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.
Myristoyl-CoA:protein N-myristoyltransferase (NMT), an essential protein in Trypanosoma brucei and Leishmania major, catalyses the covalent attachment of the fatty acid myristate to the N-terminus of a range of target proteins. In order to define the essential targets contributing to lethality in the absence of NMT activity, we have focused on the ADP-ribosylation factor (Arf) family of GTP-binding proteins, as growth arrest in Saccharomyces cerevisiae mutants with reduced NMT activity correlates with a decrease in N-myristoylated Arf proteins. We have identified nine Arf/Arls in the T. brucei and T. cruzi genomes and ten in L. major. Characterization of the T. brucei ARL1 homologue has revealed that the protein is localized in the Golgi apparatus and is expressed only in the mammalian bloodstream form of the parasite and not in the insect procyclic stage. This is the only reported example to date of a differentially expressed ARL1 homologue in any species. We have used RNA interference to demonstrate that ARL1 is essential for viability in T. brucei bloodstream parasites. Prior to cell death, depletion of ARL1 protein in bloodstream parasites results in abnormal morphology, including disintegration of the Golgi structure, multiple flagella and nuclei, and the presence of large numbers of vesicles. The cells have only a minor apparent defect in endocytosis but exocytosis of variant surface glycoprotein to the parasite surface is significantly delayed. RNA interference of ARL1 in procyclic cells has no effect on parasite growth or morphology. Our results suggest that there may be different pathways regulating Golgi structure and function in the two major life cycle stages of T. brucei.
Trypanosoma; ADP-ribosylation factor; RNA interference; Golgi proteins
Small G-proteins of the ADP-ribosylation-factor-like (Arl) subfamily have been shown to be crucial to ciliogenesis and cilia maintenance. Active Arl3 is involved in targeting and releasing lipidated cargo proteins from their carriers PDE6δ and UNC119a/b to the cilium. However, the guanine nucleotide exchange factor (GEF) which activates Arl3 is unknown. Here we show that the ciliary G-protein Arl13B mutated in Joubert syndrome is the GEF for Arl3, and its function is conserved in evolution. The GEF activity of Arl13B is mediated by the G-domain plus an additional C-terminal helix. The switch regions of Arl13B are involved in the interaction with Arl3. Overexpression of Arl13B in mammalian cell lines leads to an increased Arl3·GTP level, whereas Arl13B Joubert-Syndrome patient mutations impair GEF activity and thus Arl3 activation. We anticipate that through Arl13B’s exclusive ciliary localization, Arl3 activation is spatially restricted and thereby an Arl3·GTP compartment generated where ciliary cargo is specifically released.
Most types of cells in humans and other animals have slender, hair-like structures known as cilia that project out of the cell surface. These structures sense and respond to signals from the external environment and are crucial for organisms to develop normally. Defects in cilia can lead to many serious conditions such as Joubert syndrome, which affects the development of the brain and other organs in humans.
The Arl family of “G-proteins” play important roles in the formation and operation of cilia. They contain a section called a G-protein domain whose activity can be switched on by interactions with other proteins called guanine nucleotide exchange factors (or GEFs for short). A member of the Arl family called Arl3 is found in higher amounts in cilia than in other parts of the cell. It is involved in the transport of proteins to the cilia from other parts of the cell, but it is not known which GEFs are able to activate it.
Here, Gotthardt, Lokaj et al. used several biochemical techniques to show that another member of the Arl family called Arl13B actually acts as a GEF to activate Arl3 in cilia. Arl13B is only found in cilia and the GEF activity relies on its G-protein domain and another element at one end called a C-terminal helix. Previous studies have shown that mutations in the gene that encodes Arl13B can cause Joubert syndrome in humans. Gotthardt, Lokaj et al. found that mutant forms of Arl13B had significantly lower GEF activity than normal Arl13B proteins.
