Arf (ADP-ribosylation factor) proteins were first discovered as membrane-bound cofactors in the cholera-toxin-dependent ADP-ribosylation of Gα (Kahn and Gilman 1984). They are a family of Ras-related small GTP-binding proteins, approximately 21 kDa in size, and are expressed abundantly in all eukaryotic cells and some bacteria (Quinn 1995; Dong et al. 2007). Arfs regulate membrane trafficking and actin cytoskeleton dynamics, which are important for cellular events such as endocytosis, cell secretion, cell adhesion, cell migration, and neurite outgrowth (D’Souza-Schorey and Chavrier 2006). They act as molecular switches for many cellular pathways by cycling between inactive GDP-binding and active GTP-binding forms. Arf proteins are activated by guanine-nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs) (Quinn 1995; Donaldson and Honda 2005). A huge number of Arf GEFs and GAPs have been identified, characterized, and classified (Kahn et al. 2005; Gillingham and Munro 2007). The Arf family consists of six mammalian Arfs (Arfs1–6) (Kahn et al. 2006). Arfs 1–5 localize to and act at the Golgi, whereas Arf6 localizes to and acts at the cell surface (Donaldson and Honda 2005; Gillingham and Munro 2007). The biological functions of Arfs occur through their specific interactions with several downstream effectors (Kahn et al. 2006).
In addition to Arfs, the Arf family contains a number of Arf-like (Arl) proteins. Arl proteins are related to Arf proteins structurally but not functionally (Kahn et al. 2006). Arl proteins share 40–60% sequence identity with Arfs. In contrast to Arf proteins, Arls cannot act as cofactors in cholera-toxin-catalyzed ADP-ribosylation of Gαs, cannot activate phospholipase D (PLD), and cannot functionally complement Arf1 or Arf2 in Saccharomyces cerevisiae (Price et al. 1988; Stearns et al. 1990; Tamkun et al. 1991; Hong 1998; Sebald et al. 2003; Kahn et al. 2006). These Arls contain an amino-terminal amphipathic helix and the “interswitch” region (Pasqualato et al. 2002; Li et al. 2004; Kahn et al. 2006). In humans, there are 22 Arls (Arls1–22), which are well conserved in evolution (Hofmann and Munro 2006). Generally, membrane binding of Arl proteins occurs via an N-terminal amphipathic helix that is inserted into the lipid bilayer once activated. In addition, this N-terminal amphipathic helix contains a myristoyl or an acetyl group (Gillingham and Munro 2007).
Protein Function and Regulation of Activity
The main functions of Arl8b discovered so far include a role in lysosome motility (Hofmann and Munro 2006), delivery of endocytosed macromolecules to lysosomes (Nakae 2010), axonal transport of presynaptic cargo (Klassen 2010), neurite formation (Haraguchi et al. 2006), and chromosome segregation (Okai et al. 2004). Arl8b has been found to localize to lysosomes, and its overexpression resulted in a microtubule-dependent accumulation of lysosomes at the cell periphery (Bagshaw et al. 2006; Hofmann and Munro 2006). Wild-type Arl8 or a constitutively active (GTP-bound) mutant (Arl8-Q75L), but not a constitutively inactive (GDP-bound) mutant (Arl8-T34N), localizes to lysosomes when expressed exogenously in mammalian cells, indicating that the lysosomal localization of Arl8b depends on its guanine-nucleotide-bound status (Bagshaw et al. 2006). As previously mentioned, membrane binding of the Arf family proteins generally occurs via an N-terminal myristoyl group. However, Arl8b does not contain this motif but instead contains an N-terminally acetylated Met-Leu, which is essential for its lysosomal localization (Hofmann and Munro 2006). RNA-interference-mediated knockdown of human Nα-terminal acetyl transferase complex C (hMak3), which acetylates Met-Leu protein N termini, alters the subcellular localization of Arl8b, supporting the hypothesis that Arl8b is a hMak3 substrate in vivo (Starheim et al. 2009).
Arl8b has been shown to be involved in microtubule-related functions based on coimmunoprecipitation of Arl8 with β-tubulin and chromosomal segregation defects in Drosophila cell lines following short interfering (si)RNA-mediated knockdown of Arl8. Overexpression of Arl8 mutants that lacked the putative effector domains but still contained tubulin-binding capabilities induced micronucleus formation. The authors concluded that Arl8b may act as a molecular switch to regulate chromosomal segregation by associating with microtubules (Okai et al. 2004).
