Phosphorylation of Arl4A/D promotes their binding by the HYPK chaperone for their stable recruitment to the plasma membrane.

The Arl4 small GTPases participate in a variety of cellular events, including cytoskeleton remodeling, vesicle trafficking, cell migration, and neuronal development. Whereas small GTPases are typically regulated by their GTPase cycle, Arl4 proteins have been found to act independent of this canonical regulatory mechanism. Here, we show that Arl4A and Arl4D (Arl4A/D) are unstable due to proteasomal degradation, but stimulation of cells by fibronectin (FN) inhibits this degradation to promote Arl4A/D stability. Proteomic analysis reveals that FN stimulation induces phosphorylation at S143 of Arl4A and at S144 of Arl4D. We identify Pak1 as the responsible kinase for these phosphorylations. Moreover, these phosphorylations promote the chaperone protein HYPK to bind Arl4A/D, which stabilizes their recruitment to the plasma membrane to promote cell migration. These findings not only advance a major mechanistic understanding of how Arl4 proteins act in cell migration but also achieve a fundamental understanding of how these small GTPases are modulated by revealing that protein stability, rather than the GTPase cycle, acts as a key regulatory mechanism.

AMPK promotes Arf6 activation in a kinase-independent manner upon glucose starvation.

AMP-activated protein kinase (AMPK) is a crucial cellular nutrient and energy sensor that maintains energy homeostasis. AMPK also governs cancer cell invasion and migration by regulating gene expression and activating multiple cellular signaling pathways. ADP-ribosylation factor 6 (Arf6) can be activated via nucleotide exchange by guanine-nucleotide-exchange factors (GEFs), and its activation also regulates tumor invasion and migration. By studying GEF-mediated Arf6 activation, we have elucidated that AMPK functions as a noncanonical GEF for Arf6 in a kinase-independent manner. Moreover, by examining the physiological role of the AMPK-Arf6 axis, we have determined that AMPK activates Arf6 upon glucose starvation and 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) treatment. We have further identified the binding motif in the C-terminal regulatory domain of AMPK that is responsible for promoting Arf6 activation and, thus, inducing cell migration and invasion. These findings reveal a noncanonical role of AMPK in which its C-terminal regulatory domain serves as a GEF for Arf6 during glucose deprivation.

Cooperative recruitment of Arl4A and Pak1 to the plasma membrane contributes to sustained Pak1 activation for cell migration.

Cell migration requires the coordination of multiple signaling pathways involved in membrane dynamics and cytoskeletal rearrangement. The Arf-like small GTPase Arl4A has been shown to modulate actin cytoskeleton remodeling. However, evidence of the function of Arl4A in cell migration is insufficient. Here, we report that Arl4A acts with the serine/threonine protein kinase Pak1 to modulate cell migration through their cooperative recruitment to the plasma membrane. We first observed that Arl4A and its isoform Arl4D interact with Pak1 and Pak2 and showed that Arl4A recruits Pak1 and Pak2 to the plasma membrane. The fibronectin-induced Pak1 localization at the plasma membrane is reduced in Arl4A-depleted cells. Unexpectedly, we found that Pak1, but not Arl4A-binding-defective Pak1, can recruit a cytoplasmic myristoylation-deficient Arl4A-G2A mutant to the plasma membrane. Furthermore, we found that the Arl4A-Pak1 interaction, which is independent of Rac1 binding to Pak1, is required for Arl4A-induced cell migration. Thus, we infer that there is feedback regulation between Arl4A and Pak1, in which they mutually recruit each other to the plasma membrane for Pak1 activation, thereby modulating cell migration through direct interaction.

Multiple activities of Arl1 GTPase in the trans-Golgi network.

ADP-ribosylation factors (Arfs) and ADP-ribosylation factor-like proteins (Arls) are highly conserved small GTPases that function as main regulators of vesicular trafficking and cytoskeletal reorganization. Arl1, the first identified member of the large Arl family, is an important regulator of Golgi complex structure and function in organisms ranging from yeast to mammals. Together with its effectors, Arl1 has been shown to be involved in several cellular processes, including endosomal trans-Golgi network and secretory trafficking, lipid droplet and salivary granule formation, innate immunity and neuronal development, stress tolerance, as well as the response of the unfolded protein. In this Commentary, we provide a comprehensive summary of the Arl1-dependent cellular functions and a detailed characterization of several Arl1 effectors. We propose that involvement of Arl1 in these diverse cellular functions reflects the fact that Arl1 is activated at several late-Golgi sites, corresponding to specific molecular complexes that respond to and integrate multiple signals. We also provide insight into how the GTP-GDP cycle of Arl1 is regulated, and highlight a newly discovered mechanism that controls the sophisticated regulation of Arl1 activity at the Golgi complex.