Together, Gotthardt, Lokaj et al.’s findings provide an explanation for why Arl3 is only activated in cilia even though it is found throughout the cell. Further work is needed to understand how the activity of Arl13B is regulated.
Chlamydomonas reinhardtii; G-protein; Arf-like proteins; Guanine nucleotide exchange factor; cilium; protein trafficking; Human; Mouse; Other
ARL4D, ARL4A, and ARL4C are closely related members of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of GTPases. All three ARL4 proteins contain nuclear localization signals (NLSs) at their C-termini and are primarily found at the plasma membrane, but they are also present in the nucleus and cytoplasm. ARF function and localization depends on their controlled binding and hydrolysis of GTP. Here we show that GTP-binding-defective ARL4D is targeted to the mitochondria, where it affects mitochondrial morphology and function. We found that a portion of endogenous ARL4D and the GTP-binding-defective ARL4D mutant ARL4D(T35N) reside in the mitochondria. The N-terminal myristoylation of ARL4D(T35N) was required for its localization to mitochondria. The localization of ARL4D(T35N) to the mitochondria reduced the mitochondrial membrane potential (ΔΨm) and caused mitochondrial fragmentation. Furthermore, the C-terminal NLS region of ARL4D(T35N) was required for its effect on the mitochondria. This study is the first to demonstrate that the dysfunctional GTP-binding-defective ARL4D is targeted to mitochondria, where it subsequently alters mitochondrial morphology and membrane potential.
The Trypanosoma brucei orthologue of Arl2 is essential for viability in bloodstream form parasites. RNA interference causes inhibition of cleavage furrow formation and loss of acetylated α-tubulin.
The Arf-like (Arl) small GTPases have a diverse range of functions in the eukaryotic cell. Metazoan Arl2 acts as a regulator of microtubule biogenesis, binding to the tubulin-specific chaperone cofactor D. Arl2 also has a mitochondrial function through its interactions with BART and ANT-1, the only member of the Ras superfamily to be found in this organelle to date. In the present study, we describe characterization of the Arl2 orthologue in the protozoan parasite Trypanosoma brucei. Modulation of TbARL2 expression in bloodstream form parasites by RNA interference (RNAi) causes inhibition of cleavage furrow formation, resulting in a severe defect in cytokinesis and the accumulation of multinucleated cells. RNAi of TbARL2 also results in loss of acetylated α-tubulin but not of total α-tubulin from cellular microtubules. While overexpression of TbARL2myc also leads to a defect in cytokinesis, an excess of untagged protein has no effect on cell division, demonstrating the importance of the extreme C-terminus in correct function. TbARL2 overexpressing cells (either myc-tagged or untagged) have an increase in acetylated α-tubulin. Our data indicate that Arl2 has a fundamentally conserved role in trypanosome microtubule biogenesis that correlates with α-tubulin acetylation.
ANT-1, adenine nucleotide transporter 1; Arf, ADP-ribosylation factor; Arl, ADP-ribosylation factor-like; BART, ARF-like 2-binding protein; BSF, bloodstream form; dsRNA, double-stranded RNA; ELMO, Engulfment and Cell Motility; ELMOD, Engulfment and Cell Motility Domain; ER, endoplasmic reticulum; FAZ, flagellum attachment zone; GAP, GTPase activating protein; HRG4, human retinal gene 4; NMT, myristoyl-CoA:protein N-myristoyltransferase; PP2A, protein phosphatase 2A; RNAi, RNA interference; RP2, retinitis pigmentosa 2; Trypanosoma brucei; Arl2; Cytokinesis; Tubulin acetylation
ADP-ribosylation factor (ARF) and ARF-like (ARL) proteins are members of the ARF family, which are critical components of several different vesicular trafficking pathways. ARFs have little or no detectable GTPase activity without the assistance of a GTPase-activating protein (GAP). Here, we demonstrate that yeast Gcs1p exhibits GAP activity toward Arl1p and Arf1p in vitro, and Arl1p can interact with Gcs1p in a GTP-dependent manner. Arl1p was observed both on trans-Golgi and in cytosol and was recruited from cytosol to membranes in a GTP-dependent manner. In gcs1 mutant cells, the fraction of Arl1p in cytosol relative to trans-Golgi was less than it was in wild-type cells. Increasing Gcs1p levels returned the distribution toward that of wild-type cells. Both Arl1p and Gcs1p influenced the distribution of Imh1p, an Arl1p effector. Our data are consistent with the conclusion that Arl1p moves in a dynamic equilibrium between trans-Golgi and cytosol, and the release of Arl1p from membranes in cells requires the hydrolysis of bound GTP, which is accelerated by Gcs1p.
