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 PLC � 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 �1-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 � 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. Downloaded from jcb.rupress.org

We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway. INTRODUCTION Receptor-mediated endocytosis involves sequential passage through distinct endosomal compartments. Internalized molecules first arrive in early endosomes, where a mildly acidic luminal pH favors uncoupling of ligands and receptors The luminal pH decreases by more than a pH unit along the degradative pathway but was shown to increase along the recycling pathway in nonpolarized Chinese hamster ovary (CHO) cells . Whereas endosomes along the degradative pathway contain numerous internal membranes, recycling endosomes consist of networks of 60-nm tubules organized around the microtubule-organizing center in some cell types. Early endosomes, which are common to both pathways, exhibit a complex cisternal, tubular, and vesicular organization. Although clear morphological differences can be observed between organelles on the two legs of the endocytic pathway, the molecular basis and the functional significance of these differences are not understood. Segregation of ligands destined for degradation or recycling takes place with rapid kinetics (half-life Ͻ 3 min) in the early endosome unige.ch. © 2000 by The American Society for Cell Biology 2775 degradative pathway depends on the acidic pH of the early endosome To better characterize recycling endosomes in polarized MDCK cells at the molecular level, we have established a subcellular fractionation protocol to specifically isolate the compartment. We show that the protein composition of recycling endosomes is distinct from that of sorting endosomes, providing biochemical evidence that the two organelles are physically separated from each other. Our data show that the luminal pH of recycling endosomes in polarized cells is less acidic than that of sorting endosomes and suggest that this pH difference is due to the absence of functional vacuolar ATPase in recycling endosomes. Last, we show that lipids and proteins generally believed to associate into membrane microdomains are highly enriched in recycling endosomes, probably creating a unique lipid environment within recycling endosomes. We propose that this environment may contribute to protein and lipid sorting along the recycling and/or transcytotic pathways. MATERIALS AND METHODS Reagents M450 sheep anti-mouse Dynabeads were from Dynal (Oslo, Norway). Fluorescent probes were from Molecular Probes Europe (Leiden, The Netherlands). Human holo-Tf conjugated with HRP, apoTf, HRP, filipin, and fish skin gelatin were from Sigma (Division of Fluka Chemie, Buchs, Switzerland). Immobilized pH gradient strips were from Amersham Pharmacia Biotech (Dü bendorf, Switzerland). All chemical reagents were from Fluka Chemie or Merck (Dietikon, Switzerland). Tissue culture media were from Life Technologies (Basel, Switzerland). Antibodies The anti-myc hybridoma cell line (MYC1-9E10.2; CRL1729) was obtained from the American Type Culture Collection (Rockville, MD). Antibodies against the luminal (B3/25) and cytoplasmic (H68.4) domains of the human TfR were purchased from Boehringer Mannheim (Rotkreuz, Switzerland) and Zymed Laboratories (San Francisco, CA), respectively. Anti-ZO1 and anti-caveolin-1 antibodies were purchased from Chemicon International (Temecula, CA) and Transduction Laboratories (Basel, Switzerland), respectively. Antibodies against ␤1 and ␤2 adaptins were purchased from Sigma. Anti-peptide antibodies against p23, Rab4, and Rab7 were raised in rabbits and affinity purified as described Cells MDCK II cells were stably transfected with the pCB6 plasmid containing the human TfR with (m-hTfR cells) or without (hTfR cells) a single myc tag at its cytoplasmic N terminus. Transfection was carried out by the calcium phosphate method and was followed by selection with G418 (Life Technologies). To avoid loss of expression, cells were not used for more than 8 -10 passages. Cells were maintained as described . Unless indicated otherwise, cells were seeded at high confluence onto prewet polycarbonate filters (Corning Costar Europe, Badhoevedorp, The Netherlands) and used after 4 d in culture with daily medium changes. R. Gagescu et al. Molecular Biology of the Cell 2776 Labeling Conditions To label the plasma membrane, the basolateral side of the cells was incubated on ice with 50 g/ml Tf-HRP in internalization medium (IM) (of G-MEM, 10 mM Hepes pH 7.4, 5 mM glucose) containing 2 mg/ml BSA (IM/BSA). Unbound label was removed by two washes with ice-cold PBSϩ/BSA (5 mg/ml BSA, 1 mM CaCl 2 , 1 mM MgCl 2 ). To