Knockout of Arfrp1 leads to disruption of ARF-like1 (ARL1) targeting to the trans-Golgi in mouse embryos and HeLa cells

ADP-ribosylation factor related protein 1 (ARFRP1) is a member of the ARF-family of GTPases which operate as molecular switches in the regulation of intracellular protein traffic. Deletion of the mouse Arfrp1 gene leads to embryonic lethality during early gastrulation, suggesting that ARFRP1 is required for cell adhesion-related processes. Here we show that ARFRP1 specifically controls targeting of ARL1 and its effector Golgin-245 to the trans-Golgi. GTP-bound ARFRP1 (ARFRP1-Q79L mutant) is associated with Golgi membranes and co-localized with the GTPase ARL1. In contrast, the guanine nucleotide exchange defective ARFRP1 mutant (ARFRP1-T31N) clusters within the cytosol. ARFRP1-T31N or depletion of endogenous ARFRP1 by RNA interference disrupts the Golgi association of ARL1 and of the GRIP-domain protein Golgin-245 and alters the distribution of a trans-Golgi network marker, syntaxin 6. In contrast, the targeting of two other Golgi-associated proteins, GM130 and giantin, was unaffected. Furthermore, in Arfrp1-/ - embryos ARL1 dislocated from Golgi membranes whereas it was associated with intracellular membranes in wild-type embryos. These data suggest that lethality of Arfrp1 knockout embryos is due to a specific disruption of protein targeting, e.g., of ARL1 and Golgin-245, to the Golgi.     

ADP-ribosylation factor-like GTPase ARFRP1 is required for trans-Golgi to plasma membrane trafficking of E-cadherin

ADP-ribosylation factor-related protein 1 (ARFRP1) plays a specific role in Golgi function controlling recruitment of GRIP domain proteins and ARL1 to the trans-Golgi. Deletion of the mouse Arfrp1 gene causes embryonic lethality during early gastrulation, because epiblast cells detach from the ectodermal cell layer and do not differentiate to mesodermal tissue. Here we show that in Arfrp1(-/-) embryos E-cadherin is mistargeted to intracellular compartments, whereas in control embryos it is present at the cell surface of trophectodermal and ectodermal cells. In enterocytes of intestine-specific Arfrp1 null mutants (Arfrp1(vil)(-/-)), E-cadherin is associated with intracellular membranes, partially colocalizing with the cis-Golgi marker GM130 or with punctae close to the cell surface. In contrast, in control enterocytes E-cadherin is exclusively located in the lateral membranes. In addition, ARL1 is dislocated from Golgi membranes to the cytosol of Arfrp1(vil)(-/-) enterocytes. Depletion of endogenous ARFRP1 by RNA interference leads to a dislocation of E-cadherin from the cell surface in HeLa cells and to a reduced cell aggregation in Ltk(-)Ecad cells. ARFRP1 was coimmunoprecipitated in a complex with E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, and p120(ctn) from lysates of Madin-Darby canine kidney cells stably expressing myc-ARFRP1. These data indicate that knock-out of Arfrp1 disrupts the trafficking of E-cadherin through the Golgi and suggest an essential role of the GTPase in trans-Golgi network function.     

Altered GLUT4 trafficking in adipocytes in the absence of the GTPase Arfrp1

The GTPase ADP-ribosylation factor related protein 1 (ARFRP1) controls the recruitment of proteins such as golgin-245 to the trans-Golgi. ARFRP1 is highly expressed in adipose tissues in which the insulin-sensitive glucose transporter GLUT4 is processed through the Golgi to a specialized endosomal compartment, the insulin-responsive storage compartment from which it is translocated to the plasma membrane in response to a stimulation of cells by insulin. In order to examine the role of ARFRP1 for GLUT4 targeting, subcellular distribution of GLUT4 was investigated in adipose tissue specific Arfrp1 knockout (Arfrp1^ad−/−) mice. Immunohistochemical and ultrastructural studies of brown adipocytes demonstrated an abnormal trans-Golgi in Arfrp1^ad−/− adipocytes. In addition, in Arfrp1^ad−/− adipocytes GLUT4 protein accumulated at the plasma membrane rather than being sequestered in an intracellular compartment. A similar missorting of GLUT4 was produced by siRNA-mediated knockdown of Arfrp1 in 3T3-L1 adipocytes which was associated with significantly elevated uptake of deoxyglucose under basal conditions. Thus, Arfrp1 appears to be involved in sorting of GLUT4.     

