Membrane Biology Laboratory, Institute of Molecular and Cell Biology, Singapore
Author for correspondence (e-mail: mcbhwj{at}imcb.nus.edu.sg)
Accepted September 13, 2001
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SUMMARY |
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Key words: GTPase, Vesicular transport, Arl1, Golgi apparatus, Endoplasmic reticulum
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INTRODUCTION |
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Various GTPases are involved in vesicle formation. Sar1 regulates protein export from the ER mediated by the COPII coat protein complex (Springer et al., 1999; Aridor et al., 2001). The ADP ribosylation factor (ARF) family includes six highly homologous members (Chavrier and Goud, 1999; Boman and Kahn, 1995; Donaldson and Jackson, 2000; Moss and Vaughan, 1998). The key regulators such as guanine nucleotide exchange factors and GTPase-activating proteins for ARFs have been identified and studied extensively (Chavrier and Goud, 1999; Donaldson and Jackson, 2000; Moss and Vaughan, 1998; Jackson and Casanova, 2000). ARFs were originally identified as cofactors required for cholera-toxin-catalyzed ADP-ribosylation of the stimulatory component of adenylate cyclase Gs (Kahn and Gilman, 1984) and are important for membrane trafficking at several stages of the exocytotic/endocytotic pathway (Chavrier and Goud, 1999; Boman and Kahn, 1995; Donaldson and Jackson, 2000; Moss and Vaughan, 1998; Jackson and Casanova, 2000; Dascher and Balch, 1994; Zhang et al., 1994). ARF1 regulates COPI vesicle budding and is a component of the coat in the early secretory pathway (Serafini et al., 1991; Palmer et al., 1993). Specifically, ARF1 (in its active GTP-bound form) interacts directly with the ß subunit of coatomer (Zhao et al., 1997) and also participates in the packaging of cargo proteins into budding vesicles (Malsam et al., 1999; Stephens and Pepperkok, 2001). ARF1 and COPI also function in the endocytic pathway (Daro et al., 1997; Gu and Gruenberg, 2000). In addition, ARF1 has also been shown to participate in vesicle formation mediated by AP-1 (Robinson and Kreis, 1992; Stamnes and Rothman, 1993) and AP-3 coat protein complexes (Ooi et al., 1998) in the trans-Golgi network (TGN) and/or the endosome. ARF6 is distributed within the plasma membrane and endosomal structures and has been implicated in traffic between the surface and the endosomes (DSouza-Schorey et al., 1995; DSouza-Schorey et al., 1998; Al-Awar et al., 2000). Phosphatidylinositol 4-phosphate 5-kinase is a downstream effector of ARF6 (Honda et al., 1999).
In addition to COPI, AP-1 and AP-3, several other effectors have been identified for ARF1, including phospholipase D (PLD1) (Roth, 1999; Cockcroft et al., 1994; Brown et al., 1993), Arfaptin1, POR1/Arfaptin2 (Kanoh et al., 1997; Van Aelst et al., 1996), GGA1-3 (Boman et al., 2000; DellAngelica et al., 2000; Hirst et al., 2000) and MKLP1 (Boman et al., 1999). These effectors may interact with different residues of ARF1, and it is expected that more effectors for ARFs remain to be identified (Kuai et al., 2000).
In addition to the six ARFs, there exists a subfamily of small GTPases with homology to ARFs, referred to as ARF-like proteins (Arl). The first member (Arl1) was originally cloned from Drosophila (Tamkun et al., 1991) and is essential for normal development. The mammalian Arl1 was subsequently identified (Schurmann et al., 1994; Lowe et al., 1996) and shown to be associated with the Golgi apparatus (Lowe et al., 1996). In contrast to Drosophila Arl1, yeast Arl1 is not essential for cell growth (Lee et al., 1997). In addition, six other members (Arl2-7) of this subfamily have also been identified (Schurmann et al., 1994; Clark et al., 1993; Cavenagh et al., 1994; Jacobs et al., 1999). Arl2 appears to be cytosolic and has recently been shown to interact with a tubulin-specific chaperone known as cofactor D (Bhamidipati et al., 2000; Radcliffe et al., 2000) as well as Bart, a novel protein of unknown function (Sharer and Kahn, 1999). A recent study suggests that Arl4 is localized to nuclei and nucleoli (Lin et al., 2000). Interaction of Arl6 with Sec61ß, a subunit of the core component of ER translocan, was recently reported (Ingley et al., 1999). The exact subcellular localization of Arl3 and Arl5-7 and the function of Arl1 and Arl3-7 have not been established. In this report, we provide morphological and biochemical evidence that Arl1 regulates the structure and function of the Golgi apparatus.