label recycling endosomes, Tf conjugates (HRP or rhodamine; 50 g/ml) were prebound on ice and then internalized at 37°C for 10 min in IM/BSA. Residual plasma membrane label was removed by an ice-cold deferoxamine mesylate wash as described Fluorescence For fluorescence experiments, cells were grown on either glass coverslips or 12-mm Transclear Costar filters. When appropriate, filter-grown cells were labeled with lysine-fixable endocytic tracers before fixation. In this case, ice-cold 3% paraformaldehyde (PFA) was added to the cells and fixation was completed after 20 min at room temperature. For immunofluorescence experiments, cells were fixed with either 3% PFA for 20 min at room temperature or with methyl alcohol for 4 min at Ϫ20°C. PFA autofluorescence was quenched with 50 mM NH 4 Cl, and the cells were permeabilized with 0.05% saponin during incubation with the primary antibody. Fish skin gelatin (0.2%) was used to block unspecific binding. Cholesterol was labeled with filipin (50 g/ml) after PFA fixation as described Immunoelectron Microscopy MDCK cells were perforated and labeled exactly as described previously Subcellular Fractionation All experiments were carried out with m-hTfR and hTfR cells in parallel. Cells grown on 75-mm Costar filters were scraped with a rubber policeman, and postnuclear supernatants were prepared as described pH Measurements Subconfluent m-hTfR cells, grown on glass coverslips, were labeled either by preincubation with Tf-FITC (50 g/ml) at 4°C followed by internalization at room temperature for 5 min or by continuous internalization with Tf-FITC (25 g/ml) for 20 min at 37°C. Endosomal pH was measured by ratio fluorescence imaging as described Lipid Analysis Cells were metabolically labeled overnight with [ 32 P]orthophosphate (0.5 mCi/filter; Amersham Pharmacia Biotech) or [ 14 C]acetate (50 Ci/filter; Amersham Pharmacia Biotech), and purified endosomal fractions were prepared. Lipid extraction was carried out as described Electrophoresis Bidimensional electrophoresis was carried out as described Recycling Endosomes in MDCK Cells Vol. 11, August 2000 2777 R. Gagescu et al. Molecular Biology of the Cell 2778 RESULTS Characterization of TfR-expressing MDCK Cell Lines To characterize endosomes along the TfR recycling pathway in polarized MDCK cells, we decided to use an immunoaffinity purification protocol. We generated stable cell lines expressing human TfR, myc-tagged at the N-terminal cytoplasmic domain (m-hTfR), to allow isolation of endosomes containing m-hTfR with the anti-myc mAb 9E10 on magnetic beads coated with anti-mouse antibodies. As a control, we also prepared stable cell lines expressing untagged human TfR (hTfR), and we carried out all experiments in parallel with cells expressing comparable levels of hTfR or m-hTfR ( 35 S]Met, and samples were analyzed by high-resolution two-dimensional gel electrophoresis followed by autoradiography. The general protein pattern of the immunoisolated fraction [Bound m-hTfR (specific)] was compared with that of the unbound material sedimented at 150,000 ϫ g for 30 min [Unbound m-hTfR] to determine which proteins were specifically immunoisolated (examples are indicated with red arrowheads). The asterisk indicates the position of flotillin-1 in the same gel system. Examples of proteins that were not isolated are indicated with black arrows. Contaminants (blue arrowheads) were determined by comparison with samples from hTfR cells [Bound hTfR (unspecific)]. The position of actin, which is commonly used for orientation in this type of analysis, is indicated on the unbound m-hTfR gel. Recycling Endosomes in MDCK Cells Vol. 11, August 2000 2779 of immunoisolation. This proved necessary, because optimization tests revealed that beads without antibodies or beads coated with irrelevant antibodies, which have been commonly used by us and others as negative controls, frequently underestimate nonspecific binding to the beads, which largely depends on the quality of the antibody preparation. Both cell lines showed the characteristic features of polarized MDCK cells. Like parental cells, cells expressing hTfR or m-hTfR reached a height of ϳ12 m, and their transepithelial resistance was Ͼ200 ⍀/cm 2 . The normal polarization of the cells was further confirmed by the typical distribution of ZO1, a marker of tight junctions Isolation of TfR-positive Endosomes For immunoisolation experiments, postnuclear supernatants (PNSs) were prepared from m-hTfR and hTfR cells and incubated at 4°C with the anti-myc antibody. In a second step, PNSs from these cells were fractionated on a discontinuous sucrose gradient to remove the excess free antibody but also to prepare TfR-enriched fractions for subsequent immunoisolation. The bulk of total cellular TfR (65%) was