The yeast protein Gcs1 is a GTPase-activating protein (GAP) for Arf and Arl1 G-proteins that regulate distinct steps of vesicular transport. Absence of a GAP-independent function of Gcs1 results in dysregulated Arl1, which in turn impairs cell growth and endosomal transport. Impairing vesicle-tethering pathways removes dysregulated Arl1 and alleviates these defects.
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.
The ADP-ribosylation factor-like 2 (ARL2) GTPase and its binding
partner binder of ARL2 (BART) are ubiquitously expressed in rodent and
human tissues and are most abundant in brain. Both ARL2 and BART are
predominantly cytosolic, but a pool of each was found associated with
mitochondria in a protease-resistant form. ARL2 was found to lack
covalent N-myristoylation, present on all other members of the ARF
family, thereby preserving the N-terminal amphipathic α-helix as a
potential mitochondrial import sequence. An overlay assay was developed
to identify binding partners for the BART·ARL2·GTP complex and
revealed a specific interaction with a protein in bovine brain
mitochondria. Purification and partial microsequencing identified the
protein as an adenine nucleotide transporter (ANT). The overlay assay
was performed on mitochondria isolated from five different tissues from
either wild-type or transgenic mice deleted for ANT1. Results confirmed
that ANT1 is the predominant binding partner for the BART·ARL2·GTP
complex and that the structurally homologous ANT2 protein does not bind
the complex. Cardiac and skeletal muscle mitochondria from
mice had increased levels of ARL2, relative to that seen in
mitochondria from wild-type animals. We conclude that the amount of
ARL2 in mitochondria is subject to regulation via an ANT1-sensitive
pathway in muscle tissues.
The expansive family of metazoan ADP-ribosylation factor and ADP-ribosylation factor-like small GTPases is known to play essential roles in modulating membrane trafficking and cytoskeletal functions. Here, we present the crystal structure of ARL6, mutations in which cause Bardet-Biedl syndrome (BBS3), and reveal its unique ring-like localization at the distal end of basal bodies, in proximity to the so-called ciliary gate where vesicles carrying ciliary cargo fuse with the membrane. Overproduction of GDP- or GTP-locked variants of ARL6/BBS3 in vivo influences primary cilium length and abundance. ARL6/BBS3 also modulates Wnt signaling, a signal transduction pathway whose association with cilia in vertebrates is just emerging. Importantly, this signaling function is lost in ARL6 variants containing BBS-associated point mutations. By determining the structure of GTP-bound ARL6/BBS3, coupled with functional assays, we provide a mechanistic explanation for such pathogenic alterations, namely altered nucleotide binding. Our findings therefore establish a previously unknown role for ARL6/BBS3 in mammalian ciliary (dis)assembly and Wnt signaling and provide the first structural information for a BBS protein.
Diseases; Protein/Structure; Centriole; Signal Transduction; Subcellular Organelles; ARL6; BBS3; Bardet-Biedl Syndrome; Cilia; Small GTPase
The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the α/β- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a β-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo.