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MATERIALS AND METHODS |
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Antibodies
The Arl1 peptide-specific rabbit polyclonal antibody E6P1 has been previously described (Lowe et al., 1996) and specifically recognizes endogenous rat, hamster and human Arl1 but does not crossreact with ARF1, Arl2 or Arl3. The mouse monoclonal antibody (Mab) against a Golgi SNARE, GS28, has been described (Subramaniam et al., 1996). Mabs against ß-COP (maD) and VSV-G (P5D4) were gifts from T. Kreis (Pepperkok et al., 1993). Rabbit polyclonal antibodies against human ß-1,4-galactosyltransferase (GT) were described previously (Subramaniam et al., 1992). Mabs against -adaptin and
-adaptin were purchased from BD Transduction Laboratories. Sec31 mouse polyclonal antibodies have been described (Tang et al., 2000). The mouse monoclonal anti-ARFs antibody (clone 1D9) (Cavenagh et al., 1996), which recognizes all ARFs but does not react with Arls, was from Affinity Bioreagents, Inc.
Cell culture and transfection
A431 and CHO cells were grown in DME and RPMI media, respectively, supplemented with 10% fetal bovine serum at 37°C. The transfection was performed using either Lipofectamine (Gibco) for stable transfection of Arl1 and Arl1(Q71L) or Effectene Reagent (QIAGEN) for transient transfection of other constructs. The stable transfectants of CHO cells with pSTAR-Arl1 or pSTAR-Arl1(Q71L) were maintained in RPMI supplemented with 10% tetracycline-free fetal bovine serum (Clontech) and 1 mg/ml G418 (Gibco).
Indirect immunofluorescence microscopy
Transfected CHO cells were incubated in the presence of doxycycline (Clontech) at 8 µg/ml for 12 hours to induce the expression of transfected constructs. For Brefeldin A treatments, cells were incubated with 10 µg/ml Brefeldin A (Epicentre Technologies) at 37°C for different periods of time before fixation. Cells were washed with PBS containing 1 mM CaCl2 and 1 mM MgCl2 (PBSCM) and then fixed with methanol at 20°C for five minutes. Cells were then washed with PBSCM and incubated with antibodies in fluorescence dilution buffer (PBSCM with 5% fetal bovine serum and 2% BSA) for one hour at room temperature. After extensive washing with PBSCM, cells were incubated with the FITC-, Rhodamine- or Texas-red-conjugated secondary antibodies in fluorescence dilution buffer at room temperature for one hour. The cells were then mounted with Vectashield (Vector Laboratories) after washing. Confocal microscopy was performed with a Zeiss Axioplan II microscope equipped with a Bio-Rad MRC1024 confocal scanning laser (Bio-Rad). Typically, images shown are combinations of three to four optical sections 0.3 µm apart.
VSV-G morphological transport assay
Arl1-transfected CHO cells were grown on coverslips and induced with 8 µg/ml Doxycycline for about 12 hours. Coverslips were transferred to 3.5 cm diameter petri dishes and washed with serum-free RPMI. 400 µl RPMI (without serum) and an appropriate amount of VSVts045 virus were added to the dish. After incubation at 32°C, plus rocking, for one hour, 1.5 ml RPMI containing 10% fetal bovine serum was added and the cells were incubated for two hours at 32°C without rocking. Cycloheximide (Sigma) was added to a final concentration of 10 µg/ml, and the system was incubated for another hour before processing for indirect immunofluorecence microscopy.
Yeast two-hybrid analysis
Arl1(Q71L) and (T31N) pSTAR constructs were digested with EcoRI and BamHI and ligated into the EcoRI/BamHI sites of GAL4 DNA binding domain (GAL4-BD) vector pGBKT7 (Clontech). The resulting plasmids were used to transform yeast strain AH109 (MATa). For screening, the Arl1(Q71L) transformed AH109 yeast was mated with a pool of Y187 (MAT) (Clontech), which carried the GAL4 activation domain (GAL4-AD) fused to the human brain cDNA library (Clontech). The interaction-positive diploid yeast cells were selected on SD/-Trp/-Leu/-His/-Ade (QDO) plates. All the positive clones turned blue on X-
-gal (Clontech) QDO plate. The GAL4-AD-cDNA hybrid in pACT2 vector (Clontech) was recovered from the positive yeast cells and sequenced.