It has been demonstrated that Arl8 localizes to lysosomes and its involvement in late endosome-lysosome fusion in C. elegans. A loss of Arl8 in C. elegans caused an increase in number of late endosome-lysosome vesicles, which are smaller than those in worms expressing wild-type Arl8. In an Arl8 knockout (arl8) mutant, late endosomal vesicles are unable to fuse with lysosomes and thus a loss of Arl8 critically reduces late endosome-lysosome hybrid formation, which in turn affects the delivery of endocytosed macromolecules to lysosomes. These results suggest that Arl8 may be important in the biogenesis and function of lysosome-related organelles. In addition, homozygous arl8 worms from heterozygous arl8 mothers develop to fertile adults, whereas hermaphrodites homozygous for arl8 produce no viable embryos, suggesting that Arl8 is required maternally for embryonic development. Introduction of wild-type Arl8 or a constitutively active Arl8-Q75L mutant, but not a constitutively inactive Arl8-T34N mutant, efficiently rescued the embryonic lethality of the arl8 mutant, suggesting the importance of Arl8 in its GTP-bound active form for normal embryogenesis. Furthermore, the expression of human Arl8 proteins in C. elegans arl8 mutants could also rescue the embryonic lethality, indicating that Arl8 has a conserved role in multicellular animals (Nakae 2010).
As mentioned above, Arl8b has been shown to localize with microtubules to the spindle midzone in late mitosis and to be involved in chromosomal segregation (Okai et al. 2004). However, two later studies found no apparent mitotic spindle localization of Arl8b in mammalian cell lines other than PC12 cells tested (Hofmann and Munro 2006; Nakae 2010). The authors concluded that this role may be important in certain cell types under specific conditions.
It has also been suggested that Arl8b may have an important role in neurite formation because Arl8b, when overexpressed, accumulates at the growth cones in primary neurons and greatly affects the morphology of human embryonic kidney (HEK)293 cells by inducing the formation of long protrusions (Haraguchi et al. 2006). In the absence of Arl8, presynaptic cargo prematurely aggregates in the axons in C. elegans, indicating that Arl8 acts as a regulator of presynaptic assembly (Klassen 2010).
A study has showed higher levels of Arl8b gene expression in bovine embryos that resulted in calf delivery than in those that resulted in no pregnancy (Salilew-Wondim et al. 2010). This study highlighted the potential of Arl8b gene expression pattern in embryos as a predictor of pregnancy success in cattle. Arl8b has been identified as an endogenous reference gene (ERG) using multiplatform expression data and validated for quantitative gene expression analysis (Kwon et al. 2009). ERGs are useful in normalization of mRNA levels, which is essential for an accurate comparison of gene expression between different samples. Several studies have reported the expression variability of traditional ERGs (tERGs) such as GAPDH and ACTB in various tissues or disease status. It has been suggested that Arl8 is a better reference gene than tERGs owing to its greater expression stability.
A recent study has shown that knockdown of Arl8b (along with Arl8a) resulted in disruption of the centrifugal movement of lysosomes along microtubules and reduction in the nutrient-dependent activation of mammalian target of rapamycin (mTOR)-C1 (Korolchuk et al. 2011). In contrast, overexpression of Arl8b increased mTOR-C1 activity by increasing the number of lysosomes at the tip of cell protrusions. These findings suggest that the positioning of lysosomes regulates the mTOR-C1 activity. Inhibiting mTOR-C1 activity by restricting lysosomes transport to the cell periphery through the downregulation of Arl8b and Arl8a increases the number of autophagosomes formed even when cells are maintained in nutrient-rich conditions. Moreover, an increase in the transfer of autophagic cargo from autophagosomes to lysosomes, known as autophagic flux, and its subsequent degradation was also observed in cells with simultaneous knockdown of Arl8b and Arl8a (Korolchuk et al. 2011).
Arl8b activity is regulated through the exchange of bound GDP for GTP and hydrolysis of the bound GTP to GDP. Arl8b is active when it is bound to GTP and inactive when it is bound to GDP (Okai et al. 2004). However, it is unknown currently whether Arl8b requires GEFs for its activation and GAPs for its inactivation. No GEFs or GAPs that are specific for Arl8b have been identified so far. In addition, it is currently unknown whether Arf GEFs and GAPs are active on Arl8b.
Arl8b is N-acetylated, which is necessary for its localization, and thereby its activity in intact cells. However, it is currently unknown whether N-acetylation is required for the GTP-binding or GTPase activity of Arl8b. Several members of the Arf family require membranes or lipid vesicles and micromolar concentrations of Mg2+ for an increase in their in vitro GTP binding (Pasqualato et al. 2002). However, it is currently unknown whether Arl8b requires any of these cofactors to promote GTP binding.
Arl8b interacts with GDP and GTP (Okai et al. 2004). It has been concluded that the localization of Arl8b to lysosomes may occur only when the protein is present in its GTP-bound active state (Bagshaw et al. 2006). The authors made a constitutively GTP-bound active (Arl8-Q75L) and a constitutively GDP-bound inactive (Arl8-T34N) mutants of Arl8b. When these mutants were expressed in mammalian cells, the GTP-bound mutant localized to lysosomes whereas the GDP-bound mutant did not, indicating that the lysosomal localization of Arl8b depends on its guanine-nucleotide-bound status (Bagshaw et al. 2006).