Arls; G proteins; chaperones; microtubules; cytoskeleton
The GTPase Arf1 is the major regulator of vesicle traffic at both the cis- and trans-Golgi. Arf1 is activated at the cis-Golgi by the guanine nucleotide exchange factor (GEF) GBF1 and at the trans-Golgi by the related GEF BIG1 or its paralog, BIG2. The trans-Golgi-specific targeting of BIG1 and BIG2 depends on the Arf-like GTPase Arl1. We find that Arl1 binds to the dimerization and cyclophilin binding (DCB) domain in BIG1 and report a crystal structure of human Arl1 bound to this domain. Residues in the DCB domain that bind Arl1 are required for BIG1 to locate to the Golgi in vivo. DCB domain-binding residues in Arl1 have a distinct conformation from those in known Arl1-effector complexes, and this plasticity allows Arl1 to interact with different effectors of unrelated structure. The findings provide structural insight into how Arf1 GEFs, and hence active Arf1, achieve their correct subcellular distribution.
•Arl1 binds the N-terminal DCB domain of the Arf1-GEF BIG1•Structure of the human DCBBIG1/Arl1 complex is solved at 2.3 Å resolution•The Arl1 binding surface on the DCB domain is unrelated to known Arl1 effectors•Structure-guided mutation shows BIG1 needs Ar11 binding for Golgi targeting
The GTPase Arf1 is essential for Golgi function and is activated on the trans-Golgi by the BIG1 exchange factor. A second GTPase, Arl1, is required for BIG1 recruitment. Galindo et al. find that Arl1 binds to the DCB domain of BIG1 and determine the structure of human Arl1 bound to this domain.
The small GTPase Arl6 is implicated in the ciliopathic human genetic disorder Bardet–Biedl syndrome, acting at primary cilia in recruitment of the octomeric BBSome complex, which is required for specific trafficking events to and from the cilium in eukaryotes. Here we describe functional characterisation of Arl6 in the flagellated model eukaryote Trypanosoma brucei, which requires motility for viability. Unlike human Arl6 which has a ciliary localisation, TbARL6 is associated with electron-dense vesicles throughout the cell body following co-translational modification by N-myristoylation. Similar to the related protein ARL-3A in T. brucei, modulation of expression of ARL6 by RNA interference does not prevent motility but causes a significant reduction in flagellum length. Tubulin is identified as an ARL6 interacting partner, suggesting that ARL6 may act as an anchor between vesicles and cytoplasmic microtubules. We provide evidence that the interaction between ARL6 and the BBSome is conserved in unicellular eukaryotes. Overexpression of BBS1 leads to translocation of endogenous ARL6 to the site of exogenous BBS1 at the flagellar pocket. Furthermore, a combination of BBS1 overexpression and ARL6 RNAi has a synergistic inhibitory effect on cell growth. Our findings indicate that ARL6 in trypanosomes contributes to flagellum biogenesis, most likely through an interaction with the BBSome.
► The BBSome-associated protein ARL6 localises to vesicles in Trypanosoma brucei. ► T. brucei ARL6 is N-myristoylated. ► RNAi knockdown causes a decrease in flagellum length but does not affect motility. ► TbARL6 binds to tubulin and has a relatively low affinity for guanine nucleotides. ► The BBSome subunit BBS1 and ARL6 are functionally linked in trypanosomes.
Arf, ADP-ribosylation factor; Arl, ADP-ribosylation factor-like; Arl6ip, Arl6 interacting protein; BBS, Bardet–Biedl syndrome; BBS1, Bardet–Biedl syndrome 1 protein; BSF, bloodstream form; ConA, Concanavalin A; GEF, guanine nucleotide exchange factor; GPCR, G-protein coupled receptor; HRG4, human retinal gene 4; IFT, intraflagellar transport; ITC, isothermal titration calorimetry; MANT, N-methylanthraniloyl; MAP2, microtubule associated protein 2; NES, nuclear export signal; NLS, nuclear localisation signal; NMT, myristoyl-CoA:protein N-myristoyltransferase; PCF, procyclic form; PCM1, pericentriolar material 1; PFR, paraflagellar rod; PM, plasma membrane; RNAi, RNA interference; RP2, retinitis pigmentosa protein 2; TAP, tandem affinity purification; TiEM, transmission immuno-electron microscopy; Trypanosoma brucei; Arl6; BBSome; BBS1; Flagellum; Tubulin
Drosophila Arl2 governs neuroblast asymmetric cell division through regulation of microtubule growth and localization of Msps to centrosomes.