Unfolded protein response regulates yeast small GTPase Arl1p activation at late Golgi via phosphorylation of Arf GEF Syt1p.

ADP ribosylation factor (Arf) GTPases are key regulators of membrane traffic at the Golgi complex. In yeast, Arf guanine nucleotide-exchange factor (GEF) Syt1p activates Arf-like protein Arl1p, which was accompanied by accumulation of golgin Imh1p at late Golgi, but whether and how this function of Syt1p is regulated remains unclear. Here, we report that the inositol-requiring kinase 1 (Ire1p)-mediated unfolded protein response (UPR) modulated Arl1p activation at late Golgi. Arl1p activation was dependent on both kinase and endo-RNase activities of Ire1p. Moreover, constitutively active transcription factor Hac1p restored the Golgi localization of Arl1p and Imh1p inIRE1-deleted cells. Elucidating the mechanism of Ire1p-Hac1p axis actions, we found that it regulated phosphorylation of Syt1p, which enhances Arl1p activation, recruitment of Imh1p to the Golgi, and Syt1p interaction with Arl1p. Consistent with these findings, the induction of UPR by tunicamycin treatment increases phosphorylation of Syt1p, resulting in Arl1p activation. Thus, these findings clarify how the UPR influences the roles of Syt1p, Arl1p, and Imh1p in Golgi transport.

Mechanism of action of the flippase Drs2p in modulating GTP hydrolysis of Arl1p.

Small GTPase ADP-ribosylation factors (ARFs) are key regulators of membrane trafficking and their activities are determined by guanine-nucleotide-binding status. In Saccharomyces cerevisiae, Arl1p, an ARF-like protein, is responsible for multiple trafficking pathways at the Golgi. The GTP-hydrolysis activity of Arl1p is stimulated by its GTPase-activating protein Gcs1p, and binding with its effector Imh1p protects Arl1p from premature inactivation. However, the mechanism involved in the timing of Arl1p inactivation is unclear. Here, we demonstrate that another Arl1p effector, the lipid flippase Drs2p, is required for Gcs1p-stimulated inactivation of Arl1p. Drs2p is known to be activated by Arl1p and is involved in vesicle formation through its ability to create membrane asymmetry. We found that the flippase activity of Drs2p is required for proper membrane targeting of Gcs1p in vivo. Through modification of the membrane environment, Drs2p promotes the affinity of Gcs1p for the Golgi, where it binds to active Arl1p. Together, Imh1p and Drs2p modulate the activity of Gcs1p by timing its interaction with Arl1p, hence providing feedback regulation of Arl1p activity.

Arl1p regulates spatial membrane organization at the trans-Golgi network through interaction with Arf-GEF Gea2p and flippase Drs2p.

ADP ribosylation factors (Arfs) are the central regulators of vesicle trafficking from the Golgi complex. Activated Arfs facilitate vesicle formation through stimulating coat assembly, activating lipid-modifying enzymes and recruiting tethers and other effectors. Lipid translocases (flippases) have been implicated in vesicle formation through the generation of membrane curvature. Although there is no evidence that Arfs directly regulate flippase activity, an Arf-guanine-nucleotide-exchange factor (GEF) Gea2p has been shown to bind to and stimulate the activity of the flippase Drs2p. Here, we provide evidence for the interaction and activation of Drs2p by Arf-like protein Arl1p in yeast. We observed that Arl1p, Drs2p and Gea2p form a complex through direct interaction with each other, and each interaction is necessary for the stability of the complex and is indispensable for flippase activity. Furthermore, we show that this Arl1p-Drs2p-Gea2p complex is specifically required for recruiting golgin Imh1p to the Golgi. Our results demonstrate that activated Arl1p can promote the spatial modulation of membrane organization at the trans-Golgi network through interacting with the effectors Gea2p and Drs2p.

Competition between the golgin Imh1p and the GAP Gcs1p stabilizes activated Arl1p at the late-Golgi.