recovered at the 20/35% interface of the gradient, where the receptor was enriched Ϸ10-fold Characterization of Purified TfR-positive Endosomes We first analyzed our TfR-positive endosomal fractions for their content in proteins reported to regulate Tf recycling. Rab11 is present on early endosomal membranes and the trans-Golgi network and has been proposed to function in Tf recycling Next, we analyzed the distribution of rab5, which is known to localize to the plasma membrane and early endosomes Acidification Properties of MDCK TfR-containing Recycling Endosomes Because endocytosed TfR is expected to transit through early (sorting) endosomes before appearing in recycling endosomes, the absence of EEA1 and annexin II in our fractions was somewhat unexpected. Therefore, we decided to make use of the pH-dependent fluorescence emission of Small arrows indicate organelles that contain only dextran-OG, and arrowheads indicate organelles that contain only Tf-rhodamine. (B) Cells expressing m-hTfR were allowed to endocytose HRP from the apical medium for 10 min (pulse) or for 15 min followed by a 30-min incubation in marker-free medium (chase). Endosomes containing m-hTfR were immunoisolated as in R. Gagescu et al. Molecular Biology of the Cell 2782 FITC to follow the pathway of Tf-FITC through acidic endosomes by fluorescence ratio imaging at the level of single organelles. Indeed, it is well established that early (sorting) endosomes are acidic, but recycling endosomes were reported to be less acidic in nonpolarized CHO cells . As shown in It is well established that the acidic pH of endosomes depends on the action of the vacuolar (H ϩ )-ATPase (V-ATPase), but the mechanisms regulating vacuolar acidification are not well understood The Apical Endocytic Pathway Intersects the Tf Recycling Pathway Although mixing of apically and basolaterally internalized fluid phase tracers cannot be observed in early endosomes Raft Components in Recycling Endosomes Next, we analyzed the lipid composition of recycling endosomes after metabolic labeling of m-hTfR cells with [ 32 P]orthophosphate or [ 14 C]acetate overnight. Recycling endosomes were prepared by immunoisolation as described above. Lipids were then extracted and analyzed by TLC on silica gel plates. As shown in Sphingomyelin and other sphingolipids can transiently associate with cholesterol, presumably forming liquid-ordered phase microdomains in the plane of the membrane (reviewed by DISCUSSION In this paper, we show that recycling endosomes in polarized MDCK cells are enriched in proteins that regulate the TfR cycle but do not contain molecules involved in the dynamics of early endosomes. These observations suggest that different molecular mechanisms regulate membrane traffic in sorting and recycling endosomes. We also show that recycling endosomes in MDCK cells are less acidic than early endosomes, because they lack functional V-ATPase. Consistent with observations that the apical endocytic pathway intersects the basolateral Tf recycling pathway, we show that recycling endosomes are enriched in raft components, which are known to be abundant at the apical plasma membrane. Together, these data support the notion that apically and basolaterally endocytosed molecules meet in a common recycling endosome and suggest that selective partitioning into membrane microdomains may contribute to protein sorting. Molecular Composition Purified recycling endosomes are enriched in proteins known to regulate Tf cycling, including the v-SNARE cellubrevin and the small GTPases rab4 and to some extent rab11, in agreement with Topology and Organization We find that transit of endocytosed Tf through early endosomes is extremely rapid, because Tf exits acidic endosomes in Ͻ3 min at 37°C. Then, endocytosed Tf first appears in recycling endosomes in the basolateral cytoplasm before reaching a compartment located in the supranuclear apical portion of the cell, presumably corresponding to the ARC Acidification Properties Our observations that recycling endosomes are less acidic than early endosomes might help to explain the barrier function of epithelial cells, because recycling endosomes intersect apical and basolateral uptake routes. Some receptor-ligand complexes may escape dissociation in early endosomes, transit being very rapid. If recycling endosomes were acidic, these complexes might dissociate and, like any solute, free ligand molecules would be regurgitated in a nonpolarized manner to both sides of the monolayer. Our observations, however, suggest that uncoupling is unlikely to occur in recycling endosomes and hence that receptorbound ligand molecules that escaped from early endosomes can be recycled and undergo a second round of internalization. Reduced acidification may thus contribute to limit the delivery of endocytosed ligands to the wrong side of the epithelium. The acidic pH of endosomes depends on the action of the V-ATPase (Stevens and Forgac, 1997). Several mechanisms have been proposed to regulate the activity of this pump, including reversible dissociation of the V1 and V0 domains

