) . GTPase-activating protein (GAP) complex in vitro and in vivo. We also found that the tandem mutation Q37E/S38I prevented the binding of two Arf GAPs, but not the effector ARHGAP10, and blocked the formation of membrane protrusion and actin reorganization. Together, our results with AlF(x) and Arf6(Q37E/S38I) demonstrate the critical role of the Arf6 GAPs as effectors for Arf6-regulated actin cytoskeleton remodeling. Finally, competition experiments conducted in vivo suggest the existence of a membrane receptor for GDP-bound Arf6.     

Endocytosis of hERG Is Clathrin-Independent and Involves Arf6

The hERG potassium channel is critical for repolarisation of the cardiac action potential. Reduced expression of hERG at the plasma membrane, whether caused by hereditary mutations or drugs, results in long QT syndrome and increases the risk of ventricular arrhythmias. Thus, it is of fundamental importance to understand how the density of this channel at the plasma membrane is regulated. We used antibodies to an extracellular native or engineered epitope, in conjunction with immunofluorescence and ELISA, to investigate the mechanism of hERG endocytosis in recombinant cells and validated the findings in rat neonatal cardiac myocytes. The data reveal that this channel undergoes rapid internalisation, which is inhibited by neither dynasore, an inhibitor of dynamin, nor a dominant negative construct of Rab5a, into endosomes that are largely devoid of the transferrin receptor. These results support a clathrin-independent mechanism of endocytosis and exclude involvement of dynamin-dependent caveolin and RhoA mechanisms. In agreement, internalised hERG displayed marked overlap with glycosylphosphatidylinositol-anchored GFP, a clathrin-independent cargo. Endocytosis was significantly affected by cholesterol extraction with methyl-β-cyclodextrin and inhibition of Arf6 function with dominant negative Arf6-T27N-eGFP. Taken together, we conclude that hERG undergoes clathrin-independent endocytosis via a mechanism involving Arf6.     

The small G-protein Arf6GTP recruits the AP-2 adaptor complex to membranes

The small GTP-binding protein ADP-ribosylation factor 6 (Arf6) is involved in plasma membrane/endosomes trafficking. However, precisely how the activation of Arf6 regulates vesicular transport is still unclear. Here, we show that, in vitro, recombinant Arf6GTP recruits purified clathrin-adaptor complex AP-2 (but not AP-1) onto phospholipid liposomes in the absence of phosphoinositides. We also show that phosphoinositides and Arf6 tightly cooperate to translocate AP-2 to the membrane. In vivo, Arf6GTP (but not Arf6GDP) was found associated to AP-2. The expression of the GTP-locked mutant of Arf6 leads to the plasma membrane redistribution of AP-2 in Arf6GTP-enriched areas. Finally, we demonstrated that the expression of the GTP-locked mutant of Arf6 inhibits transferrin receptor internalization without affecting its recycling. Altogether, our results demonstrated that Arf6GTP interacts specifically with AP-2 and promotes its membrane recruitment. These findings strongly suggest that Arf6 plays a major role in clathrin-mediated endocytosis by directly controlling the assembly of the AP-2/clathrin coat.     

Arf6 promotes cell proliferation via the PLD-mTORC1 and p38MAPK pathways

The small G-protein ADP-ribosylation factor 6 (Arf6) belongs to the Ras GTPases superfamily and is mostly known for its actin remodeling functions and involvement in the processes of plasma membrane reorganization and vesicular transport. The majority of data indicates that Arf6 contributes to cancer progression through activation of cell motility and invasion. Alternatively, we found that the expression of a wild-type or a constitutively active Arf6 does not influence tumor cell motility and invasion but instead significantly stimulates cell proliferation and activates phospholipase D (PLD). Conversely the expression of a mutant Arf6 (Arf6N48I), that is, unable to interact with PLD has no effect on proliferation but promotes motility, invasion, and matrix degradation by uPA extracellular proteinase. Studying the mechanisms of Arf6-dependent stimulation of cell proliferation, we found some signaling pathways contributing to Arf6 promitogenic activity. Namely, we showed that Arf6 in a PLD-mTORC1-dependent manner activates S6K1 kinase, a well-known regulator of mitogen-stimulated translation initiation. Furthermore, we demonstrated an Arf6-dependent phosphorylation of mTORC1 downstream targets, 4E-BP1 and ribosomal S6 protein, confirming an existence of Arf6-PLD-mTORC1-S6K1/4E-BP1 signaling pathway and also demonstrated its impact on proliferation stimulation. Next, we found that Arf6 activation potentiates Erk1/2 and p38MAP kinases phosphorylation. Surprisingly, p38 opposite to Erk1/2 significantly contributes to Arf6-dependent proliferation increase promoting S6 ribosomal protein phosphorylation at Ser235/236 residues. Therefore, we demonstrated Arf6 proliferation stimulating activity and revealed PLD-mTORC1 and p38MAP kinase as Arf6 partners mediating promitogenic activity. These results highlight a new aspect of Arf6 functioning in cancer cell biology.     