Embryonic lethality caused by apoptosis during gastrulation in mice lacking the gene of the ADP-ribosylation factor-related protein 1

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a membrane-associated GTPase with significant similarity to the family of ARFs. We have recently shown that ARFRP1 interacts with the Sec7 domain of the ARF-specific guanine nucleotide exchange factor Sec7-1/cytohesin and inhibits the ARF/Sec7-dependent activation of phospholipase D in a GTP-dependent manner. In order to further analyze the function of ARFRP1, we cloned the mouse Arfrp1 gene and generated Arfrp1 null-mutant mice by gene targeting in embryonic stem cells. Heterozygous Arfrp1 mutants developed normally, whereas homozygosity for the mutant allele led to embryonic lethality. Cultured homozygous Arfrp1 null-mutant blastocysts were indistinguishable from wild-type blastocysts. In vivo, they implanted and formed egg cylinder stage embryos that appeared normal until day 5. Between embryonic days 6 and 7, however, apoptotic cell death of epiblast cells occurred in the embryonic ectoderm during gastrulation, as was shown by histological analysis combined with terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling. Epiblast cells that would normally differentiate to mesodermal cells detached from the ectodermal cell layer and were dispersed into the proamniotic cavity. In contrast, the development of extraembryonic structures appeared unaffected. Our results demonstrate that ARFRP1 is necessary for early embryonic development during gastrulation.     

An Arabidopsis GRIP domain protein locates to the trans-Golgi and binds the small GTPase ARL1

GRIP domain proteins are a class of golgins that have been described in yeast and animals. They locate to the trans-Golgi network and are thought to play a role in endosome-to-Golgi trafficking. The Arabidopsis GRIP domain protein, AtGRIP, fused to the green fluorescent protein (GFP), locates to Golgi stacks but does not exactly co-locate with the Golgi marker sialyl transferase (ST)-mRFP, nor with the t-SNAREs Memb11, SYP31 and BS14a. We conclude that the location of AtGRIP is further to the trans side of the stack than STtmd-mRFP. The 185-aa C-terminus of AtGRIP containing the GRIP domain targeted GFP to the Golgi, although a proportion of the fusion protein was still found in the cytosol. Mutation of a conserved tyrosine (Y717) to alanine in the GRIP domain disrupted Golgi localization. ARL1 is a small GTPase required for Golgi targeting of GRIP domain proteins in other systems. An Arabidopsis ARL1 homologue was isolated and shown to target to Golgi stacks. The GDP-restricted mutant of ARL1, AtARL1-T31N, was observed to locate partially to the cytosol, whereas the GTP-restricted mutant AtARL1-Q71L labelled the Golgi and a population of small structures. Increasing the levels of AtARL1 in epidermal cells increased the proportion of GRIP-GFP fusion protein on Golgi stacks. We show, moreover, that AtARL1 interacted with the GRIP domain in a GTP-dependent manner in vitro in affinity chromatography and in the yeast two-hybrid system. This indicates that AtGRIP and AtARL1 interact directly. We conclude that the pathway involving ARL1 and GRIP domain golgins is conserved in plants.     

Interaction of Arl1-GTP with GRIP domains recruits autoantigens Golgin-97 and Golgin-245/p230 onto the Golgi

A cellular role and the mechanism of action for small GTPase Arl1 have been defined. Arl1-GTP interacts with the GRIP domains of Golgin-97 and Golgin-245, a process dependent on conserved residues of the GRIP domains that are important for Golgi targeting. The switch II region of Arl1 confers the specificity of this interaction. Arl1-GTP mediates Golgi recruitment of Golgin-97 in a switch II-dependent manner, whereas tethering Arl1-GTP onto endosomes can mediate endosomal targeting of Golgin-97. Golgin-97 and Golgin-245 are dissociated from the Golgi when Arl1 is knocked-down by its siRNA. Arl1-GTP thus functions to recruit Golgin-97 and Golgin-245 onto the Golgi via interacting with their GRIP domains.     