Golgins play diverse roles in regulating the structure and function of the Golgi. The yeast golgin Imh1p is targeted to the trans-Golgi network (TGN) through interaction of its GRIP domain with GTP-bound Arl1p. Recycling of Arl1p and Imh1p to the cytosol requires the hydrolysis of GTP bound to Arl1p; however, the point at which GTP hydrolysis occurs remains unknown. Here, we report that self-interaction of Imh1p plays a role in modulating spatial inactivation of Arl1p. Deletion of IMH1 in yeast decreases the amount of the GTP-bound Arl1p and results in less Arl1p residing on the TGN. Biochemically, purified Imh1p competes with Gcs1p, an Arl1p GTPase-activating protein (GAP), for binding to Arl1p, thus interfering with the GAP activity of Gcs1p toward Arl1p. Furthermore, we demonstrate that the self-interaction of Imh1p attenuates the Gcs1p-dependent GTP hydrolysis of Arl1p. Thus, we propose that the golgin Imh1p serves as a feedback regulator to modulate the GTP hydrolysis of Arl1p.

The RNA helicase Dhh1p cooperates with Rbp1p to promote porin mRNA decay via its non-conserved C-terminal domain.

The yeast RNA helicase Dhh1p has been shown to associate with components of mRNA decay and is involved in mRNA decapping and degradation. An RNA-binding protein, Rbp1p, is known to bind to the 3'-UTR of porin (POR1) mRNA, and induces mRNA decay by an uncharacterized mechanism. Here, we show that Dhh1p can associate with POR1 mRNA and specifically promote POR1 mRNA decay via its interaction with Rbp1p. As compared to its mammalian homolog RCK/p54/DDX6, Dhh1p has a unique and long extension at its C-terminus. Interestingly, this non-conserved C-terminal region of Dhh1p is required for interaction with Rbp1p and modulating Rbp1p-mediated POR1 mRNA decay. Notably, expression of a C-terminal 81-residue deleted Dhh1p can fully complement the growth defect of a dhh1Δ strain and retains its function in regulating the mRNA level of an RNA-binding protein Edc1p. Moreover, mammalian DDX6 became capable of interacting with Rbp1p and could confer Rbp1p-mediated POR1 mRNA decay in the dhh1Δ strain upon fusion to the C-terminal unique region of Dhh1p. Thus, we propose that the non-conserved C-terminus of Dhh1p plays a role in defining specific interactions with mRNA regulatory factors that promote distinct mRNA decay.

ARL4A acts with GCC185 to modulate Golgi complex organization.

ADP-ribosylation factor-like protein 4A (ARL4A) is a developmentally regulated member of the ARF/ARL GTPase family. The primary structure of ARL4A is very similar to that of other ARF/ARL molecules, but its function remains unclear. The trans-Golgi network golgin GCC185 is required for maintenance of Golgi structure and distinct endosome-to-Golgi transport. We show here that GCC185 acts as a new effector for ARL4 to modulate Golgi organization. ARL4A directly interacts with GCC185 in a GTP-dependent manner. Sub-coiled-coil regions of the CC2 domain of GCC185 are required for the interaction between GCC185 and ARL4A. Depletion of ARL4A reproduces the GCC185-depleted phenotype, causing fragmentation of the Golgi compartment and defects in endosome-to-Golgi transport. GCC185 and ARL4A localize to the Golgi independently of each other. Deletion of the ARL4A-interacting region of GCC185 results in inability to maintain Golgi structure. Depletion of ARL4A impairs the interaction between GCC185 and cytoplasmic linker-associated proteins 1 and 2 (CLASP1 and CLASP2, hereafter CLASPs) in vivo, and abolishes the GCC185-mediated Golgi recruitment of these CLASPs, which is crucial for the maintenance of Golgi structure. In summary, we suggest that ARL4A alters the integrity of the Golgi structure by facilitating the interaction of GCC185 with CLASPs.

The SNARE-associated protein Sft2 functions in Imh1-mediated SNARE recycling transport upon ER stress.

