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Arf GTPases are central regulators of the Golgi complex, which serves as the nexus of membrane-trafficking pathways in eukaryotic cells. Arf proteins recruit dozens of effectors to modify membranes, sort cargos, and create and tether transport vesicles, and are therefore essential for orchestrating Golgi trafficking. The regulation of Arf activity is controlled by the action of Arf-GEFs which activate via nucleotide exchange, and Arf-GAPs which inactivate via nucleotide hydrolysis. The localization dynamics of Arf GTPases and their Arf-GAPs during Golgi maturation have not been reported. Here we use the budding yeast model to examine the temporal localization of the Golgi Arf-GAPs. We also determine the mechanisms used by the Arf-GAP Age2 to localize to the Golgi. We find that the catalytic activity of Age2 and a conserved sequence in the unstructured C-terminal domain of Age2 are both required for Golgi localization. This sequence is predicted to form an amphipathic helix and mediates direct binding of Age2 to membranes in vitro. We also report the development of a probe for sensing active Arf1 in living cells and use this probe to characterize the temporal dynamics of Arf1 during Golgi maturation.

The mechanistic target of rapamycin (mTOR) kinase regulates a major signaling pathway in eukaryotic cells. In addition to regulation of mTORC1 at lysosomes, mTORC1 is also localized at other locations. However, little is known about the recruitment and activation of mTORC1 at nonlysosomal sites. To identify regulators of mTORC1 recruitment to nonlysosomal compartments, novel interacting partners with the mTORC1 subunit, Raptor, were identified using immunoprecipitation and mass spectrometry. We show that one of the interacting partners, Arf5, is a novel regulator of mTORC1 signaling at plasma membrane ruffles. Arf5-GFP localizes with endogenous mTOR at PI3,4P2-enriched membrane ruffles together with the GTPase required for mTORC1 activation, Rheb. Knockdown of Arf5 reduced the recruitment of mTOR to membrane ruffles. The activation of mTORC1 at membrane ruffles was directly demonstrated using a plasma membrane-targeted mTORC1 biosensor, and Arf5 was shown to enhance the phosphorylation of the mTORC1 biosensor substrate. In addition, endogenous Arf5 was shown to be required for rapid activation of mTORC1-mediated S6 phosphorylation following nutrient starvation and refeeding. Our findings reveal a novel Arf5-dependent pathway for recruitment and activation of mTORC1 at plasma membrane ruffles, a process relevant for spatial and temporal regulation of mTORC1 by receptor and nutrient stimuli.

ELMODs are a family of three mammalian paralogues that display GTPase-activating protein (GAP) activity toward a uniquely broad array of ADP-ribosylation factor (ARF) family GTPases that includes ARF-like (ARL) proteins. ELMODs are ubiquitously expressed in mammalian tissues, highly conserved across eukaryotes, and ancient in origin, being present in the last eukaryotic common ancestor. We described functions of ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs) in the regulation of cell division, microtubules, ciliogenesis, and mitochondrial fusion. Here, using similar strategies with the paralogues ELMOD1 and ELMOD3, we identify novel functions and locations of these cell regulators and compare them to those of ELMOD2, allowing the determination of functional redundancy among the family members. We found strong similarities in phenotypes resulting from deletion of either Elmod1 or Elmod3 and marked differences from those arising in Elmod2 deletion lines. Deletion of either Elmod1 or Elmod3 results in the decreased ability of cells to form primary cilia, loss of a subset of proteins from cilia, and accumulation of some ciliary proteins at the Golgi, predicted to result from compromised traffic from the Golgi to cilia. These phenotypes are reversed upon activating mutant expression of either ARL3 or ARL16, linking their roles to ELMOD1/3 actions.

ELMOD2 is a GTPase-activating protein with uniquely broad specificity for ARF family GTPases. We previously showed that it acts with ARL2 in mitochondrial fusion and microtubule stability and with ARF6 during cytokinesis. Mouse embryonic fibroblasts deleted for ELMOD2 also displayed changes in cilia-related processes including increased ciliation, multiciliation, ciliary morphology, ciliary signaling, centrin accumulation inside cilia, and loss of rootlets at centrosomes with loss of centrosome cohesion. Increasing ARL2 activity or overexpressing Rootletin reversed these defects, revealing close functional links between the three proteins. This was further supported by the findings that deletion of Rootletin yielded similar phenotypes, which were rescued upon increasing ARL2 activity but not ELMOD2 overexpression. Thus, we propose that ARL2, ELMOD2, and Rootletin all act in a common pathway that suppresses spurious ciliation and maintains centrosome cohesion. Screening a number of markers of steps in the ciliation pathway supports a model in which ELMOD2, Rootletin, and ARL2 act downstream of TTBK2 and upstream of CP110 to prevent spurious release of CP110 and to regulate ciliary vesicle docking. These data thus provide evidence supporting roles for ELMOD2, Rootletin, and ARL2 in the regulation of ciliary licensing.

The components and subprocesses underlying the formation of COPI-coated vesicles at the Golgi are well understood. The coating cascade is initiated after the small GTPase Arf1 is activated by the Sec7 domain-containing guanine nucleotide exchange factor GBF1 (Golgi brefeldin A resistant guanine nucleotide exchange factor 1). This causes a conformational shift within Arf1 that facilitates stable association of Arf1 with the membrane, a process required for subsequent recruitment of the COPI coat. Although we have atomic-level knowledge of Arf1 activation by Sec7 domain-containing GEFs, our understanding of the biophysical processes regulating Arf1 and GBF1 dynamics is limited. We used fluorescence recovery after photobleaching data and kinetic Monte Carlo simulation to assess the behavior of Arf1 and GBF1 during COPI vesicle formation in live cells. Our analyses suggest that Arf1 and GBF1 associate with Golgi membranes independently, with an excess of GBF1 relative to Arf1. Furthermore, the GBF1-mediated Arf1 activation is much faster than GBF1 cycling on/off the membrane, suggesting that GBF1 is regulated by processes other than its interactions Arf1. Interestingly, modeling the behavior of the catalytically inactive GBF1/E794K mutant stabilized on the membrane is inconsistent with the formation of a stable complex between it and an endogenous Arf1 and suggests that GBF1/E794K is stabilized on the membrane independently of complex formation.