Hepatic trans-Golgi action coordinated by the GTPase ARFRP1 is crucial for lipoprotein lipidation and assembly

The liver is a major organ in whole body lipid metabolism and malfunctioning can lead to various diseases including dyslipidemia, fatty liver disease, and type 2 diabetes. Triglycerides and cholesteryl esters are packed in the liver as very low density lipoproteins (VLDLs). Generation of these lipoproteins is initiated in the endoplasmic reticulum and further maturation likely occurs in the Golgi. ADP-ribosylation factor-related protein 1 (ARFRP1) is a small trans-Golgi-associated guanosine triphosphatase (GTPase) that regulates protein sorting and is required for chylomicron lipidation and assembly in the intestine. Here we show that the hepatocyte-specific deletion of Arfrp1 (Arfrp1^liv−/−) results in impaired VLDL lipidation leading to reduced plasma triglyceride levels in the fasted state as well as after inhibition of lipoprotein lipase activity by Triton WR-1339. In addition, the concentration of ApoC3 that comprises 40% of protein mass of secreted VLDLs is markedly reduced in the plasma of Arfrp1^liv−/− mice but accumulates in the liver accompanied by elevated triglycerides. Fractionation of Arfrp1^liv−/− liver homogenates reveals more ApoB48 and a lower concentration of triglycerides in the Golgi compartments than in the corresponding fractions from control livers. In conclusion, ARFRP1 and the Golgi apparatus play an important role in lipoprotein maturation in the liver by influencing lipidation and assembly of proteins to the lipid particles.     

ADP-ribosylation factors (ARFs) and ARF-like 1 (ARL1) have both specific and shared effectors: characterizing ARL1-binding proteins

Despite the 40-60% identity between ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins, distinct functional roles have been inferred from findings that ARLs lack the biochemical or genetic activities characteristic of ARFs. The potential for functional overlap between ARFs and ARLs was examined by comparing effects of expression on intact cells and the ability to bind effectors. Expression of [Q71L]ARL1 in mammalian cells led to altered Golgi structure similar to, but less dramatic than, that reported previously for [Q71L]ARF1. Two previously identified partners of ARFs, MKLP1 and Arfaptin2/POR1, also bind ARL1 but not ARL2 or ARL3. Two-hybrid screens of human cDNA libraries with dominant active mutants of human ARL1, ARL2, and ARL3 identified eight different but overlapping sets of binding partners. Specific interactions between ARL1 and two binding proteins, SCOCO and Golgin-245, are defined and characterized in more detail. Like ARFs and ARL1, the binding of SCOCO to Golgi membranes is rapidly reversed by brefeldin A, suggesting the presence of a brefeldin A-sensitive ARL1 exchange factor. These data reveal a complex network of interactions between GTPases in the ARF family and their effectors and reveal a potential for cross-talk not demonstrated previously.     

The ARF-like GTPases Arl1p and Arl3p act in a pathway that interacts with vesicle-tethering factors at the Golgi apparatus