In this study, we have documented an essential role for ADP-ribosylation factor 6 (ARF6) in cell surface remodeling in response to physiological stimulus and in the down regulation of stress fiber formation. We demonstrate that the G-protein-coupled receptor agonist bombesin triggers the redistribution of ARF6-and Rac1-containing endosomal vesicles to the cell surface. This membrane redistribution was accompanied by cortical actin rearrangements and was inhibited by dominant negative ARF6, implying that bombesin is a physiological trigger of ARF6 activation. Furthermore, these studies provide a new model for bombesininduced Rac1 activation that involves ARF6-regulated endosomal recycling. The bombesin-elicited translocation of vesicular ARF6 was mimicked by activated G␣q and was partially inhibited by expression of RGS2, which down regulates Gq function. This suggests that Gq functions as an upstream regulator of ARF6 activation. The ARF6-induced peripheral cytoskeletal rearrangements were accompanied by a depletion of stress fibers. Moreover, cells expressing activated ARF6 resisted the formation of stress fibers induced by lysophosphatidic acid. We show that the ARF6-dependent inhibition of stress fiber formation was due to an inhibition of RhoA activation and was overcome by expression of a constitutively active RhoA mutant. The latter observations demonstrate that activation of ARF6 down regulates Rho signaling. Our findings underscore the potential roles of ARF6, Rac1, and RhoA in the coordinated regulation of cytoskeletal remodeling. The ADP-ribosylation factor (ARF) proteins comprise a group of five Ras-related GTPases that are thought to function as regulators of membrane traffic. In vitro, the ARF proteins function as cofactors in the cholera toxin-catalyzed ADP-ribosylation of Gs (21, 36), hence its name, and have also been shown to stimulate the activity of phospholipase D Immunoelectron microscopy observations in CHO cells have revealed that expression of the GTP-bound constitutively activated mutant of ARF6, ARF6(Q67L), induced an elaboration of the plasma membrane and a depletion of recycling endosomal vesicles. In contrast, the expression of the dominant negative mutant of ARF6, ARF6(T27N), resulted in sequestration of ARF6 in the perinuclear recycling endosome, the distribution of which partially overlapped with that of transferrin receptors and cellubrevin (10). These findings, together with the observation that ARF6(T27N) expression inhibited the recycling of ligands to the plasma membrane, led to the speculation that nucleotide exchange of ARF6 triggered the redistribution of endosomal membrane to the cell surface The Rho family GTPases regulate the assembly and organization of the actin cytoskeleton and have more recently also been implicated in the regulation of transcriptional activation, cell cycle progression, and cell transformation (15, 53). In fibroblasts, activation of Cdc42 and Rac results in the formation of filopodia and lamellipodia, respectively (23, 39, 48), whereas Rho activation induces the formation of stress fibers (49). These GTPases may also function in a hierarchical signaling cascade in which the activation of Cdc42 leads to the activation of Rac, which in turn activates Rho (38). We had previously shown that ARF6-mediated peripheral actin rearrangements were regulated by POR1, a Rac1-interacting protein that plays a role in Rac1-induced membrane ruffling (9, 53). As previously observed for Rac1, ARF6 in its GTP-bound conformation interacted with POR1 and deletion mutants of POR1 blocked ARF6-mediated cytoskeletal rearrangements. The dominant negative mutants of either GTPase, ARF6 or Rac1, did not interfere with actin rearrangements mediated by the other, which lead us to conclude that ARF6 and Rac1 functioned in parallel rather than on a linear signaling pathway. In addition to interacting with POR1, Rac1 and ARF6 have been implicated in regulated secretion in mast cells and adrenal chromaffin cells, respectively (13, 41), and they both inhibit receptor-mediated endocytosis

Abstract. Small GTP-binding proteins such as ADPribosylation factor 1 (ARF1) and Sar1p regulate the membrane association of coat proteins involved in intracellular membrane trafficking. ARF1 controls the clathrin coat adaptor AP-1 and the nonclathrin coat COPI, whereas Sar1p controls the nonclathrin coat COPII. In this study, we demonstrate that membrane association of the recently described AP-3 adaptor is regulated by ARF1. Association of AP-3 with membranes in vitro was enhanced by GTP�S and inhibited by brefeldin A (BFA), an inhibitor of ARF1 guanine nucleotide exchange. In addition, recombinant myristoylated ARF1 promoted association of AP-3 with membranes. The role of ARF1 in vivo was examined by assessing AP-3 subcellular localization when the intracellular