The action of guanine nucleotide exchange factors (GEFs) on the ADP-ribosylation factor (ARF) family of small GTPases initiates intracellular transport pathways. This role requires ARF GEFs to be recruited from the cytosol to intracellular membrane compartments. An ARF GEF known as General receptor for 3-phosphoinositides 1 (Grp1) is recruited to the plasma membrane through its pleckstrin homology (PH) domain that recognizes phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here, we find that the phosphorylation of Grp1 induces its PH domain to recognize instead phosphatidylinositol 4-phosphate (PI4P). This phosphorylation also releases an autoinhibitory mechanism that results in the coil-coil (CC) domain of Grp1 engaging two peripheral membrane proteins of the recycling endosome. Because the combination of these actions results in Grp1 being recruited preferentially to the recycling endosome rather than to the plasma membrane, our findings reveal the complexity of recruitment mechanisms that need to be coordinated in localizing an ARF GEF to an intracellular compartment to initiate a transport pathway. Our elucidation is also remarkable for having revealed that phosphoinositide recognition by a PH domain can be switched through its phosphorylation.

ADP-ribosylation factor (Arf)-like 4D (Arl4D), one of the Arf-like small GTPases, functions in the regulation of cell morphology, cell migration, and actin cytoskeleton remodeling. End-binding 1 (EB1) is a microtubule (MT) plus-end tracking protein that preferentially localizes at the tips of the plus ends of growing MTs and at the centrosome. EB1 depletion results in many centrosome-related defects. Here, we report that Arl4D promotes the recruitment of EB1 to the centrosome and regulates MT nucleation. We first showed that Arl4D interacts with EB1 in a GTP-dependent manner. This interaction is dependent on the C-terminal EB homology region of EB1 and partially dependent on an SxLP motif of Arl4D. We found that Arl4D colocalized with γ-tubulin in centrosomes and the depletion of Arl4D resulted in a centrosomal MT nucleation defect. We further demonstrated that abolishing Arl4D-EB1 interaction decreased MT nucleation rate and diminished the centrosomal recruitment of EB1 without affecting MT growth rate. In addition, Arl4D binding to EB1 increased the association between the p150 subunit of dynactin and the EB1, which is important for MT stabilization. Together, our results indicate that Arl4D modulates MT nucleation through regulation of the EB1-p150 association at the centrosome.

ELMOD2 is a ∼32 kDa protein first purified by its GTPase-activating protein (GAP) activity toward ARL2 and later shown to have uniquely broad specificity toward ARF family GTPases in in vitro assays. To begin the task of defining its functions in cells, we deleted ELMOD2 in immortalized mouse embryonic fibroblasts and discovered a number of cellular defects, which are reversed upon expression of ELMOD2-myc. We show that these defects, resulting from the loss of ELMOD2, are linked to two different pathways and two different GTPases: with ARL2 and TBCD to support microtubule nucleation from centrosomes and with ARF6 in cytokinesis. These data highlight key aspects of signaling by ARF family GAPs that contribute to previously underappreciated sources of complexity, including GAPs acting from multiple sites in cells, working with multiple GTPases, and contributing to the spatial and temporal control of regulatory GTPases by serving as both GAPs and effectors.

Reactive oxygen species (ROS)-induced oxidative stress has been associated with diseases such as amyotrophic lateral sclerosis, stroke, and cancer. While the effect of ROS on mitochondria and endoplasmic reticulum (ER) has been well documented, its consequence on the Golgi apparatus is less well understood. In this study, we characterized the Golgi structure and function in HeLa cells after exposure to hydrogen peroxide (H 2 O 2 ), a reagent commonly used to introduce ROS to cells. Treatment of cells with 1 mM H 2 O 2 for 10 min resulted in the degradation of Arl1 and dissociation of GRIP domain-containing proteins Golgin-97 and Golgin-245 from the trans -Golgi. This effect could be rescued by treatment of cells with a ROS scavenger N -acetyl cysteine or protease inhibitors. Structurally, H 2 O 2 treatment reduced the number of cisternal membranes per Golgi stack, suggesting a loss of trans -Golgi cisternae. Functionally, H 2 O 2 treatment inhibited both anterograde and retrograde protein transport, consistent with the loss of membrane tethers on the trans -Golgi cisternae. This study revealed membrane tethers at the trans -Golgi as novel specific targets of ROS in cells.

Guanine nucleotide exchange factors (GEFs) are the initiators of signaling by every regulatory GTPase, which in turn act to regulate a wide array of essential cellular processes. To date, each family of GTPases is activated by distinct families of GEFs. Bidirectional membrane trafficking is regulated by ADP-ribosylation factor (ARF) GTPases and the development throughout eukaryotic evolution of increasingly complex systems of such traffic required the acquisition of a functionally diverse cohort of ARF GEFs to control it. We performed phylogenetic analyses of ARF GEFs in eukaryotes, defined by the presence of the Sec7 domain, and found three subfamilies (BIG, GBF1, and cytohesins) to have been present in the ancestor of all eukaryotes. The four other subfamilies (EFA6/PSD, IQSEC7/BRAG, FBX8, and TBS) are opisthokont, holozoan, metazoan, and alveolate/haptophyte specific, respectively, and each is derived from cytohesins. We also identified a cytohesin-derived subfamily, termed ankyrin repeat-containing cytohesin, that independently evolved in amoebozoans and members of the SAR and haptophyte clades. Building on evolutionary data for the ARF family GTPases and their GTPase--activating proteins allowed the generation of hypotheses about ARF GEF protein function(s) as well as a better understanding of the origins and evolution of cellular complexity in eukaryotes.

The integrity of the Golgi and trans -Golgi network (TGN) is disrupted by brefeldin A (BFA), which inhibits the Golgi-localized BFA-sensitive factor (GBF1) and brefeldin A-inhibited guanine nucleotide-exchange factors (BIG1 and BIG2). Using a cellular replacement assay to assess GBF1 functionality without interference from the BIGs, we show that GBF1 alone maintains Golgi architecture; facilitates secretion; activates ADP-ribosylation factor (ARF)1, 3, 4, and 5; and recruits ARF effectors to Golgi membranes. Unexpectedly, GBF1 also supports TGN integrity and recruits numerous TGN-localized ARF effectors. The impact of the catalytic Sec7 domain (Sec7d) on GBF1 functionality was assessed by swapping it with the Sec7d from ARF nucleotide-binding site opener (ARNO)/cytohesin-2, a plasma membrane GEF reported to activate all ARFs. The resulting chimera (GBF1-ARNO-GBF1 [GARG]) targets like GBF1, supports Golgi/TGN architecture, and facilitates secretion. However, unlike GBF1, GARG activates all ARFs (including ARF6) at the Golgi/TGN and recruits additional ARF effectors to the Golgi/TGN. Our results have general implications: 1) GEF's targeting is independent of Sec7d, but Sec7d influence the GEF substrate specificity and downstream effector events; 2) all ARFs have access to all membranes, but are restricted in their distribution by the localization of their activating GEFs; and 3) effector association with membranes requires the coincidental presence of activated ARFs and specific membrane identifiers.