Asymmetric division of neural stem cells is a fundamental strategy to balance their self-renewal and differentiation. It is long thought that microtubules are not essential for cell polarity in asymmetrically dividing Drosophila melanogaster neuroblasts (NBs; neural stem cells). Here, we show that Drosophila ADP ribosylation factor like-2 (Arl2) and Msps, a known microtubule-binding protein, control cell polarity and spindle orientation of NBs. Upon arl2 RNA intereference, Arl2-GDP expression, or arl2 deletions, microtubule abnormalities and asymmetric division defects were observed. Conversely, overactivation of Arl2 leads to microtubule overgrowth and depletion of NBs. Arl2 regulates microtubule growth and asymmetric division through localizing Msps to the centrosomes in NBs. Moreover, Arl2 regulates dynein function and in turn centrosomal localization of D-TACC and Msps. Arl2 physically associates with tubulin cofactors C, D, and E. Arl2 functions together with tubulin-binding cofactor D to control microtubule growth, Msps localization, and NB self-renewal. Therefore, Arl2- and Msps-dependent microtubule growth is a new paradigm regulating asymmetric division of neural stem cells.
The small G proteins of the Arf family play critical roles in membrane trafficking and cytoskeleton organization. However, the function of some members of the family remains poorly understood including Arl5 which is widely conserved in eukaryotes. Humans have two closely related Arl5 paralogues (Arl5a and Arl5b), and both Arl5a and Arl5b localize to the trans-Golgi with Arl5b being involved in retrograde traffic from endosomes to the Golgi apparatus. To investigate the function of Arl5, we have used Drosophila melanogaster as a model system. We find that the single Arl5 orthologue in Drosophila also localizes to the trans-Golgi, but flies lacking the Arl5 gene are viable and fertile. By using both liposome and column based affinity chromatography methods we find that Arl5 interacts with the Golgi-associated retrograde protein (GARP) complex that acts in the tethering of vesicles moving from endosomes to the trans-Golgi network (TGN). In Drosophila tissues the GARP complex is partially displaced from the Golgi when Arl5 is absent, and the late endosomal compartment is enlarged. In addition, in HeLa cells GARP also becomes cytosolic upon depletion of Arl5b. These phenotypes are consistent with a role in endosome-to-Golgi traffic, but are less severe than loss of GARP itself. Thus it appears that Arl5 is one of the factors that directs the recruitment of the GARP complex to the trans-Golgi, and this function is conserved in both flies and humans.
Membrane traffic; Golgi apparatus; Arf family GTPase
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 α-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.
We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.
We show Arl13b is localized to the ciliary membrane and regulates tubulin modifications and ciliary length in vitro. Significantly, we found that Smoothened is enriched in Arl13b null fibroblasts, even without Sonic hedgehog stimulation, but that Glis are not similarly enriched.
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.
By exploiting NK cell LROs (known as lytic granules) as a model, a new role is defined for Arl8b in regulating motility and exocytosis of lytic granules of NK cells. Not only lytic granules but also the MTOC is unable to polarize toward the immune synapse formed between the NK cell and its target in Arl8b-depleted NK cells.
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.
A novel Arl13b effector is identified. Sec8 and Sec5 exocyst subunits are shown to interact with Arl13b in a GTP-dependent manner. This interaction is synergistic in phenotypes caused by impaired ciliogenesis during zebrafish development. Similar to Sec10, Arl13b depletion in mouse kidneys inhibits ciliogenesis and leads to the formation of cysts.
