Domain architecture of the atypical Arf-family GTPase Arl13b involved in cilia formation

Arl13b is an atypical Arf/Arl-family GTPase consisting of an extending large C-terminal region (C domain) and Arf-homologous GTP-binding motifs in the N terminus (N domain). Although Arl13b appears to be involved in cilia formation, its precise function and roles of the domains remain unknown. Here, we show the unique domain architecture of Arl13b by analyzing the relationship between its biochemical properties and cilia formation. Arl13b binds guanine nucleotides and specifically localizes to cilia. The ciliary localization of Arl13b requires both N and C domains but is independent of its guanine nucleotide-binding ability. Arl13b is capable of self-associating via N domain, and overexpression of N domain inhibits not only cilia formation but also the maintenance of pre-generated cilia. These findings suggest that N and C domains of Arl13b cooperatively regulate its ciliary localization and that N domain-dependent self-association of Arl13b may be important for its function in cilia biogenesis.     

Molecular views of Arf-like small GTPases in cilia and ciliopathies

The primary cilia are microtubule-based organelles that protrude from most of the eukaryotic cells. Recognized as the cell's antenna, primary cilium functions as a signaling hub for many physiologically and developmentally important signaling cascades. Ciliary dysfunction causes a wide spectrum of syndromic human genetic diseases collectively termed “ciliopathies”. Mounting evidences have shown that various small GTPases have been implicated in the context of cilia as well as human ciliopathies. However, how these small GTPases affect cilia formation and function remains poorly understood. Here we review and discuss the ciliary role of three Arf-like small GTPases (Arls), Arl3, Arl6, and Arl13b.     

Rabs and other small GTPases in ciliary transport

The non-motile primary cilium is a single, microtubule-based hair-like projection that emanates from most, if not all, non-dividing mammalian cells. Enriched in a variety of signalling receptors and accessories, the cilium mediates crucial sensory and regulatory functions during development and postnatal tissue homoeostasis. Maintenance of ciliary morphology and function requires continuous IFT (intraflagellar transport), and recent findings have shed light on some molecular details of how ciliogenesis is dependent on targeted exocytic membrane trafficking from the Golgi. The ARL [Arf (ADP ribosylation factor)-related] small GTPase Arf4 functions in TGN (trans-Golgi network) sorting of cilia-targeted rhodopsin into carrier vesicles, while Arl6 (Arf-like 6) and Arl13b regulate aspects of ciliary transport and IFT. Ciliogenesis and ciliary functions are also regulated by small Rabs. Rab8a, in conjunction with Rab11a, and via its interaction with a multitude of proteins associated with the ciliary basal body and axoneme/membrane, appears to be critical for ciliogenesis. Rab8's close homologue Rab10 may also play a ciliogenic role in some cells. Rab23, the depletion or inactivation of which affects cilia formation, may regulate specific ciliary protein targeting and turnover, particularly those involved in Shh (Sonic hedgehog) signalling. Recent findings have also implicated Ran, a small GTPase better known for nuclear import, in ciliary targeting of the KIF17 motor protein. We highlight and discuss recent findings on how Rabs and other small GTPases mediate ciliogenesis and ciliary traffic.     

Valproic acid-inducible Arl4D and cytohesin-2/ARNO, acting through the downstream Arf6, regulate neurite outgrowth in N1E-115 cells

The mood-stabilizing agent valproic acid (VPA) potently promotes neuronal differentiation. As yet, however, little is known about the underlying molecular mechanism. Here, we show that VPA upregulates cytohesin-2 and mediates neurite outgrowth in N1E-115 neuroblastoma cells. Cytohesin-2 is the guanine-nucleotide exchange factor (GEF) for small GTPases of the Arf family; it regulates many aspects of cellular functions including morphological changes. Treatment with the specific cytohesin family inhibitor SecinH3 or knockdown of cytohesin-2 with its siRNA results in blunted induction of neurite outgrowth in N1E-115 cells. The outgrowth is specifically inhibited by siRNA knockdown of Arf6, but not by that of Arf1. Furthermore, VPA upregulates Arl4D, an Arf-like small GTPase that has recently been identified as the regulator that binds to cytohesin-2. Arl4D knockdown displays an inhibitory effect on neurite outgrowth resulting from VPA, while expression of constitutively active Arl4D induces outgrowth. We also demonstrate that the addition of cell-permeable peptide, coupling the cytohesin-2-binding region of Arl4D into cells, reduces the effect of VPA. Thus, Arl4D is a previously unknown regulator of neurite formation through cytohesin-2 and Arf6, providing another example that the functional interaction of two different small GTPases controls an important cellular function.     