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The ADP-ribosylation factors (Arfs) are six proteins within the larger Arf family and Ras superfamily that regulate membrane traffic. Arfs all share numerous biochemical activities and have very similar specific activities. The use of dominant mutants and brefeldin A has been important to the discovery of the cellular functions of Arfs but lack specificity between Arf isoforms. We developed small interference RNA constructs capable of specific depletion of each of the cytoplasmic human Arfs to examine the specificity of Arfs in live cells. No single Arf was required for any step of membrane traffic examined in HeLa cells. However, every combination of the double knockdowns of Arf1, Arf3, Arf4, and Arf5 yielded a distinct pattern of defects in secretory and endocytic traffic, demonstrating clear specificity for Arfs at multiple steps. These results suggest that the cooperation of two Arfs at the same site may be a general feature of Arf signaling and provide candidates at several cellular locations that when paired with data on the localization of the many different Arf guanine nucleotide exchange factors, Arf GTPase activating proteins, and effectors will aid in the description of the mechanisms of specificity in this highly conserved and primordial family of regulatory GTPases.

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The eukaryotic family of ADP-ribosylation factor (Arf) GTPases plays a key role in the regulation of protein trafficking, and guanine-nucleotide exchange is crucial for Arf function. Exchange is stimulated by members of another family of proteins characterized by a 200-amino acid Sec7 domain, which alone is sufficient to catalyze exchange on Arf. Here, we analyzed the phylogeny of Sec7-domain–containing proteins in seven model organisms, representing fungi, plants, and animals. The phylogenetic tree has seven main groups, of which two include members from all seven model systems. Three groups are specific for animals, whereas two are specific for fungi. Based on this grouping, we propose a phylogenetically consistent set of names for members of the Sec7-domain family. Each group, except for one, contains proteins with known Arf exchange activity, implying that all members of this family have this activity. Contrary to the current convention, the sensitivity of Arf exchange activity to the inhibitor brefeldin A probably cannot be predicted by group membership. Multiple alignment reveals group-specific domains outside the Sec7 domain and a set of highly conserved amino acids within it. Determination of the importance of these conserved elements in Arf exchange activity and other cellular functions is now possible.

Abstract. The GTP-binding protein ADP-ribosylation factor 6 (Arf6) regulates endosomal membrane trafficking and the actin cytoskeleton in the cell periphery. GTPase-activating proteins (GAPs) are critical regulators of Arf function, controlling the return of Arf to the inactive GDP-bound state. Here, we report the identification and characterization of two Arf6 GAPs, ACAP1 and ACAP2. Together with two previously described Arf GAPs, ASAP1 and PAP, they can be grouped into a protein family defined by several common structural motifs including coiled coil, pleckstrin homology, Arf GAP, and three complete ankyrin-repeat domains. All contain phosphoinositide-dependent GAP activity. ACAP1 and ACAP2 are widely expressed and occur together in the various cultured cell lines we examined.

Cholera toxin (CT) and related AB 5 toxins bind to glycolipids at the plasma membrane and are then transported in a retrograde manner, first to the Golgi and then to the endoplasmic reticulum (ER). In the ER, the catalytic subunit of CT is translocated into the cytosol, resulting in toxicity. Using fluorescence microscopy, we found that CT is internalized by multiple endocytic pathways. Inhibition of the clathrin-, caveolin-, or Arf6-dependent pathways by overexpression of appropriate dominant mutants had no effect on retrograde traffic of CT to the Golgi and ER, and it did not affect CT toxicity. Unexpectedly, when we blocked all three endocytic pathways at once, although fluorescent CT in the Golgi and ER became undetectable, CT-induced toxicity was largely unaffected. These results are consistent with the existence of an additional retrograde pathway used by CT to reach the ER.