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.
Certain residues within proteins are highly conserved across very distantly related organisms, yet their (presumably critical) structural or mechanistic roles are completely unknown. To obtain clues regarding such residues within Arf and Arf-like (Arf/Arl) GTPases--which function as on/off switches regulating vesicle trafficking, phospholipid metabolism and cytoskeletal remodeling--I apply a new sampling procedure for comparative sequence analysis, termed multiple category Bayesian Partitioning with Pattern Selection (mcBPPS).
The mcBPPS sampler classified sequences within the entire P-loop GTPase class into multiple categories by identifying those evolutionarily-divergent residues most likely to be responsible for functional specialization. Here I focus on categories of residues that most distinguish various Arf/Arl GTPases from other GTPases. This identified residues whose specific roles have been previously proposed (and in some cases corroborated experimentally and that thus serve as positive controls), as well as several categories of co-conserved residues whose possible roles are first hinted at here. For example, Arf/Arl/Sar GTPases are most distinguished from other GTPases by a conserved aspartate residue within the phosphate binding loop (P-loop) and by co-conserved residues nearby that, together, can form a network of salt-bridge and hydrogen bond interactions centered on the GTPase active site. Residues corresponding to an N-[VI] motif that is conserved within Arf/Arl GTPases may play a role in the interswitch toggle characteristic of the Arf family, whereas other, co-conserved residues may modulate the flexibility of the guanine binding loop. Arl8 GTPases conserve residues that strikingly diverge from those typically found in other Arf/Arl GTPases and that form structural interactions suggestive of a novel interswitch toggle mechanism.
This analysis suggests specific mutagenesis experiments to explore mechanisms underlying GTP hydrolysis, nucleotide exchange and interswitch toggling within Arf/Arl GTPases. More generally, it illustrates how the mcBPPS sampler can complement traditional evolutionary analyses by providing an objective, quantitative and statistically rigorous way to explore protein functional-divergence in molecular detail. Because the sampler classifies the input sequences at the same time, it can be used to generate subgroup profiles, in which functionally-divergent categories of residues are annotated automatically.
This article was reviewed by Frank Eisenhaber, L Aravind and Daniel Gaston (nominated by Eric Bapteste). For the full reviews, go to the Reviewers' comments section.
This unit describes techniques and approaches that can be used to study the functions of the ADP-ribosylation factor (Arf) GTP-binding proteins in cells. There are 6 mammalian Arfs and many more Arf-like proteins (Arls) and these proteins are conserved in eukaryotes from yeast to man. Like all GTPases, Arfs cycle between GDP-bound, inactive and GTP-bound active conformations, facilitated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that catalyze GTP binding and hydrolysis respectively. Here we describe approaches that can be taken to examine the localization and function of Arf and Arl proteins in cells (Protocol 1). We also provide a simple protocol for measuring activation (GTP-binding) of specific Arf proteins in cells using a pull-down assay (Protocol 2). We then discuss approaches that can be taken to assess function of GEFs and GAPs in cells (Protocol 3).
Arf; GTP-binding proteins; guanine nucleotide exchange factors; GTPase activating proteins
Members of the ADP-ribosylation factor (Arf) family of small GTP-binding (G) proteins regulate several aspects of membrane trafficking, such as vesicle budding, tethering and cytoskeleton organization. Arf family members, including Arf-like (Arl) proteins have been implicated in several essential cellular functions, like cell spreading and migration. These functions are used by cancer cells to disseminate and invade the tissues surrounding the primary tumor, leading to the formation of metastases. Indeed, Arf and Arl proteins, as well as their guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) have been found to be abnormally expressed in different cancer cell types and human cancers. Here, we review the current evidence supporting the involvement of Arf family proteins and their GEFs and GAPs in cancer progression, focusing on 3 different mechanisms: cell-cell adhesion, integrin internalization and recycling, and actin cytoskeleton remodeling.