Macropinocytosis is an actin-driven form of clathrin-independent endocytosis that generates an enlarged structure, the macropinosome. Although many studies focus on signaling molecules and phosphoinositides involved in initiating macropinocytosis, the commitment to forming a macropinosome and the handling of that membrane have not been studied in detail. Here we show in HT1080 cells, a human fibrosarcoma cell line, a requirement for microtubules, dynein, the JIP3 microtubule motor scaffold protein, and Arf6, a JIP3 interacting protein, for the formation and inward movement of the macropinosome. While actin and myosin II also play critical roles in the formation of ruffling membrane, microtubules provide an important tract for initiation, sealing, and transport of the macropinosome through the actin- and myosin-rich lamellar region.

The Arf and Rab/Ypt GTPases coordinately regulate membrane traffic and organelle structure by regulating vesicle formation and fusion. Ample evidence has indicated that proteins in these two families may function in parallel or complementarily; however, the manner in which Arf and Rab/Ypt proteins perform interchangeable functions remains unclear. In this study, we report that a Golgi-localized Arf, Arl1, could suppress Ypt6 dysfunction via its effector golgin, Imh1, but not via the lipid flippase Drs2. Ypt6 is critical for the retrograde transport of vesicles from endosomes to the trans-Golgi network (TGN), and its mutation leads to severe protein mislocalization and growth defects. We first overexpress the components of the Arl3-Syt1-Arl1-Imh1 cascade and show that only Arl1 and Imh1 can restore endosome-to-TGN trafficking in ypt6-deleted cells. Interestingly, increased abundance of Arl1 or Imh1 restores localization of the tethering factor Golgi associated retrograde-protein (GARP) complex to the TGN in the absence of Ypt6. We further show that the N-terminal domain of Imh1 is critical for restoring GARP localization and endosome-to-TGN transport in ypt6-deleted cells. Together, our results reveal the mechanism by which Arl1-Imh1 facilitates the recruitment of GARP to the TGN and compensates for the endosome-to-TGN trafficking defects in dysfunctional Ypt6 conditions.

Cell migration is a highly regulated event that is initiated by cell membrane protrusion and actin reorganization. Robo1, a single-pass transmembrane receptor, is crucial for neuronal guidance and cell migration. ADP-ribosylation factor (Arf)-like 4A (Arl4A), an Arf small GTPase, functions in cell morphology, cell migration, and actin cytoskeleton remodeling; however, the molecular mechanisms of Arl4A in cell migration are unclear. Here, we report that the binding of Arl4A to Robo1 modulates cell migration by promoting Cdc42 activation. We found that Arl4A interacts with Robo1 in a GTP-dependent manner and that the Robo1 amino acid residues 1394-1398 are required for this interaction. The Arl4A-Robo1 interaction is essential for Arl4A-induced cell migration and Cdc42 activation but not for the plasma membrane localization of Robo1. In addition, we show that the binding of Arl4A to Robo1 decreases the association of Robo1 with the Cdc42 GTPase-activating protein srGAP1. Furthermore, Slit2/Robo1 binding down-regulates the Arl4A-Robo1 interaction in vivo, thus attenuating Cdc42-mediated cell migration. Therefore, our study reveals a novel mechanism by which Arl4A participates in Slit2/Robo1 signaling to modulate cell motility by regulating Cdc42 activity.

Hemidesmosomes (HDs) are epithelial-specific cell-matrix adhesions that stably anchor the intracellular keratin network to the extracellular matrix. Although their main role is to protect the epithelial sheet from external mechanical strain, how HDs respond to mechanical stress remains poorly understood. Here we identify a pathway essential for HD remodeling and outline its role with respect to α6β4 integrin recycling. We find that α6β4 integrin chains localize to the plasma membrane, caveolae, and ADP-ribosylation factor-6+ (Arf6+) endocytic compartments. Based on fluorescence recovery after photobleaching and endocytosis assays, integrin recycling between both sites requires the small GTPase Arf6 but neither caveolin1 (Cav1) nor Cavin1. Strikingly, when keratinocytes are stretched or hypo-osmotically shocked, α6β4 integrin accumulates at cell edges, whereas Cav1 disappears from it. This process, which is isotropic relative to the orientation of stretch, depends on Arf6, Cav1, and Cavin1. We propose that mechanically induced HD growth involves the isotropic flattening of caveolae (known for their mechanical buffering role) associated with integrin diffusion and turnover., (© 2018 Osmani et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