Biochemical characterization of missense mutations in the Arf/Arl-family small GTPase Arl6 causing Bardet-Biedl syndrome

Bardet-Biedl syndrome (BBS) is a pleiotropically genetic disorder, whose etiology is linked to cilia. Mutations in the Arf/Arl-family GTPase Arl6 have been recently shown to be responsible for BBS type 3. Here we show that BBS mutations alter the guanine nucleotide-binding properties of Arl6. Specifically, substitution of 31st Threonine to Arginine selectively abrogates the GTP-binding ability of Arl6 without affecting GDP-binding/dissociating properties. Furthermore, all the BBS mutations in Arl6 result in low expression of the mutant proteins, which can be restored by the inhibition of the proteasome. These findings implicate that Arl6 mutants are destabilized and eliminated by the proteasome in cells, probably due to the altered nucleotide-binding properties.     

Specificity of Arl2/Arl3 signaling is mediated by a ternary Arl3-effector-GAP complex

Arl2 and Arl3, members of the Arf subfamily of small G proteins, are believed to be involved in ciliary and microtubule-dependent processes. Recently, we could identify RP2, responsible for a variant of X-linked retinitis pigmentosa, as the Arl3-specific GAP. Here, we have characterized Arl2/3 interactions. We show the formation of a ternary complex between Arl3, its cognate GAP RP2 and its retinal effector HRG4. This complex seems to be important for photoreceptor function.     

The small G protein Arl1 directs the trans-Golgi-specific targeting of the Arf1 exchange factors BIG1 and BIG2

The small G protein Arf1 regulates Golgi traffic and is activated by two related types of guanine nucleotide exchange factor (GEF). GBF1 acts at the cis-Golgi, whereas BIG1 and its close paralog BIG2 act at the trans-Golgi. Peripheral membrane proteins such as these GEFs are often recruited to membranes by small G proteins, but the basis for specific recruitment of Arf GEFs, and hence Arfs, to Golgi membranes is not understood. In this paper, we report a liposome-based affinity purification method to identify effectors for small G proteins of the Arf family. We validate this with the Drosophila melanogaster Arf1 orthologue (Arf79F) and the related class II Arf (Arf102F), which showed a similar pattern of effector binding. Applying the method to the Arf-like G protein Arl1, we found that it binds directly to Sec71, the Drosophila ortholog of BIG1 and BIG2, via an N-terminal region. We show that in mammalian cells, Arl1 is necessary for Golgi recruitment of BIG1 and BIG2 but not GBF1. Thus, Arl1 acts to direct a trans-Golgi-specific Arf1 GEF, and hence active Arf1, to the trans side of the Golgi.     

PH Domain-Arf G Protein Interactions Localize the Arf-GEF Steppke for Cleavage Furrow Regulation in Drosophila

The recruitment of GDP/GTP exchange factors (GEFs) to specific subcellular sites dictates where they activate small G proteins for the regulation of various cellular processes. Cytohesins are a conserved family of plasma membrane GEFs for Arf small G proteins that regulate endocytosis. Analyses of mammalian cytohesins have identified a number of recruitment mechanisms for these multi-domain proteins, but the conservation and developmental roles for these mechanisms are unclear. Here, we report how the pleckstrin homology (PH) domain of the Drosophila cytohesin Steppke affects its localization and activity at cleavage furrows of the early embryo. We found that the PH domain is necessary for Steppke furrow localization, and for it to regulate furrow structure. However, the PH domain was not sufficient for the localization. Next, we examined the role of conserved PH domain amino acid residues that are required for mammalian cytohesins to bind PIP3 or GTP-bound Arf G proteins. We confirmed that the Steppke PH domain preferentially binds PIP3 in vitro through a conserved mechanism. However, disruption of residues for PIP3 binding had no apparent effect on GFP-Steppke localization and effects. Rather, residues for binding to GTP-bound Arf G proteins made major contributions to this Steppke localization and activity. By analyzing GFP-tagged Arf and Arf-like small G proteins, we found that Arf1-GFP, Arf6-GFP and Arl4-GFP, but not Arf4-GFP, localized to furrows. However, analyses of embryos depleted of Arf1, Arf6 or Arl4 revealed either earlier defects than occur in embryos depleted of Steppke, or no detectable furrow defects, possibly because of redundancies, and thus it was difficult to assess how individual Arf small G proteins affect Steppke. Nonetheless, our data show that the Steppke PH domain and its conserved residues for binding to GTP-bound Arf G proteins have substantial effects on Steppke localization and activity in early Drosophila embryos.     