Vesicular trafficking involving SNARE proteins play a crucial role in the delivery of cargo to the target membrane. Arf-like protein 1 (Arl1) is an important regulator of the endosomal trans-Golgi network (TGN) and secretory trafficking. In yeast, ER stress-enhances Arl1 activation and Golgin Imh1 recruitment to the late-Golgi. Although Arl1 and Imh1 are critical for GARP-mediated endosomal SNARE-recycling transport in response to ER stress, their downstream effectors are unknown. Here, we report that the SNARE-associated protein Sft2 acts downstream of the Arl1-Imh1 axis to regulate SNARE recycling upon ER stress. We first demonstrated that Sft2 is required for Tlg1/Snc1 SNARE-recycling transport under tunicamycin-induced ER stress. Interestingly, we found that Imh1 regulates Tlg2 retrograde transport to the late-Golgi under ER stress, which in turn is required for Sft2 targeting to the late-Golgi. We further showed that Sft2 with 40 amino acids deleted from the N-terminus exhibits defective mediation of SNARE recycling and decreased association with Tlg1 under ER stress. Finally, we demonstrated that Sft2 is required for GARP-dependent endosome-to-Golgi transport in the absence of Rab protein Ypt6. This study highlights Sft2 as a critical downstream effector of the Arl1-Imh1 axis, mediating the endosome-to-Golgi transport of SNAREs.

Endosomal Arl4A attenuates EGFR degradation by binding to the ESCRT-II component VPS36.

Ligand-induced epidermal growth factor receptor (EGFR) endocytosis followed by endosomal EGFR signaling and lysosomal degradation plays important roles in controlling multiple biological processes. ADP-ribosylation factor (Arf)-like protein 4 A (Arl4A) functions at the plasma membrane to mediate cytoskeletal remodeling and cell migration, whereas its localization at endosomal compartments remains functionally unknown. Here, we report that Arl4A attenuates EGFR degradation by binding to the endosomal sorting complex required for transport (ESCRT)-II component VPS36. Arl4A plays a role in prolonging the duration of EGFR ubiquitinylation and deterring endocytosed EGFR transport from endosomes to lysosomes under EGF stimulation. Mechanistically, the Arl4A-VPS36 direct interaction stabilizes VPS36 and ESCRT-III association, affecting subsequent recruitment of deubiquitinating-enzyme USP8 by CHMP2A. Impaired Arl4A-VPS36 interaction enhances EGFR degradation and clearance of EGFR ubiquitinylation. Together, we discover that Arl4A negatively regulates EGFR degradation by binding to VPS36 and attenuating ESCRT-mediated late endosomal EGFR sorting.

The Arf family GTPase Arl4A complexes with ELMO proteins to promote actin cytoskeleton remodeling and reveals a versatile Ras-binding domain in the ELMO proteins family.

The prototypical DOCK protein, DOCK180, is an evolutionarily conserved Rac regulator and is indispensable during processes such as cell migration and myoblast fusion. The biological activity of DOCK180 is tightly linked to its binding partner ELMO. We previously reported that autoinhibited ELMO proteins regulate signaling from this pathway. One mechanism to activate the ELMO-DOCK180 complex appears to be the recruitment of this complex to the membrane via the Ras-binding domain (RBD) of ELMO. In the present study, we aimed to identify novel ELMO-interacting proteins to further define the molecular events capable of controlling ELMO recruitment to the membrane. To do so, we performed two independent interaction screens: one specifically interrogated an active GTPase library while the other probed a brain cDNA library. Both methods converged on Arl4A, an Arf-related GTPase, as a specific ELMO interactor. Biochemically, Arl4A is constitutively GTP-loaded, and our binding assays confirm that both wild-type and constitutively active forms of the GTPase associate with ELMO. Mechanistically, we report that Arl4A binds the ELMO RBD and acts as a membrane localization signal for ELMO. In addition, we report that membrane targeting of ELMO via Arl4A promotes cytoskeletal reorganization including membrane ruffling and stress fiber disassembly via an ELMO-DOCK1800-Rac signaling pathway. We conclude that ELMO is capable of interacting with GTPases from Rho and Arf families, leading to the conclusion that ELMO contains a versatile RBD. Furthermore, via binding of an Arf family GTPase, the ELMO-DOCK180 is uniquely positioned at the membrane to activate Rac signaling and remodel the actin cytoskeleton.

Syt1p promotes activation of Arl1p at the late Golgi to recruit Imh1p.

In yeast, Arl3p recruits Arl1p GTPase to regulate Golgi function and structure. However, the molecular mechanism involved in regulating activation of Arl1p at the Golgi is unknown. Here, we show that Syt1p promoted activation of Arl1p and recruitment of a golgin protein, Imh1p, to the Golgi. Deletion of SYT1 resulted in the majority of Arl1p being distributed diffusely throughout the cytosol. Overexpression of Syt1p increased Arl1p-GTP production in vivo and the Syt1-Sec7 domain promoted nucleotide exchange on Arl1p in vitro. Syt1p function required the N-terminal region, Sec7 and PH domains. Arl1p, but not Arl3p, interacted with Syt1p. Localization of Syt1p to the Golgi did not require Arl3p. Unlike arl1Δ or arl3Δ mutants, syt1Δ did not show defects in Gas1p transport, cell wall integrity or vacuolar structure. These findings reveal that activation of Arl1p is regulated in part by Syt1p, and imply that Arl1p activation, by using more than one GEF, exerts distinct biological activities at the Golgi compartment.