Arf6 Regulation of Gyrating‐Clathrin

‘Gyrating‐’ or ‘G’‐clathrin are coated endocytic structures located near peripheral sorting endosomes (SEs), which exhibit highly dynamic but localized movements when visualized by live‐cell microscopy. They have been implicated in recycling of transferrin from the sorting endosome directly to the cell surface, but there is no information about their formation or regulation. We show here that G‐clathrin comprise a minority of clathrin‐coated structures in the cell periphery and are brefeldin A (BFA)‐resistant. Arf6‐GTP substantially increases G‐clathrin levels, probably by lengthening coated bud lifetimes as suggested by photobleaching and photoactivation results, and an Arf6(Q67L)‐GTP mutant bearing an internal GFP tag can be directly visualized in G‐clathrin structures in live cells. Upon siRNA‐mediated depletion of Arf6 or expression of Arf6(T27N), G‐clathrin levels rise and are primarily Arf1‐dependent, yet still BFA‐resistant. However, BFA‐sensitive increased G‐clathrin levels are observed upon acute incubation with cytohesin inhibitor SecinH3, indicating a shift in GEF usage. Depletion of both Arf6 and Arf1 abolishes G‐clathrin, and results in partial inhibition of fast transferrin recycling consistent with the latter's participation in this pathway. Collectively, these results demonstrate that the dynamics of G‐clathrin primarily requires completion of the Arf6 guanine nucleotide cycle, but can be regulated by multiple Arf and GEF proteins, reflecting both overlapping mechanisms operative in their regulation and the complexity of processes involved in endosomal sorting.     

The structural GDP/GTP cycle of human Arf6

The small GTP-binding protein Arf6 coordinates membrane traffic at the plasma membrane with aspects of cytoskeleton organization. This function does not overlap with that of other members of the ADP-ribosylation factor (Arf) family, although their switch regions, which are their major sites of interaction with regulators and effectors, have virtually identical sequences. Here we report the crystal structure of full-length, non-myristoylated human Arf6 bound to GTPγS. Unlike their GDP-bound forms, the active forms of Arf6 and Arf1 are very similar. Thus, the switch regions are discriminatory elements between Arf isoforms in their inactive but not in their active forms, a property that may generalize to other families of small G proteins. This suggests that GTP-bound Arfs may establish specific interactions outside the switch regions and/or be recognized in their cellular context rather than as isolated proteins. The structure also allows further insight into the lack of spontaneous GTPase activity of Arf proteins.     

NMR structural studies of the myristoylated N-terminus of ADP ribosylation factor 6 (Arf6)

Arf proteins are guanine nucleotide binding proteins that are implicated in endocytotic pathways and vesicle trafficking. The two widely studied isoforms of Arf proteins (Arf1 and Arf6) have different cellular functions and localizations but similar structures. Arf proteins have an N-terminal helix with a covalently bound myristoyl group. Except structural models, there are no three dimensional structures of the myristoylated N-terminal peptide or the intact myristoylated Arf proteins. However, understanding the role of both the myristoyl group and the N-terminal helix based on the details of their molecular structures is of great interest. In the solution structure of myristoylated N-terminal peptide of Arf6 described here, the myristoyl group folds toward the N-terminus to interact with the hydrophobic residues in particular, the phenyl ring. Also, the structure of the dodecylphosphocholine (DPC) micelle-bound of the peptide together with paramagnetic studies showed that the myristoyl group is inserted into the micelle while residues V4-G10 interact with the surface of the micelle. The structural differences between the unbound and micelle-bound myristoylated N-terminal peptide of Arf6 involves the myristoyl group and the side chains of the hydrophobic residues.     

Internalization of exogenous ADP-ribosylation factor 6 (Arf6) proteins into cells

Endogenous Arf6 is a myristoylated protein mainly involved in endosomal membrane traffic and structural organization at the plasma membrane. It has been shown that Arf6 mediates cancer cell invasion and shedding of plasma membrane microvesicles derived from tumor cells. In this article, we determined that Arf6 proteins both in the GDP and GTPγS bound forms can enter cells when simply added in the cell culture medium without requiring the myristoyl group. The GTPγS bound can enter cells at a faster rate than the GDP-bound Arf6. Despite the role of the endogenous Arf6 in endocytosis and membrane trafficking, the internalization of exogenous Arf6 may involve non-endocytic processes. As protein therapeutics is becoming important in medicine, we examined the effect of the uptake of Arf6 proteins on cellular functions and determined that exogenous Arf6 inhibits proliferation, invasion, and migration of cells. Future studies of the internalization of Arf6 mutants will reveal key residues that play a role in the internalization of Arf6 and its interaction and possible structural conformations bound to the plasma membrane.     

Characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements

Clathrin-independent endocytosis internalizes plasma membrane proteins that lack cytoplasmic sequences recognized by clathrin adaptor proteins. There is evidence for different clathrin-independent pathways but whether they share common features has not been systematically tested. Here, we examined whether CD59, an endogenous glycosylphosphatidyl inositol-anchored protein (GPI-AP), and major histocompatibility protein class I (MHCI), an endogenous, integral membrane protein, entered cells through a common mechanism and followed a similar itinerary. At early times of internalization, CD59 and MHCI were found in the same Arf6-associated endosomes before joining clathrin cargo proteins such as transferrin in common sorting endosomes. CD59 and MHCI, but not transferrin, also were observed in the Arf6-associated tubular recycling membranes. Endocytosis of CD59 and MHCI required free membrane cholesterol because it was inhibited by filipin binding to the cell surface. Expression of active Arf6 stimulated endocytosis of GPI-APs and MHCI to the same extent and led to their accumulation in Arf6 endosomes that labeled intensely with filipin. This blocked delivery of GPI-APs and MHCI to early sorting endosomes and to lysosomes for degradation. Endocytosis of transferrin was not affected by any of these treatments. These observations suggest common mechanisms for endocytosis without clathrin.