Arf,Arl, Arp and Sar proteins: a family of GTP-binding proteins with a structural device for 'front-back' communication

Arf proteins are important regulators of cellular traffic and the founding members of an expanding family of homologous proteins and genomic sequences. They depart from other small GTP-binding proteins by a unique structural device, which we call the 'interswitch toggle', that implements front–back communication from the N-terminus to the nucleotide binding site. Here we define the sequence and structural determinants that propagate information across the protein and identify them in all of the Arf family proteins other than Arl6 and Arl4/Arl7. The positions of these determinants lead us to propose that Arf family members with the interswitch toggle device are activated by a bipartite mechanism acting on opposite sides of the protein. The presence of this communication device might provide a more useful basis for unifying Arf homologs as a family than do the cellular functions of these proteins, which are mostly unrelated. We review available genomic sequences and functional data from this perspective, and identify a novel subfamily that we call Arl8.     

Arl2 and Arl3 regulate different microtubule-dependent processes

Arl2 and Arl3 are closely related members of the Arf family of regulatory GTPases that arose from a common ancestor early in eukaryotic evolution yet retain extensive structural, biochemical, and functional features. The presence of Arl3 in centrosomes, mitotic spindles, midzones, midbodies, and cilia are all supportive of roles in microtubule-dependent processes. Knockdown of Arl3 by siRNA resulted in changes in cell morphology, increased acetylation of alpha-tubulin, failure of cytokinesis, and increased number of binucleated cells. We conclude that Arl3 binds microtubules in a regulated manner to alter specific aspects of cytokinesis. In contrast, an excess of Arl2 activity, achieved by expression of the [Q70L]Arl2 mutant, caused the loss of microtubules and cell cycle arrest in M phase. Initial characterization of the underlying defects suggests a defect in the ability to polymerize tubulin in the presence of excess Arl2 activity. We also show that Arl2 is present in centrosomes and propose that its action in regulating tubulin polymerization is mediated at centrosomes. Somewhat paradoxically, no phenotypes were observed Arl2 expression was knocked down or Arl3 activity was increased in HeLa cells. We conclude that Arl2 and Arl3 have related but distinct roles at centrosomes and in regulating microtubule-dependent processes.     

The Arl4 family of small G proteins can recruit the cytohesin Arf6 exchange factors to the plasma membrane

The small GTPase Arf6 regulates endocytosis, actin dynamics, and cell adhesion, and one of its major activators is the exchange factor Arf nucleotide-binding site opener (ARNO), also called cytohesin-2 [1, 2]. ARNO must be recruited from the cytosol to the plasma membrane in order to activate Arf6, and in addition to a Sec7 nucleotide-exchange domain it contains a C-terminal pleckstrin homology (PH) domain that binds phosphoinositides [3, 4]. ARNO and its three relatives, cytohesin-1, Grp1/cytohesin-3, and cytohesin-4, are expressed as two splice variants, with either two or three glycines in a loop in the phosphoinositide-binding pocket of the PH domain [5, 6]. The diglycine form binds PtdIns(3,4,5)P(3) with high affinity and mediates recruitment of cytohesins to the plasma membrane in response to insulin and growth factors [7, 8]. However, the triglycine form has only micromolar affinity for both PtdIns(3,4,5)P(3) and PtdIns(4,5)P(2), affinities that are insufficient to confer membrane recruitment, raising the question of how the triglycine forms of cytohesins are regulated [5, 9]. Here we show that three related Arf-like GTPases of unknown function, Arl4a, Arl4c, and Arl4d, are able to recruit ARNO and other cytohesins to the plasma membrane by binding to their PH domains irrespective of whether they are in the diglycine or triglycine form. The Arl4 family thus defines a signal-transduction pathway that can mediate the plasma-membrane recruitment of cytohesins independently of a requirement for the generation of PtdIns(3,4,5)P(3).     

Arf-like GTPases: not so Arf-like after all

ADP-ribosylation factor (Arf) GTP-binding proteins are among the best-characterized members of the Ras superfamily of GTPases, with well-established functions in membrane-trafficking pathways. A recent watershed of genomic and structural information has identified a family of conserved related proteins: the Arf-like (Arl) GTPases. The best-characterized Arl protein, Arl2, regulates the folding of beta tubulin, and recent data suggest that Arl1 and Arf-related protein 1 (ARFRP1) are localized to the trans-Golgi network (TGN), where they function, in part, to regulate the tethering of endosome-derived transport vesicles. Other Arl proteins are localized to the cytosol, nucleus, cytoskeleton and mitochondria, which indicates that Arl proteins have diverse roles that are distinct from the known functions of traditional Arf GTPases.     