The ARLs are a diverse family of GTPases that are related to ADP-ribosylation factors (ARFs), but whose function is poorly understood. There are at least ten ARLs in humans, two of which have homologs in the yeast Saccharomyces cerevisiae (ARL1/Arl1p and ARFRP1/Arl3p). The function of ARFRP1 is unknown, but mammalian ARL1 has recently been found to interact with a number of effectors including the GRIP domain that is present in a family of Golgi-localized long coiled-coil proteins. We find that in yeast, the intracellular targeting of Imh1p, the only yeast GRIP domain protein, is dependent on both Arl1p and Arl3p, but not on the ARF proteins. A recombinant form of the Imh1p GRIP domain binds to Arl1p in a GTP-dependent manner, but not to Arl3p. Yeast also contain a relative of SCOCO, a protein proposed to bind human ARL1, but this yeast protein, Slo1p, appears to bind Arl3p rather than Arl1p in vitro. However, Imh1p is not the sole effector of Arl1p since affinity chromatography of cytosol with immobilized Arl1p:GTP revealed an interaction with the GARP/VFT complex that is thought to act in the tethering of vesicles to the Golgi apparatus. Finally, we find that Arl3p is required in vivo for the targeting of Arl1p, explaining its requirement for the normal distribution of Imh1p.     

Autoantigen Golgin-97, an effector of Arl1 GTPase, participates in traffic from the endosome to the trans-golgi network

The precise cellular function of Arl1 and its effectors, the GRIP domain Golgins, is not resolved, despite our recent understanding that Arl1 regulates the membrane recruitment of these Golgins. In this report, we describe our functional study of Golgin-97. Using a Shiga toxin B fragment (STxB)-based in vitro transport assay, we demonstrated that Golgin-97 plays a role in transport from the endosome to the trans-Golgi network (TGN). The recombinant GRIP domain of Golgin-97 as well as antibodies against Golgin-97 inhibited the transport of STxB in vitro. Membrane-associated Golgin-97, but not its cytosolic pool, was required in the in vitro transport assay. The kinetic characterization of inhibition by anti-Golgin-97 antibody in comparison with anti-Syntaxin 16 antibody established that Golgin-97 acts before Syntaxin 16 in endosome-to-TGN transport. Knock down of Golgin-97 or Arl1 by their respective small interference RNAs (siRNAs) also significantly inhibited the transport of STxB to the Golgi in vivo. In siRNA-treated cells with reduced levels of Arl1, internalized STxB was instead distributed peripherally. Microinjection of Golgin-97 antibody led to the fragmentation of Golgi apparatus and the arrested transport to the Golgi of internalized Cholera toxin B fragment. We suggest that Golgin-97 may function as a tethering molecule in endosome-to-TGN retrograde traffic.     

Targeting of the Arf-like GTPase Arl3p to the Golgi requires N-terminal acetylation and the membrane protein Sys1p

The GTPase Arl3p is required to recruit a second GTPase, Arl1p, to the Golgi in Saccharomyces cerevisiae. Arl1p binds to the GRIP domain, which is present in a number of long coiled-coil proteins or 'golgins'. Here we show that Arl3p is not myristoylated like most members of the Arf family, but is instead amino-terminally acetylated by the NatC complex. Targeting of Arl3p also requires a Golgi membrane protein Sys1p. The human homologues of Arl3p (Arf-related protein 1 (ARFRP1)) and Sys1p (hSys1) can be isolated in a complex after chemical cross-linking. This suggests that the targeting of ARFRP1/Arl3p to the Golgi is mediated by a direct interaction between its acetylated N terminus and Sys1p/hSys1.     

Multilayer interactions determine the Golgi localization of GRIP golgins

Golgin-97, RanBP2alpha, Imh1p and p230/golgin-245 (GRIP) domain golgins are targeted to the Golgi membrane through their GRIP domains. By analyzing more than 30 mutants of golgin-97 and golgin-245 GRIP domains for their properties of dimerization, interaction with ARF like protein 1 (Arl1)-GTP and Golgi targeting, we found hierarchically organized three-tier interactions governing the Golgi targeting of GRIP domain golgins. GRIP domain self-dimerization is necessary for bivalent interaction with Arl1-GTP. Unexpectedly, however, these two interactions are not sufficient for Golgi targeting, as a third group of residues, including positive-charged arginine between alpha1 and alpha2 and hydrophobic residues C-terminal to the GRIP domain, turn out to be essential. Surface plasmon resonance analysis indicates that GRIP domain interacts directly with membrane lipid, partially through the third group of residues such as W744 of golgin-97. This third tier of interaction with the membrane could be mediated by non-specific hydrophobic and electrostatic forces.     