As described above, Arl8b regulates cargo trafficking to lysomoes in C. elegans and mammalian cells. Depletion of Arl8b impairs the delivery of both fluid-phase (dextran and LDL) and membrane-bound (CD1d and MHC class II) cargo to the lysosomes (Michelet et al. 2015). It was revealed that Arl8b in its active GTP-bound state interacts with SKIP (Sif-A and kinesin-interacting protein), which has a binding site for the light chain of kinesin-1 (KIF-1). SKIP and KIF-1 are crucial for the kinesin-dependent plus-end movement of lysosomes (Ishida et al. 2015). Arl8b GTPase has also been shown to bind and recruit homotypic fusion and protein sorting (HOPS) complexes to the vacuolar membranes (Gillingham et al. 2014; Khatter et al. 2015). Consistent with this, depletion of Arl8b has been shown to impair localization of HOPS subunits. Arl8b binding to the HOPS subunit Vps41 is required for epidermal growth factor receptor (EGFR) trafficking to lysosomes and its lysosomal degradation (Khatter et al. 2015).
Arl8b associates with β-tubulin independent of its guanine-nucleotide-bound status or the absence of its effector domain (Okai et al. 2004). Interaction between Arl8b and β-tubulin was shown by coimmunoprecipitation from the extracts of HeLa cells overexpressing Arl8b. In addition, several interacting proteins for Arl8b have been identified in the Biogrid, IntAct, HPRD, Bind, UniProtKB, MINT, and STRING databases. However, these interactions are yet to be confirmed.
It has been demonstrated that short-term feeding of a high beef-tallow diet to mice results in the downregulation of Arl8b mRNA in the brain (Haraguchi et al. 2006). Arl8b is shown to be critical factor for Natural Killer (NK) cell-mediated cytotoxicity. It has been suggested that Arl8b drives the polarization of lytic granules and microtubule-organizing centers toward the immune synapse between effector NK lymphocytes and target cells (Tuli et al. 2013).
A recent study identified two mechanisms by which Arl8b regulates cancer progression. In prostate cancer cells, Arl8 depletion has been shown to result in juxtanuclear lysosome aggregation, which in turn affects the invasiveness of the cancer cells. In addition, Arl8b facilitates lipid hydrolysis to maintain efficient metabolism for a proliferative capacity in low nutrient environment (Dykes et al. 2016).
Major Sites of Expression and Subcellular Localization
Arl8b is ubiquitously expressed in various tissues and cell lines. Expression in human tissues includes the brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung, and peripheral blood leukocytes. However, higher expression levels were observed in brain, heart, skeletal muscle, kidney, and placenta (Okai et al. 2004). Arl8b has also been shown to be expressed in cell lines such as human HeLa, HEK293, YT-INDY cells and DU145, rat PC12, mouse NIH3T3 and neuro2A, and mast cells (Okai et al. 2004).
Arl8b has been identified as a lysosomal membrane protein by proteomic analysis (Bagshaw et al. 2006). The lysosomal localization of Arl8b is supported by its colocalization with the lysosomal markers CD63, Lamp1, Lamp2, and NPC1 when expressed exogenously in mammalian cells (Bagshaw et al. 2006; Hofmann and Munro 2006; Nakae 2010). In addition, Arl8b has been shown to change its localization from lysosomes to the midzone of spindle during mitosis (Okai et al. 2004). However, two later studies disproved this by showing Arl8b localization to lysosomes during mitosis (Hofmann and Munro 2006; Klassen et al. 2010; Nakae 2010). In neuronal cells, Arl8b localizes to the protruded core and the perinuclear regions (Haraguchi et al. 2006).
Arl8b is N-terminally acetylated, which is essential for its lysosome localization (Hofmann and Munro 2006). Arl8b becomes cytosolic when three hydrophobic amino acids in the N-terminal amphipathic helix are mutated (I5A, L8A, and F12A), which does not affect the N-acetylation of Arl8b (Hofmann and Munro 2006). This study indicates that the lysosomal localization of Arl8b may require not only the N-acetylation but also an intact N-terminal amphipathic helix region.
A rabbit polyclonal antibody raised against the carboxy-terminal peptide (CLIQHSKSRRS) of human Arl8b and Arl8a was used in the recognition of Arl8 proteins by Western blot (Okai et al. 2004).
A rabbit antiserum raised against residues 18–186 of Drosophila Arl8 was used for the detection of endogenous Drosophila Arl8 by Western blot (Hofmann and Munro 2006).
An anti-Arl8 antibody was generated by immunizing rabbits with a synthetic peptide (CDITLQWLIDHSKAQR) corresponding to the C terminus of C. elegans Arl8 and used in the identification of Arl8 protein by Western blot (Nakae 2010).