Arl5b is a Golgi-localised small G protein involved in the regulation of retrograde transport

Regulation of membrane transport is controlled by small G proteins, which include members of the Rab and Arf families. Whereas the role of the classic Arf family members are well characterized, many of the Arf-like proteins (Arls) remain poorly defined. Here we show that Arl5a and Arl5b are localised to the trans-Golgi in mammalian cells, and furthermore have identified a role for Arl5b in the regulation of retrograde membrane transport from endosomes to the trans-Golgi network (TGN). The constitutively active Arl5b (Q70L)-GFP mutant was localised efficiently to the Golgi in HeLa cells whereas the dominant-negative Arl5b (T30N)-GFP mutant was dispersed throughout the cytoplasm and resulted in perturbation of the Golgi apparatus. Stable HeLa cells expressing GFP-tagged Arl5b (Q70L) showed an increased rate of endosome-to-Golgi transport of the membrane cargo TGN38 compared with control HeLa cells. Depletion of Arl5b by RNAi resulted in an alteration in the intracellular distribution of mannose-6-phosphate receptor, and significantly reduced the endosome-to-TGN transport of the membrane cargo TGN38 and of Shiga toxin, but had no affect on the anterograde transport of the cargo E-cadherin. Collectively these results suggest that Arl5b is a TGN-localised small G protein that plays a key role in regulating transport along the endosome-TGN pathway.     

Properties of the interaction of Arf-like protein 2 with PDEdelta

Arf-like proteins (Arl) share certain characteristic features with the Arf subfamily of Ras superfamily proteins, but their function is unknown. Here, we show by a variety of spectroscopic techniques that Arl2, unlike most other Ras-related proteins, has micromolar rather than picomolar affinity for nucleotides. As a consequence of low affinity, nucleotide dissociation rates are rather fast, arguing that it is not regulated by guanine nucleotide exchange factors. Arl2 is isolated as prey in a yeast double hybrid screen using phosphodiesterase 6delta (PDEdelta) as bait. This interaction is dependent on GTP, and the binding of PDEdelta substantially stabilizes GTP binding, increasing affinity and decreasing dissociation rates by a similar factor. Among all Arl proteins tested, PDEdelta only interacted with the closely related proteins Arl2 and Arl3, strongly suggesting that Arl2/3 are specific regulators of PDEdelta.     

Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4

Dysfunctions of primary cilia and cilia‐derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans‐Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase‐activating protein ASAP1. ASAP1 mediates GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates budding of post‐TGN carriers, along with FIP3 and Rab11. The Arf4 mutant I46D, impaired in ASAP1‐mediated GTP hydrolysis, causes aberrant rhodopsin trafficking and cytoskeletal and morphological defects resulting in retinal degeneration in transgenic animals. As the VxPx motif is present in other ciliary membrane proteins, the Arf4‐based targeting complex is most likely a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.     

Mutations in the cilia gene ARL13B lead to the classical form of Joubert syndrome

Joubert syndrome (JS) and related disorders are a group of autosomal-recessive conditions sharing the "molar tooth sign" on axial brain MRI, together with cerebellar vermis hypoplasia, ataxia, and psychomotor delay. JS is suggested to be a disorder of cilia function and is part of a spectrum of disorders involving retinal, renal, digital, oral, hepatic, and cerebral organs. We identified mutations in ARL13B in two families with the classical form of JS. ARL13B belongs to the Ras GTPase family, and in other species is required for ciliogenesis, body axis formation, and renal function. The encoded Arl13b protein was expressed in developing murine cerebellum and localized to the cilia in primary neurons. Overexpression of human wild-type but not patient mutant ARL13B rescued the Arl13b scorpion zebrafish mutant. Thus, ARL13B has an evolutionarily conserved role mediating cilia function in multiple organs.     

Structural basis for Arl3‐specific release of myristoylated ciliary cargo from UNC119

Access to the ciliary membrane for trans‐membrane or membrane‐associated proteins is a regulated process. Previously, we have shown that the closely homologous small G proteins Arl2 and Arl3 allosterically regulate prenylated cargo release from PDEδ. UNC119/HRG4 is responsible for ciliary delivery of myristoylated cargo. Here, we show that although Arl3 and Arl2 bind UNC119 with similar affinities, only Arl3 allosterically displaces cargo by accelerating its release by three orders of magnitude. Crystal structures of Arl3 and Arl2 in complex with UNC119a reveal the molecular basis of specificity. Contrary to previous structures of GTP‐bound Arf subfamily proteins, the N‐terminal amphipathic helix of Arl3·GppNHp is not displaced by the interswitch toggle but remains bound on the surface of the protein. Opposite to the mechanism of cargo release on PDEδ, this induces a widening of the myristoyl binding pocket. This leads us to propose that ciliary targeting of myristoylated proteins is not only dependent on nucleotide status but also on the cellular localization of Arl3.