Autophagy in Saccharomyces cerevisiae requires the monomeric GTP-binding proteins,Arl1 and Ypt6

Macroautophagy/autophagy is a cellular degradation process that sequesters organelles or proteins into a double-membrane structure called the phagophore; this transient compartment matures into an autophagosome, which then fuses with the lysosome or vacuole to allow hydrolysis of the cargo. Factors that control membrane traffic are also essential for each step of autophagy. Here we demonstrate that 2 monomeric GTP-binding proteins in Saccharomyces cerevisiae, Arl1 and Ypt6, which belong to the Arf/Arl/Sar protein family and the Rab family, respectively, and control endosome-trans-Golgi traffic, are also necessary for starvation-induced autophagy under high temperature stress. Using established autophagy-specific assays we found that cells lacking either ARL1 or YPT6, which exhibit synthetic lethality with one another, were unable to undergo autophagy at an elevated temperature, although autophagy proceeds normally at normal growth temperature; specifically, strains lacking one or the other of these genes are unable to construct the autophagosome because these 2 proteins are required for proper traffic of Atg9 to the phagophore assembly site (PAS) at the restrictive temperature. Using degron technology to construct an inducible arl1Δ ypt6Δ double mutant, we demonstrated that cells lacking both genes show defects in starvation-inducted autophagy at the permissive temperature. We also found Arl1 and Ypt6 participate in autophagy by targeting the Golgi-associated retrograde protein (GARP) complex to the PAS to regulate the anterograde trafficking of Atg9. Our data show that these 2 membrane traffic regulators have novel roles in autophagy.     

GMx33 associates with the trans-Golgi matrix in a dynamic manner and sorts within tubules exiting the Golgi

The trans-Golgi matrix consists of a group of proteins dynamically associated with the trans-Golgi and thought to be involved in anterograde and retrograde Golgi traffic, as well as interactions with the cytoskeleton and maintenance of the Golgi structure. GMx33 is localized to the cytoplasmic face of the trans-Golgi and is also present in a large cytoplasmic pool. Here we demonstrate that GMx33 is dynamically associated with the trans-Golgi matrix, associating and dissociating with the Golgi in seconds. GMx33 can be locked onto the trans-Golgi matrix by GTPgammaS, indicating that its association is regulated in a GTP-dependent manner like several other Golgi matrix proteins. Using live-cell imaging we show that GMx33 exits the Golgi associated with tubules and within these tubules GMx33 segregates from transmembrane proteins followed by fragmentation of the tubules into smaller tubules and vesicles. Within vesicles produced by an in vitro budding reaction, GMx33 remains segregated in a matrixlike tail region that sometimes contains Golgin-245. This trans-matrix often links a few vesicles together. Together these data suggest that GMx33 is a member of the trans-Golgi matrix and offer clues regarding the role of the trans-Golgi matrix in sorting and exit from the Golgi.     

Dual Targeting of Osh1p, a Yeast Homologue of Oxysterol-binding Protein, to both the Golgi and the Nucleus-Vacuole Junction

Oxysterol binding protein (OSBP) is the only protein known to bind specifically to the group of oxysterols with potent effects on cholesterol homeostasis. Although the function of OSBP is currently unknown, an important role is implicated by the existence of multiple homologues in all eukaryotes so far examined. OSBP and a subset of homologues contain pleckstrin homology (PH) domains. Such domains are responsible for the targeting of a wide range of proteins to the plasma membrane. In contrast, OSBP is a peripheral protein of Golgi membranes, and its PH domain targets to the trans-Golgi network of mammalian cells. In this article, we have characterized Osh1p, Osh2p, and Osh3p, the three homologues of OSBP in Saccharomyces cerevisiae that contain PH domains. Examination of a green fluorescent protein (GFP) fusion to Osh1p revealed a striking dual localization with the protein present on both the late Golgi, and in the recently described nucleus-vacuole (NV) junction. Deletion mapping revealed that the PH domain of Osh1p specified targeting to the late Golgi, and an ankyrin repeat domain targeting to the NV junction, the first such targeting domain identified for this structure. GFP fusions to Osh2p and Osh3p showed intracellular distributions distinct from that of Osh1p, and their PH domains appear to contribute to their differing localizations.     