At the Golgi complex, the biosynthetic sorting center of the cell, the Arf GTPases are responsible for coordinating vesicle formation. The Arf-GEFs activate Arf GTPases and are therefore the key molecular decision-makers for trafficking from the Golgi. In Saccharomyces cerevisiae , three conserved Arf-GEFs function at the Golgi: Sec7, Gea1, and Gea2. Our group has described the regulation of Sec7, the trans -Golgi Arf-GEF, through autoinhibition, positive feedback, dimerization, and interactions with a suite of small GTPases. However, we lack a clear understanding of the regulation of the early Golgi Arf-GEFs Gea1 and Gea2. Here we demonstrate that Gea1 and Gea2 prefer neutral over anionic membrane surfaces in vitro, consistent with their localization to the early Golgi. We illustrate a requirement for a critical mass of either Gea1 or Gea2 for cell growth under stress conditions. We show that the C-terminal domains of Gea1 and Gea2 toggle roles in the cytosol and at the membrane surface, preventing membrane binding in the absence of a recruiting interaction but promoting maximum catalytic activity once recruited. We also identify the small GTPase Ypt1 as a recruiter for Gea1 and Gea2. Our findings illuminate core regulatory mechanisms unique to the early Golgi Arf-GEFs., (© 2017 Gustafson and Fromme. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Changes in cell morphology and the physical forces that occur during migration are generated by a dynamic filamentous actin cytoskeleton. The ADP-ribosylation factor-like 4C (Arl4C) small GTPase acts as a molecular switch to regulate morphological changes and cell migration, although the mechanism by which this occurs remains unclear. Here we report that Arl4C functions with the actin regulator filamin-A (FLNa) to modulate filopodium formation and cell migration. We found that Arl4C interacted with FLNa in a GTP-dependent manner and that FLNa IgG repeat 22 is both required and sufficient for this interaction. We also show that interaction between FLNa and Arl4C is essential for Arl4C-induced filopodium formation and increases the association of FLNa with Cdc42-GEF FGD6, promoting cell division cycle 42 (Cdc42) GTPase activation. Thus our study revealed a novel mechanism, whereby filopodium formation and cell migration are regulated through the Arl4C-FLNa-mediated activation of Cdc42., (© 2017 Chiang et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Soluble αβ-tubulin heterodimers are maintained at high concentration inside eukaryotic cells, forming pools that fundamentally drive microtubule dynamics. Five conserved tubulin cofactors and ADP ribosylation factor-like 2 regulate the biogenesis and degradation of αβ-tubulins to maintain concentrated soluble pools. Here I describe a revised model for the function of three tubulin cofactors and Arl2 as a multisubunit GTP-hydrolyzing catalytic chaperone that cycles to promote αβ-tubulin biogenesis and degradation. This model helps explain old and new data indicating these activities enhance microtubule dynamics in vivo via repair or removal of αβ-tubulins from the soluble pools., (© 2017 Al-Bassam. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Macrophages and dendritic cells exposed to lipopolysaccharide (LPS) convert their lysosomes from small, punctate organelles into a network of tubules. Tubular lysosomes have been implicated in phagosome maturation, retention of fluid phase, and antigen presentation. There is a growing appreciation that lysosomes act as sensors of stress and the metabolic state of the cell through the kinase mTOR. Here we show that LPS stimulates mTOR and that mTOR is required for LPS-induced lysosome tubulation and secretion of major histocompatibility complex II in macrophages and dendritic cells. Specifically, we show that the canonical phosphatidylinositol 3-kinase-Akt-mTOR signaling pathway regulates LPS-induced lysosome tubulation independently of IRAK1/4 and TBK. Of note, we find that LPS treatment augmented the levels of membrane-associated Arl8b, a lysosomal GTPase required for tubulation that promotes kinesin-dependent lysosome movement to the cell periphery, in an mTOR-dependent manner. This suggests that mTOR may interface with the Arl8b-kinesin machinery. To further support this notion, we show that mTOR antagonists can block outward movement of lysosomes in cells treated with acetate but have no effect in retrograde movement upon acetate removal. Overall our work provides tantalizing evidence that mTOR plays a role in controlling lysosome morphology and trafficking by modulating microtubule-based motor activity in leukocytes., (© 2016 Saric et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Arl13b belongs to the ADP-ribosylation factor family within the Ras superfamily of regulatory GTPases. Mutations in Arl13b cause Joubert syndrome, which is characterized by congenital cerebellar ataxia, hypotonia, oculomotor apraxia, and mental retardation. Arl13b is highly enriched in cilia and is required for ciliogenesis in multiple organs. Nevertheless, the precise role of Arl13b remains elusive. Here we report that the exocyst subunits Sec8, Exo70, and Sec5 bind preferentially to the GTP-bound form of Arl13b, consistent with the exocyst being an effector of Arl13b. Moreover, we show that Arl13b binds directly to Sec8 and Sec5. In zebrafish, depletion of arl13b or the exocyst subunit sec10 causes phenotypes characteristic of defective cilia, such as curly tail up, edema, and abnormal pronephric kidney development. We explored this further and found a synergistic genetic interaction between arl13b and sec10 morphants in cilia-dependent phenotypes. Through conditional deletion of Arl13b or Sec10 in mice, we found kidney cysts and decreased ciliogenesis in cells surrounding the cysts. Moreover, we observed a decrease in Arl13b expression in the kidneys from Sec10 conditional knockout mice. Taken together, our results indicate that Arl13b and the exocyst function together in the same pathway leading to functional cilia., (© 2016 Seixas et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Although TrkA postendocytic sorting significantly influences neuronal cell survival and differentiation, the molecular mechanism underlying TrkA receptor sorting in the recycling or degradation pathways remains poorly understood. Here we demonstrate that Golgi-localized, γ adaptin-ear-containing ADP ribosylation factor-binding protein 3 (GGA3) interacts directly with the TrkA cytoplasmic tail through an internal DXXLL motif and mediates the functional recycling of TrkA to the plasma membrane. We find that GGA3 depletion by siRNA delays TrkA recycling, accelerates TrkA degradation, attenuates sustained NGF-induced Akt activation, and reduces cell survival. We also show that GGA3's effect on TrkA recycling is dependent on the activation of Arf6. This work identifies GGA3 as a key player in a novel DXXLL-mediated endosomal sorting machinery that targets TrkA to the plasma membrane, where it prolongs the activation of Akt signaling and survival responses., (© 2015 Li et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Cby1, the mammalian orthologue of the Drosophila Chibby protein, localizes to mature centrioles, is important for ciliogenesis in multiciliated airway epithelia in mice, and antagonizes canonical Wnt signaling via direct regulation of β-catenin. We report that deletion of the mouse Cby1 gene results in cystic kidneys, a phenotype common to ciliopathies, and that Cby1 facilitates the formation of primary cilia and ciliary recruitment of the Joubert syndrome protein Arl13b. Localization of Cby1 to the distal end of mature centrioles depends on the centriole protein Ofd1. Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium. The amount of centriole-localized Ahi1, but not Ofd1, is reduced in Cby1(-/-) cells. This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1(-/-) cells., (© 2014 Lee et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Oligodendrocyte precursor cells differentiate to produce myelin sheaths that insulate axons to ensure fast propagation of action potentials. Many aspects of differentiation are regulated by multiple extracellular signals. However, their intracellular signalings remain elusive. We show that Rab35 and its effector, ACAP2, a GTPase-activating protein that switches off Arf6 activity, negatively regulate oligodendrocyte morphological differentiation. Knockdown of Rab35 or ACAP2 with their respective small interfering RNAs promotes differentiation. As differentiation initiates, the activities of Rab35 and ACAP2 are down-regulated. The activity of Arf6, in contrast, is up-regulated. Arf6 knockdown inhibits differentiation, indicating that Rab35 and ACAP2 negatively regulate differentiation by down-regulating Arf6. Importantly, as differentiation proceeds, the activity of cytohesin-2, a guanine nucleotide exchange factor that switches on Arf6 activity, is up-regulated. Pharmacological inhibition of cytohesin-2 inhibits differentiation, suggesting that cytohesin-2 promotes differentiation by activating Arf6. Furthermore, using oligodendrocyte-neuronal cocultures, we find that knockdown of Rab35 or ACAP2 promotes myelination, whereas inhibition of cytohesin-2 or knockdown of Arf6 inhibits myelination. Thus Rab35/ACAP2 and cytohesin-2 antagonistically control oligodendrocyte differentiation and myelination through Arf6 regulation, presenting a unique small GTPase on/off switching mechanism.

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell-mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.