actin cytoskeleton remodeling; Arf; Arl; cell adhesion; cell migration; integrin recycling; small GTP-binding proteins
ADP-ribosylation-like factor 6 interacting protein 5 (Arl6ip5), which belongs to the prenylated rab-acceptor-family, has an important role in exocytic protein trafficking, glutathione metabolism and involves in cancer progression. However, its expression pattern and functional role in bone are unknown. Here we demonstrate that Arl6ip5 knock-out mice (Arl6ip5 Δ2/Δ2) show marked decrease of bone mineral density, trabecular bone volume and trabecular thickness. Histomorphometric studies reveal that bone formation parameters are decreased but bone resorption parameters and mRNA level of osteoclast-specific markers are increased in Arl6ip5Δ2/Δ2 mice. In osteoblast, we demonstrate that Arl6ip5 abundantly expresses in osteoblastic cells and is regulated by bone metabolism-related hormones and growth factors. In vitro analysis reveals that osteoblast proliferation and differentiation are impaired in Arl6ip5 knocked-down and deficient primary osteoblast. Arl6ip5 is also found to function as an ER calcium regulator and control calmodulin signaling for osteoblast proliferation. Moreover, Arl6ip5 insufficiency in osteoblast induces ER stress and enhances ER stress-mediated apoptosis. CCAAT/enhancer-binding protein homologous protein (Chop) is involved in the regulation of apoptosis and differentiation in Arl6ip5 knocked-down osteoblasts. For osteoclastogenesis, Arl6ip5 insufficiency in osteoclast precursors has no effect on osteoclast formation. However, knocked-down osteoblastic Arl6ip5 induces receptor activator of nuclear factor-κB ligand (RANKL) expression and enhances osteoclastogenesis. In addition, ER stress and Chop are involved in the RANKL expression in Arl6ip5 knocked-down osteoblasts. In conclusion, we demonstrate that Arl6ip5 is a novel regulator of bone formation in osteoblasts.
The BBSome is a coat-like ciliary trafficking complex composed of proteins mutated in Bardet-Biedl syndrome (BBS). A critical step in BBSome-mediated sorting is recruitment of the BBSome to membranes by the GTP-bound Arf-like GTPase ARL6. We have determined crystal structures of C.reinhardtii ARL6-GDP, ARL6-GTP and the ARL6-GTP-BBS1 complex. The structures demonstrate how ARL6-GTP binds the BBS1 β-propeller at blades 1 and 7 and explain why GTP–but not GDP–bound ARL6 can recruit the BBSome to membranes. Single point mutations in the ARL6-GTP-BBS1 interface abolish the interaction of ARL6 with the BBSome and prevent the import of BBSomes into cilia. Furthermore, we show that BBS1 with the M390R mutation, responsible for 30% of all reported BBS disease cases, fails to interact with ARL6-GTP providing a molecular rationale for patient pathologies.
The BBSome is a coat-like ciliary trafficking complex composed of proteins mutated in Bardet-Biedl syndrome (BBS). A critical step in BBSome-mediated sorting is recruitment of the BBSome to membranes by the GTP–bound Arf-like GTPase ARL6. We have determined crystal structures of C. reinhardtii ARL6–GDP, ARL6–GTP and the ARL6–GTP–BBS1 complex. The structures demonstrate how ARL6–GTP binds the BBS1 β-propeller at blades 1 and 7 and explain why GTP– but not GDP–bound ARL6 can recruit the BBSome to membranes. Single point mutations in the ARL6–GTP–BBS1 interface abolish the interaction of ARL6 with the BBSome and prevent the import of BBSomes into cilia. Furthermore, we show that BBS1 with the M390R mutation, responsible for 30% of all reported BBS disease cases, fails to interact with ARL6–GTP providing a molecular rationale for patient pathologies.