E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by golgin-97

E-cadherin is a cell-cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane-bound carriers for the post-Golgi exocytosis of E-cadherin have not been characterized. Green fluorescent protein (GFP)-tagged E-cadherin (Ecad-GFP) is transported from the trans-Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. Here, we examine the association of golgins with tubular carriers containing E-cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full-length proteins and were coexpressed with Ecad-GFP. The GRIP domains of p230/golgin-245 and golgin-97 had overlapping but nonidentical distributions on the TGN; both domains were on TGN-derived tubules but only the golgin-97 GRIP domain coincided with Ecad-GFP tubules in live cells. When the Arl1-binding endogenous golgins, p230/golgin-245 and golgin-97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad-GFP was inhibited. siRNA knockdown of golgin-97 also inhibited trafficking of Ecad-GFP. Thus, the GRIP domains of p230/golgin-245 and golgin-97 bind discriminately to distinct membrane subdomains of the TGN. Golgin-97 is identified as a selective and essential component of the tubulovesicular carriers transporting E-cadherin out of the TGN.     

Loss of the Atp2c1 secretory pathway Ca(2+)-ATPase (SPCA1) in mice causes Golgi stress, apoptosis, and midgestational death in homozygous embryos and squamous cell tumors in adult heterozygotes

Loss of one copy of the human ATP2C1 gene, encoding SPCA1 (secretory pathway Ca(2+)-ATPase isoform 1), causes Hailey-Hailey disease, a skin disorder. We performed targeted mutagenesis of the Atp2c1 gene in mice to analyze the functions of this Golgi membrane Ca(2+) pump. Breeding of heterozygous mutants yielded a normal Mendelian ratio among embryos on gestation day 9.5; however, null mutant (Spca1(-/-)) embryos exhibited growth retardation and did not survive beyond gestation day 10.5. Spca1(-/-) embryos had an open rostral neural tube, but hematopoiesis and cardiovascular development were ostensibly normal. Golgi membranes of Spca1(-/-) embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased Golgi biogenesis. The number of Golgi-associated vesicles was also increased, and rough endoplasmic reticulum had fewer ribosomes. Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipid by the Golgi. Adult heterozygous mice appeared normal and exhibited no evidence of Hailey-Hailey disease; however, aged heterozygotes had an increased incidence of squamous cell tumors of keratinized epithelial cells of the skin and esophagus. These data show that loss of the Golgi Ca(2+) pump causes Golgi stress, expansion of the Golgi, increased apoptosis, and embryonic lethality and demonstrates that SPCA1 haploinsufficiency causes a genetic predisposition to cancer.     

ARL1 Plays a Role in the Binding of the GRIP Domain of a Peripheral Matrix Protein to the Golgi Apparatus in Plant Cells

ARF GTPases play a central role in regulating membrane dynamics and protein transport in eukaryotic cells. ARF-like (ARL) proteins are close relatives of the ARF regulators of vesicular transport, but their function in plant cells is poorly characterized. Here, by means of live cell imaging and site-directed mutagenesis, we have investigated the cellular function of the plant GTPase ARL1. We provide direct evidence for a role of this ARL family member in the association of a plant golgin with the plant Golgi apparatus. Our data reveal the existence of key residues within the conserved GRIP-domain of the golgin and within the GTPase ARL1 that are central to ARL1–GRIP interaction. Mutations of these residues abolish the interaction of GRIP with the GTP-bound ARL1 and induce a redistribution of GRIP into the cytosol. This indicates that the localization of GRIP to the Golgi apparatus is strongly influenced by the interaction of GRIP with Golgi-localized ARL1. Our results assign a cellular role to a member of the Arabidopsis ARL family in the plant secretory pathway and propose mechanisms for localization of peripheral golgins to the plant Golgi apparatus. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.