The regulation and signaling pathways involved in the invasive growth of yeast have been studied extensively because of their general applicability to fungal pathogenesis. Bud2p, which functions as a GTPase-activating protein (GAP) for Bud1p/Rsr1p, is required for appropriate budding patterns and filamentous growth. The regulatory mechanisms leading to Bud2p activation, however, are poorly understood. In this study, we report that ADP-ribosylation factor 3p (Arf3p) acts as a regulator of Bud2p activation during invasive growth. Arf3p binds directly to the N-terminal region of Bud2p and promotes its GAP activity both in vitro and in vivo. Genetic analysis shows that deletion of BUD1 suppresses the defect of invasive growth in arf3Δ or bud2Δ cells. Lack of Arf3p, like that of Bud2p, causes the intracellular accumulation of Bud1p-GTP. The Arf3p-Bud2p interaction is important for invasive growth and facilitates the Bud2p-Bud1p association in vivo. Finally, we show that under glucose depletion-induced invasion conditions in yeast, more Arf3p is activated to the GTP-bound state, and the activation is independent of Arf3p guanine nucleotide-exchange factor Yel1p. Thus we demonstrate that a novel spatial activation of Arf3p plays a role in regulating Bud2p activation during glucose depletion-induced invasive growth.

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.

Glucose is a master regulator of cell behavior in the yeast Saccharomyces cerevisiae. It acts as both a metabolic substrate and a potent regulator of intracellular signaling cascades. Glucose starvation induces the transient delocalization and then partial relocalization of clathrin adaptors at the trans-Golgi network and endosomes. Although these localization responses are known to depend on the protein kinase A (PKA) signaling pathway, the molecular mechanism of this regulation is unknown. Here we demonstrate that PKA and the AMP-regulated kinase regulate adaptor localization through changes in energy metabolism. We show that genetic and chemical manipulation of intracellular ATP levels cause corresponding changes in adaptor localization. In permeabilized cells, exogenous ATP is sufficient to induce adaptor localization. Furthermore, we reveal distinct energy-dependent steps in adaptor localization: a step that requires the ADP-ribosylation factor ARF, an ATP-dependent step that requires the phosphatidyl-inositol-4 kinase Pik1, and third ATP-dependent step for which we provide evidence but for which the mechanism is unknown. We propose that these energy-dependent mechanisms precisely synchronize membrane traffic with overall proliferation rates and contribute a crucial aspect of energy conservation during acute glucose starvation.

Epithelial cysts comprise the structural units of the glandular epithelium. Although glandular inversion in epithelial tumors is thought to be a potential mechanism for the establishment of metastatic disease, little is known about the morphogenic cues and signaling pathways that govern glandular polarity and organization. Using organotypic cultures of Madin-Darby canine kidney cells in reconstituted basement membrane, we show that cellular depletion of the small GTP-binding protein ARF6 promotes the formation of inverted cysts, wherein the apical cell membrane faces the cyst exterior, and the basal domain faces the central lumen, while individual cell polarity is maintained. These cysts are also defective in interactions with laminin at the cyst-matrix interface. This inversion of glandular orientation is accompanied by Rac1 inactivation during early cystogenesis, and temporal activation of Rac1 is sufficient to recover the normal cyst phenotype. In an unnatural collagen I microenvironment, ARF6-depleted, inverted epithelial cysts exhibit some loss of cell polarity, a marked increase in Rho activation and Rac1 inactivation, and striking rearrangement of the surrounding collagen I matrix. These studies demonstrate the importance of ARF6 as a critical determinant of glandular orientation and the matrix environment in dictating structural organization of epithelial cysts.

Small guanosine triphosphatase (GTPase) ADP-ribosylation factors (Arfs) regulate membrane traffic and actin reorganization under the strict control of GTPase-activating proteins (GAPs). ARAP1 (Arf GAP with Rho GAP domain, ankyrin repeat, and PH domain 1) is an Arf GAP molecule with multiple PH domains that recognize phosphatidylinositol 3,4,5-trisphosphate. We found that growth factor stimulation induced localization of ARAP1 to an area of the plasma membrane inside the ring structure of circular dorsal ruffles (CDRs). Moreover, expression of ARAP1 increased the size of the CDR filamentous-actin ring in an Arf GAP activity-dependent manner, whereas smaller CDRs were formed by ARAP1 knockdown. In addition, expression of a dominant-negative mutant of Arf1 and Arf5, the substrates of ARAP1, expanded the size of CDRs, suggesting that the two Arf isoforms regulate ring structure downstream of ARAP1. Therefore our results reveal a novel molecular mechanism of CDR ring size control through the ARAP1-Arf1/5 pathway.

Cell function requires the integration of cytoskeletal organization and membrane trafficking. Small GTP-binding proteins are key regulators of these processes. We find that EPI64, an apical microvillar protein with a Tre-2/Bub2/Cdc16 (TBC) domain that stabilizes active Arf6 and has RabGAP activity, regulates Arf6-dependent membrane trafficking. Expression of EPI64 in HeLa cells induces the accumulation of actin-coated vacuoles, a distinctive phenotype seen in cells expressing constitutively active Arf6. Expression of EPI64 with defective RabGAP activity does not induce vacuole formation. Coexpression of Rab8a suppresses the vacuole phenotype induced by EPI64, and EPI64 expression lowers the level of Rab8-GTP in cells, strongly suggesting that EPI64 has GAP activity toward Rab8a. JFC1, an effector for Rab8a, colocalizes with and binds directly to a C-terminal region of EPI64. Together this region and the N-terminal TBC domain of EPI64 are required for the accumulation of vacuoles. Through analysis of mutants that uncouple JFC1 from either EPI64 or from Rab8-GTP, our data suggest a model in which EPI64 binds JFC1 to recruit Rab8a-GTP for deactivation by the RabGAP activity of EPI64. We propose that EPI64 regulates membrane trafficking both by stabilizing Arf6-GTP and by inhibiting the recycling of membrane through the tubular endosome by decreasing Rab8a-GTP levels.

Patterning of the Drosophila pupal eye is characterized by precise cell movements. In this paper, we demonstrate that these movements require an Arf regulatory cycle that connects surface receptors to actin-based movement. dArf6 activity-regulated by the Arf GTPase-activating proteins (ArfGAPs) dAsap and dArfGAP3 and the Arf GTP exchange factors Schizo and dPsd-promoted large cellular extensions; time-lapse microscopy indicated that these extensions presage cell rearrangements into correct epithelial niches. During this process, the Drosophila eye also requires interactions between surface Neph1/nephrin adhesion receptors Roughest and Hibris, which bind the adaptor protein Cindr (CD2AP). We provide evidence that Cindr forms a physical complex with dArfGAP3 and dAsap. Our data suggest this interaction sequesters ArfGAP function to liberate active dArf6 elsewhere in the cell. We propose that a Neph1/nephrin-Cindr/ArfGAP complex accumulates to limit local Arf6 activity and stabilize adherens junctions. Our model therefore links surface adhesion via an Arf6 regulatory cascade to dynamic modeling of the cytoskeleton, accounting for precise cell movements that organize the functional retinal field. Further, we demonstrate a similar relationship between the mammalian Cindr orthologue CD2AP and Arf6 activity in cell motility assays. We propose that this Cindr/CD2AP-mediated regulation of Arf6 is a widely used mechanism in emerging epithelia.