A polyclonal antibody raised against the C-terminal region of Arl8b was used in Western blot analysis to indicate that the protein is highly enriched on phagosomes in macrophages (Garin et al. 2001). However, neither the immunogen sequence nor animal used for raising the antibody was reported in the publication.
There are several anti-Arl8b antibodies that are commercially available, which could be used for studying Arl8b expression in tissues and cell lines. Four rabbit polyclonal antibodies were raised against the C terminus (SAB1104167) and the center (SAB2106054, SAB2106055, and SAB1104166) and a mouse monoclonal (SAB1403281) clone 1A2 was raised against the full length of Arl8b. These antibodies are available from Sigma and can be used for the detection of protein of human origin by Western blot, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry.
Six rabbit polyclonal antibodies generated against synthetic peptide corresponding to internal regions of human ARL8b (LS-B5831, LS-C379507, LS-C410403, LS-C111980, LS-C155630, and LS-C111981) are available from Lifespan Biosciences. These antibodies can be used for the detection of endogenous ARL8b by Western blot. Abnova supplies a mouse monoclonal antibody, clone 1A2 (H00055207-M01), raised against a full-length recombinant human ARL8, which can be used for the detection of recombinant protein by ELISA. An anti-Arl8b rabbit polyclonal antibody (13049-1-AP) available from ProteinTech group is suitable for recognition of Arl8b by Western blot and ELISA. Although no publications currently site any of these commercially available antibodies, the product data sheets supplied by companies contain evidence of their efficiency in the methods outlined above.
Phenotypes and Splice Variants
No studies on the phenotypic effects of Arl8b have been carried out in mammals because of the lack of Arl8b knockout mice. However, loss of Arl8 in C. elegans results in an increase in the number of late endosomal-lysosomal compartments that are small in size and inhibition of viable embryo production by hermaphrodites (Nakae 2010). Knockdown of Arl8b in Drosophila S2 cells with siRNA resulted in chromosomal missegregation but no inhibition of mitotic progression (Okai et al. 2004). These abnormalities were also observed in HeLa cells following introduction of the dominant-negative mutants, Arl8b-T34N and Arl8b-N130I, into the cell. Therefore, the function of Arl8b in chromosome segregation must be conserved in animals. In addition, a recent study has shown that simultaneous knockdown of Arl8b and Arl8a additively enhanced autophagosome-lysosome fusion in HeLa cells (Korolchuk et al. 2011).
The human Arl8b gene is located on chromosome 3 and has eight exons. Nine splice variants have been predicted to be formed through alternative splicing of Arl8b, which differ from each other in length and amino acid sequence. However, it is not yet known whether the splice variants of Arl8b are functionally active or whether their expression levels differ.
Arl8b is a member of the Arf family of small GTP-binding proteins, which regulate membrane trafficking in eukaryotic cells. Arl8b is 186 amino acids in length and has a molecular weight of approximately 22 kDa. The protein contains an amino-terminally acetylated amphipathic helix, a putative effector domain, and GTP-binding domains. Like other small GTP-binding proteins, Arl8b acts as a molecular switch by cycling between the GDP-bound inactive and the GTP-bound active forms. Arl8b is primarily localized to lysosomes in its GTP-bound form. The N-terminal acetylation of Arl8b is also important for its localization to lysosomes. The main functions of Arl8b discovered so far include a role in microtubule-dependent accumulation of lysosomes at the cell periphery, delivery of endocytosed macromolecules to lysosomes, axonal transport of presynaptic cargo, neurite formation, and chromosome segregation. Recent studies also highlighted the potential of Arl8b as an endogenous reference gene for quantitative gene expression analysis and the use of its gene expression pattern in embryos as a predictor of pregnancy success in cattle. Arl8b has been shown to associate with β-tubulin independent of its guanine-nucleotide-bound status or the effector domain. It also interacts with several other proteins, but the interactions have not yet been confirmed. Arl8b is ubiquitously expressed in tissues and is especially high in brain, heart, skeletal muscle, kidney, liver, placenta, and lung and cell lines. Furthermore, Arl8b mRNA expression is downregulated in the brains of mice fed a high-fat diet. Studies on the phenotypic effects of loss of Arl8 in Caenorhabditis elegans revealed that Arl8 is required for embryonic development and late endosome-lysosome fusion. The human Arl8b gene is located on chromosome 3 and its alternative splicing is predicted to generate nine splice variants. To date, two Arl8b effectors (HOPS and SKIP) have been discovered, and both have shown to play a role in lysosome motility and trafficking. These effectors play a crucial role in vesicle fusion, which could act as a target for evasion strategies acted by pathogenic microorganisms. There is no doubt that future studies on Arl8b will better our understanding in lysosome positioning in health and disease.
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