Golgin Imh1 and GARP complex cooperate to restore the impaired SNARE recycling transport induced by ER stress.

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR), which acts through various mechanisms to reduce ER stress. While the UPR has been well studied for its effects on the ER, its impact on the Golgi is less understood. The Golgi complex receives transport vesicles from the endosome through two types of tethering factors: long coiled-coil golgin and the multisubunit Golgi-associated retrograde protein (GARP) complex. Here, we report that ER stress increases the phosphorylation of golgin Imh1 to maintain the GARP-mediated recycling of the SNAREs Snc1 and Tlg1. We also identify a specific function of the Golgi affected by ER stress and elucidate a homeostatic response to restore this function, which involves both an Ire1-dependent and a MAP kinase Slt2/ERK2-dependent mechanism. Furthermore, our findings advance a general understanding of how two different types of tethers act cooperatively to mediate a transport pathway.

ARL4D recruits cytohesin-2/ARNO to modulate actin remodeling.

ARL4D is a developmentally regulated member of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of Ras-related GTPases. Although the primary structure of ARL4D is very similar to that of other ARF/ARL molecules, its function remains unclear. Cytohesin-2/ARF nucleotide-binding-site opener (ARNO) is a guanine nucleotide-exchange factor (GEF) for ARF, and, at the plasma membrane, it can activate ARF6 to regulate actin reorganization and membrane ruffling. We show here that ARL4D interacts with the C-terminal pleckstrin homology (PH) and polybasic c domains of cytohesin-2/ARNO in a GTP-dependent manner. Localization of ARL4D at the plasma membrane is GTP- and N-terminal myristoylation-dependent. ARL4D(Q80L), a putative active form of ARL4D, induced accumulation of cytohesin-2/ARNO at the plasma membrane. Consistent with a known action of cytohesin-2/ARNO, ARL4D(Q80L) increased GTP-bound ARF6 and induced disassembly of actin stress fibers. Expression of inactive cytohesin-2/ARNO(E156K) or small interfering RNA knockdown of cytohesin-2/ARNO blocked ARL4D-mediated disassembly of actin stress fibers. Similar to the results with cytohesin-2/ARNO or ARF6, reduction of ARL4D suppressed cell migration activity. Furthermore, ARL4D-induced translocation of cytohesin-2/ARNO did not require phosphoinositide 3-kinase activation. Together, these data demonstrate that ARL4D acts as a novel upstream regulator of cytohesin-2/ARNO to promote ARF6 activation and modulate actin remodeling.

Arl4D-EB1 interaction promotes centrosomal recruitment of EB1 and microtubule growth.

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.

ADP-ribosylation factor-like 4A interacts with Robo1 to promote cell migration by regulating Cdc42 activation.

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.

Action of Arl1 GTPase and golgin Imh1 in Ypt6-independent retrograde transport from endosomes to the trans-Golgi network.

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.

ARF GTPases and their GEFs and GAPs: concepts and challenges.

Detailed structural, biochemical, cell biological, and genetic studies of any gene/protein are required to develop models of its actions in cells. Studying a protein family in the aggregate yields additional information, as one can include analyses of their coevolution, acquisition or loss of functionalities, structural pliability, and the emergence of shared or variations in molecular mechanisms. An even richer understanding of cell biology can be achieved through evaluating functionally linked protein families. In this review, we summarize current knowledge of three protein families: the ARF GTPases, the guanine nucleotide exchange factors (ARF GEFs) that activate them, and the GTPase-activating proteins (ARF GAPs) that have the ability to both propagate and terminate signaling. However, despite decades of scrutiny, our understanding of how these essential proteins function in cells remains fragmentary. We believe that the inherent complexity of ARF signaling and its regulation by GEFs and GAPs will require the concerted effort of many laboratories working together, ideally within a consortium to optimally pool information and resources. The collaborative study of these three functionally connected families (≥70 mammalian genes) will yield transformative insights into regulation of cell signaling.