Arl13b, a ciliary protein within the ADP-ribosylation factor family and Ras superfamily of GTPases, is required for ciliary structure but has poorly defined ciliary functions. In this paper, we further characterize the role of Arl13b in cilia by examining mutant cilia in vitro and determining the localization and dynamics of Arl13b within the cilium. Previously, we showed that mice lacking Arl13b have abnormal Sonic hedgehog (Shh) signaling; in this study, we show the dynamics of Shh signaling component localization to the cilium are disrupted in the absence of Arl13b. Significantly, we found Smoothened (Smo) is enriched in Arl13b-null cilia regardless of Shh pathway stimulation, indicating Arl13b regulates the ciliary entry of Smo. Furthermore, our analysis defines a role for Arl13b in regulating the distribution of Smo within the cilium. These results suggest that abnormal Shh signaling in Arl13b mutant embryos may result from defects in protein localization and distribution within the cilium.

Primary cilia regulate epithelial differentiation and organ function. Failure of mutant polycystins to localize to cilia abolishes flow-stimulated calcium signaling and causes autosomal dominant polycystic kidney disease. We identify a conserved amino acid sequence, KVHPSST, in the C-terminus of polycystin-1 (PC1) that serves as a ciliary-targeting signal. PC1 binds a multimeric protein complex consisting of several GTPases (Arf4, Rab6, Rab11) and the GTPase-activating protein (GAP), ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) in the Golgi, which facilitates vesicle budding and Golgi exocytosis. A related N-terminal ciliary-targeting sequence in polycystin-2 similarly binds Arf4. Deletion of the extreme C-terminus of PC1 ablates Arf4 and ASAP1 binding and prevents ciliary localization of an integral membrane CD16.7-PC1 chimera. Interactions are confirmed for chimeric and endogenous proteins through quantitated in vitro and cell-based approaches. PC1 also complexes with Rab8; knockdown of trafficking regulators Arf4 or Rab8 functionally blocks CD16.7-PC1 trafficking to cilia. Mutations in rhodopsin disrupt a similar signal and cause retinitis pigmentosa, while Bardet-Biedl syndrome, primary open-angle glaucoma, and tumor cell invasiveness are linked to dysregulation of ASAP1 or Rab8 or its effectors. In this paper, we provide evidence for a conserved GTPase-dependent ciliary-trafficking mechanism that is shared between epithelia and neurons, and is essential in ciliary-trafficking and cell homeostasis.

Small monomeric G proteins regulated in part by GTPase-activating proteins (GAPs) are molecular switches for several aspects of vesicular transport. The yeast Gcs1 protein is a dual-specificity GAP for ADP-ribosylation factor (Arf) and Arf-like (Arl)1 G proteins, and also has GAP-independent activities. The absence of Gcs1 imposes cold sensitivity for growth and endosomal transport; here we present evidence that dysregulated Arl1 may cause these impairments. We show that gene deletions affecting the Arl1 or Ypt6 vesicle-tethering pathways prevent Arl1 activation and membrane localization, and restore growth and trafficking in the absence of Gcs1. A mutant version of Gcs1 deficient for both ArfGAP and Arl1GAP activity in vitro still allows growth and endosomal transport, suggesting that the function of Gcs1 that is required for these processes is independent of GAP activity. We propose that, in the absence of this GAP-independent regulation by Gcs1, the resulting dysregulated Arl1 prevents growth and impairs endosomal transport at low temperatures. In cells with dysregulated Arl1, an increased abundance of the Arl1 effector Imh1 restores growth and trafficking, and does so through Arl1 binding. Protein sequestration at the trans-Golgi membrane by dysregulated, active Arl1 may therefore be the mechanism of inhibition.

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.

Organelle morphology of the endomembrane system is critical for optimal organelle function. ADP ribosylation factors (Arfs), a family of small GTPases, are required for maintaining the structure of the Golgi and endosomes. What determines the discontinuous nature of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) as tubulovesicular clusters is unknown. In search of morphological determinants for the ERGIC, we found that a double knockdown of Arf1+Arf4 induced dynamic ERGIC tubules that connect ERGIC clusters, indicating that the tubules mediated lateral intraERGIC traffic. Tubule formation was inhibited by an antagonist of group VI calcium-independent phospholipase A₂ (PLA2G6) and by silencing the A isoform of PLA2G6 (PLA2G6-A). Arf1+Arf4 depletion altered the expression of PLA2G6-A splice variants and relocalized PLA2G6-A from the cytosol to ERGIC clusters and tubules, suggesting that the enzyme became locally active. We show that changes in Arf1 can modulate the activity of PLA2G6-A. We propose that a concerted action of Arf1, Arf4, and PLA2G6-A controls the architecture of the ERGIC in a way that is predicted to impact the rate and possibly the destination of cargos. Our findings have identified key components in the molecular mechanism underlying the regulation of tubules in the ERGIC and uncover tubular carriers as tightly controlled machinery.

Myoblast fusion is an essential step during myoblast differentiation that remains poorly understood. M-cadherin-dependent pathways that signal through Rac1 GTPase activation via the Rho-guanine nucleotide exchange factor (GEF) Trio are important for myoblast fusion. The ADP-ribosylation factor (ARF)6 GTPase has been shown to bind to Trio and to regulate Rac1 activity. Moreover, Loner/GEP(100)/BRAG2, a GEF of ARF6, has been involved in mammalian and Drosophila myoblast fusion, but the specific role of ARF6 has been not fully analyzed. Here, we show that ARF6 activity is increased at the time of myoblast fusion and is required for its implementation in mouse C2C12 myoblasts. Specifically, at the onset of myoblast fusion, ARF6 is associated with the multiproteic complex that contains M-cadherin, Trio, and Rac1 and accumulates at sites of myoblast fusion. ARF6 silencing inhibits the association of Trio and Rac1 with M-cadherin. Moreover, we demonstrate that ARF6 regulates myoblast fusion through phospholipase D (PLD) activation and phosphatidylinositol 4,5-bis-phosphate production. Together, these data indicate that ARF6 is a critical regulator of C2C12 myoblast fusion and participates in the regulation of PLD activities that trigger both phospholipids production and actin cytoskeleton reorganization at fusion sites.

Caveolin-1 and caveolae are often lost in cancer. We found that levels of caveolin-1 and polymerase I and transcript release factor (PTRF)/cavin-1 correlated closely in a panel of cancer and normal cells. Caveolin-1 reexpression in cancer cells lacking both proteins induced formation of long membrane tubules rarely seen in normal cells. PTRF/cavin-1 inhibited tubule formation when coexpressed with caveolin-1 in these cells, whereas suppression of PTRF/cavin-1 expression in cells that normally expressed both genes stimulated tubule formation by endogenous caveolin-1. Caveolin-1 tubules shared several features with previously described Rab8 tubules. Coexpressed Rab8 and caveolin-1 labeled the same tubules (as did EHD proteins), and synergized to promote tubule formation, whereas a dominant-interfering Rab8 mutant inhibited caveolin-1 tubule formation. Both overexpression and inhibition of dynamin-2 reduced the abundance of caveolin-1 tubules. Caveolin-1 reexpression in SK-BR-3 breast cancer cells also induced formation of short membrane tubules close to cortical actin filaments, which required actin filaments but not microtubules. Actomyosin-induced tension destabilized both long and short tubules; they often snapped and resolved to small vesicles. Actin filament depolymerization or myosin II inhibition reduced tension and stabilized tubules. These data demonstrate a new function for PTRF/cavin-1, a new functional interaction between caveolin-1 and Rab8 and that actomyosin interactions can induce tension on caveolin-1-containing membranes.

The delivery of proteins and organelles to the vacuole by autophagy involves membrane rearrangements that result in the formation of large vesicles called autophagosomes. The mechanism underlying autophagosome biogenesis and the origin of the membranes composing these vesicles remains largely unclear. We have investigated the role of the Golgi complex in autophagy and have determined that in yeast, activation of ADP-ribosylation factor (Arf)1 and Arf2 GTPases by Sec7, Gea1, and Gea2 is essential for this catabolic process. The two main events catalyzed by these components, the biogenesis of COPI- and clathrin-coated vesicles, do not play a critical role in autophagy. Analysis of the sec7 strain under starvation conditions revealed that the autophagy machinery is correctly assembled and the precursor membrane cisterna of autophagosomes, the phagophore, is normally formed. However, the expansion of the phagophore into an autophagosome is severely impaired. Our data show that the Golgi complex plays a crucial role in supplying the lipid bilayers necessary for the biogenesis of double-membrane vesicles possibly through a new class of transport carriers or a new mechanism.

Tumor development in glandular tissues is associated with structural alterations in the hollow ducts and spherical structures that comprise such tissues. We describe a signaling axis involving sustained activation of the GTP-binding protein, ARF6, that provokes dramatic changes in the organization of epithelial cysts, reminiscent of tumorigenic glandular phenotypes. In reconstituted basement membrane cultures of renal epithelial cysts, enhanced ARF6 activation induces the formation of cell-filled glandular structures with multiple lumens and disassembled cadherin-based cell-cell contacts. All of these alterations are accompanied by growth factor receptor internalization into signaling endosomes and reversed by blocking ARF6 activation or receptor endocytosis. Receptor localization in signaling endosomes results in hyperactive extracellular signal-regulated kinase signaling leading to Bcl-2 stabilization and aberrant cysts. Similarly, formation of hyperproliferative and disorganized mammary acini induced by chronic stimulation of colony-stimulating factor 1 receptor is coupled to endogenous ARF6 activation and constitutive receptor internalization and is reversed by ARF6 inhibition. These findings identify a previously unrecognized link between ARF6-regulated receptor internalization and events that drive dramatic alterations in cyst morphogenesis providing new mechanistic insight into the molecular processes that can promote epithelial glandular disruption.

It is widely assumed that class I and II Arfs function interchangeably throughout the Golgi complex. However, we report here that in vivo, Arf3 displays several unexpected properties. Unlike other Golgi-localized Arfs, Arf3 associates selectively with membranes of the trans-Golgi network (TGN) in a manner that is both temperature-sensitive and uniquely dependent on guanine nucleotide exchange factors of the BIGs family. For example, BIGs knockdown redistributed Arf3 but not Arf1 from Golgi membranes. Furthermore, shifting temperature to 20 degrees C, a temperature known to block cargo in the TGN, selectively redistributed Arf3 from Golgi membranes. Arf3 redistribution occurred slowly, suggesting it resulted from a change in membrane composition. Arf3 knockdown and overexpression experiments suggest that redistribution is not responsible for the 20 degrees C block. To investigate in more detail the mechanism for Arf3 recruitment and temperature-dependent release, we characterized several mutant forms of Arf3. This analysis demonstrated that those properties are readily separated and depend on pairs of residues present at opposite ends of the protein. Furthermore, phylogenetic analysis established that all four critical residues were absolutely conserved and unique to Arf3. These results suggest that Arf3 plays a unique function at the TGN that likely involves recruitment by a specific receptor.

ARFs are small GTPases that regulate vesicular trafficking, cell shape, and movement. ARFs are subject to extensive regulation by a large number of accessory proteins. The many different accessory proteins are likely specialized to regulate ARF signaling during particular processes. ARNO/cytohesin 2 is an ARF-activating protein that promotes cell migration and cell shape changes. We report here that protein-protein interactions mediated by the coiled-coil domain of ARNO are required for ARNO induced motility. ARNO lacking the coiled-coil domain does not promote migration and does not induce ARF-dependent Rac activation. We find that the coiled-coil domain promotes the assembly of a multiprotein complex containing both ARNO and the Rac-activating protein Dock180. Knockdown of either GRASP/Tamalin or IPCEF, two proteins known to bind to the coiled-coil of ARNO, prevents the association of ARNO and Dock180 and prevents ARNO-induced Rac activation. These data suggest that scaffold proteins can regulate ARF dependent processes by biasing ARF signaling toward particular outputs.

ADP-ribosylation factor (Arf) 6 activity is crucially involved in the regulation of E-cadherin-based cell-cell adhesions. Erythropoietin-producing hepatocellular carcinoma (Eph)-family receptors recognize ligands, namely, ephrins, anchored to the membrane of apposing cells, and they mediate cell-cell contact-dependent events. Here, we found that Arf6 activity is down-regulated in Madin-Darby canine kidney cells, which is dependent on cell density and calcium ion concentration, and we provide evidence of a novel signaling pathway by which ligand-activated EphA2 suppresses Arf6 activity. This EphA2-mediated suppression of Arf6 activity was linked to the induction of cell compaction and polarization, but it was independent of the down-regulation of extracellular signal-regulated kinase 1/2 kinase activity. We show that G protein-coupled receptor kinase-interacting protein (Git) 1 and noncatalytic region of tyrosine kinase (Nck) 1 are involved in this pathway, in which ligand-activated EphA2, via its phosphorylated Tyr594, binds to the Src homology 2 domain of Nck1, and then via its Src homology 3 domain binds to the synaptic localizing domain of Git1 to suppress Arf6 activity. We propose a positive feedback loop in which E-cadherin-based cell-cell contacts enhance EphA-ephrinA signaling, which in turn down-regulates Arf6 activity to enhance E-cadherin-based cell-cell contacts as well as the apical-basal polarization of epithelial cells.

GCC185, a trans-Golgi network-localized protein predicted to assume a long, coiled-coil structure, is required for Rab9-dependent recycling of mannose 6-phosphate receptors (MPRs) to the Golgi and for microtubule nucleation at the Golgi via CLASP proteins. GCC185 localizes to the Golgi by cooperative interaction with Rab6 and Arl1 GTPases at adjacent sites near its C terminus. We show here by yeast two-hybrid and direct biochemical tests that GCC185 contains at least four additional binding sites for as many as 14 different Rab GTPases across its entire length. A central coiled-coil domain contains a specific Rab9 binding site, and functional assays indicate that this domain is important for MPR recycling to the Golgi complex. N-Terminal coiled-coils are also required for GCC185 function as determined by plasmid rescue after GCC185 depletion by using small interfering RNA in cultured cells. Golgi-Rab binding sites may permit GCC185 to contribute to stacking and lateral interactions of Golgi cisternae as well as help it function as a vesicle tether.

Golgi-localized, gamma-Ear-containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. To examine the roles of the Ggas and AP-1 in TGN-PVC transport, we used a cell-free assay that measures delivery to the PVC of either Kex2p or a chimeric protein (K-V), in which the Vps10p cytosolic tail replaces the Kex2p tail. Either antibody inhibition or dominant-negative Gga2p completely blocked K-V transport but only partially blocked Kex2p transport. Deletion of APL2, encoding the beta subunit of AP-1, did not affect K-V transport but partially blocked Kex2p transport. Residual Kex2p transport seen with apl2Delta membranes was insensitive to dominant-negative Gga2p, suggesting that the apl2Delta mutation causes Kex2p to localize to a compartment that precludes Gga-dependent trafficking. These results suggest that yeast Ggas facilitate the specific and direct delivery of Vps10p and Kex2p from the TGN to the PVC and that AP-1 modulates Kex2p trafficking through a distinct pathway, presumably involving the early endosome.

ADP-ribosylation factors (Arfs) and Arf GTPase-activating proteins (GAPs) are key regulators of membrane trafficking and the actin cytoskeleton. The Arf GAP ASAP1 contains an N-terminal BAR domain, which can induce membrane tubulation. Here, we report that the BAR domain of ASAP1 can also function as a protein binding site. Two-hybrid screening identified FIP3, which is a putative Arf6- and Rab11-effector, as a candidate ASAP1 BAR domain-binding protein. Both coimmunoprecipitation and in vitro pulldown assays confirmed that ASAP1 directly binds to FIP3 through its BAR domain. ASAP1 formed a ternary complex with Rab11 through FIP3. FIP3 binding to the BAR domain stimulated ASAP1 GAP activity against Arf1, but not Arf6. ASAP1 colocalized with FIP3 in the pericentrosomal endocytic recycling compartment. Depletion of ASAP1 or FIP3 by small interfering RNA changed the localization of transferrin receptor, which is a marker of the recycling endosome, in HeLa cells. The depletion also altered the trafficking of endocytosed transferrin. These results support the conclusion that ASAP1, like FIP3, functions as a component of the endocytic recycling compartment.

Despite extensive work on ADP-ribosylation factor (Arf) 1 at the Golgi complex, the functions of Arf2-5 in the secretory pathway, or for that of any Arf at the ER-Golgi intermediate compartment (ERGIC) remain uncharacterized. Here, we examined the recruitment of fluorescently tagged Arf1, -3, -4, and -5 onto peripheral ERGIC. Live cell imaging detected Arfs on peripheral puncta that also contained Golgi-specific brefeldin A (BFA) resistance factor (GBF) 1 and the ERGIC marker p58. Unexpectedly, BFA did not promote corecruitment of Arfs with GBF1 either at the Golgi complex or the ERGIC, but it uncovered striking differences between Arf1,3 and Arf4,5. Although Arf1,3 quickly dissociated from all endomembranes after BFA addition, Arf4,5 persisted on ERGIC structures, even after redistribution of GBF1 to separate compartments. The GDP-arrested Arf4(T31N) mutant localized to the ERGIC, even with BFA and Exo1 present. In addition, loss of Arf x GTP after treatment with Exo1 caused rapid release of all Arfs from the Golgi complex and led to GBF1 accumulation on both Golgi and ERGIC membranes. Our results demonstrate that GDP-bound Arf4,5 associate with ERGIC membranes through binding sites distinct from those responsible for GBF1 recruitment. Furthermore, they provide the first evidence that GBF1 accumulation on membranes may be caused by loss of Arf x GTP, rather than the formation of an Arf x GDP x BFA x GBF1 complex.

BIG2 and BIG1 are closely related guanine-nucleotide exchange factors (GEFs) for ADP-ribosylation factors (ARFs) and are involved in the regulation of membrane traffic through activating ARFs and recruiting coat protein complexes, such as the COPI complex and the AP-1 clathrin adaptor complex. Although both ARF-GEFs are associated mainly with the trans-Golgi network (TGN) and BIG2 is also associated with recycling endosomes, it is unclear whether BIG2 and BIG1 share some roles in membrane traffic. We here show that knockdown of both BIG2 and BIG1 by RNAi causes mislocalization of a subset of proteins associated with the TGN and recycling endosomes and blocks retrograde transport of furin from late endosomes to the TGN. Similar mislocalization and protein transport block, including furin, were observed in cells depleted of AP-1. Taken together with previous reports, these observations indicate that BIG2 and BIG1 play redundant roles in trafficking between the TGN and endosomes that involves the AP-1 complex.

Phosphatidylinositol 4-phosphate (PI(4)P) is a key regulator of membrane transport required for the formation of transport carriers from the trans-Golgi network (TGN). The molecular mechanisms of PI(4)P signaling in this process are still poorly understood. In a search for PI(4)P effector molecules, we performed a screen for synthetic lethals in a background of reduced PI(4)P and found the gene GGA2. Our analysis uncovered a PI(4)P-dependent recruitment of the clathrin adaptor Gga2p to the TGN during Golgi-to-endosome trafficking. Gga2p recruitment to liposomes is stimulated both by PI(4)P and the small GTPase Arf1p in its active conformation, implicating these two molecules in the recruitment of Gga2p to the TGN, which ultimately controls the formation of clathrin-coated vesicles. PI(4)P binding occurs through a phosphoinositide-binding signature within the N-terminal VHS domain of Gga2p resembling a motif found in other clathrin interacting proteins. These data provide an explanation for the TGN-specific membrane recruitment of Gga2p.

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.