The coding sequence of mouse ARF1 (EST clone, GenBank Accession Number: AA266176) was cloned into the EcoRI/BamHI sites of pGBKT7. The corresponding mutants ARF1(T31N) and (Q71L) were generated in pBGKT7 by PCR mutagenesis and subsequently transformed into AH109 yeast cells. The coding region of human GGA1 was derived from EST clone (GenBank Accession Number: BF347585) and cloned into the GAL4-AD fusion vector pGADT7 (Clontech). The coding sequence of POR1 in pACT2 was recovered from one of the positive clones through screening. Mutant Arl1- and ARF1-containing AH109 cells were mated to POR1 and GGA1 transformed Y187 yeast cells, respectively. After selection on SD/-Trp/-Leu medium, the diploid yeast cells were assayed on QDO plates containing 20 µg/ml X--gal (Clontech). The positive interacting diploid yeast cells grew and turned blue in three to four days.
In vitro binding assay
To facilitate cloning, the MCS of pGEX-KG (Amersham-Phamacia) was modified to place the EcoRI site in the same reading frame as in pGBKT7 vector. A small linker with EcoRI, NheI and BamHI sites made by the annealing of two oligonucleotides (5'-GATCAGAATTCGCTAGCGGATCCTTAAC-3' and 5'-AATTGTTAAGGATCCGCTAGCGAATTCT-3') was ligated with EcoRI- and BamHI-digested pGEX-KG to make a MCS modified vector - pGEB. Arl1(T31N), Arl1(Q71L) and wild-type ARF1 were cloned into pGEB. The recombinant GST fusion protein was produced in Escherichia coli DH5 and affinity purified by Glutathione Sepharose 4B (Amersham-Pharmacia) as previously described (Lowe at al., 1996). The GDP and GTP
S exchange reactions were performed as previously described (Christoforidis and Zerial, 2000). The POR1 pACT2 construct was digested by EcoRI/XhoI (blunt ended) and subsequently ligated into EcoRI/BamHI (blunt ended)-digested pBSK vector (Stratagene). S35-methionine (Amersham-Phamacia)-labeled POR1 and GGA1 were obtained by in vitro translation of POR1/pBSK and GGA1/pGADT7 constructs using the TNT T7 Quick Coupled Transcription/Translation System (Promega) according to the manufacturers protocol. 60 µg guanine nucleotide-exchanged GST fusion proteins were incubated in Nucleotide Stabilizing Buffer (NS Buffer) containing 2 mg/ml BSA and 100 µM GDP or GTP
S at 4°C for one hour to block the beads before the addition of 10 µl in vitro translated POR1 or GGA1. After over night incubation at 4°C, the beads of each reaction were washed three times with NS buffer. Proteins bound to the beads were eluted by boiling in 2 x SDS sample buffer and resolved by 12% SDS-PAGE. Autoradiography was performed with the PhosphoImager (Molecular Dynamics).
EM immunogold labeling
This was performed as described previously (Griffiths, 1993).
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RESULTS |
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Traffic through the Golgi apparatus is inhibited by Arl1(Q71L)
We next examined the effect of expression of Arl1(Q71L) on protein trafficking along the secretory pathway. Specifically, we analyzed the transport of the envelope protein (VSV-G) of VSVts045. Cells were infected with VSVts045 at 32°C for one hour and then incubated for another two hours to allow sufficient VSV-G protein to be produced, followed by an additional one hour incubation in the presence of cycloheximide to chase VSV-G along the pathway. As shown (Fig. 9B,E,H), VSV-G was almost completely transported to the cell surface in cells not expressing Arl1(Q71L). However, the transport of VSV-G to the surface was essentially abolished in cells expressing Arl1(Q71L) (Fig. 9A-C), and VSV-G now accumulated in the expanded Golgi-like structure marked by Arl1(Q71L). These results suggest that export of VSV-G from the ER mediated by COPII coat proteins occurred normally, although subsequent transport through the Golgi and/or from the Golgi to the surface was blocked. Similarly, VSV-G transport was arrested upon overexpression of Arl1(Q71L)-EGFP (Fig. 9D-F), although its effect was not as potent as that of Arl1(Q71L). The effect of the activated form of Arl1 on VSV-G transport was also dependent on myristoylation because VSV-G was transported normally to the cell surface in cells overexpressing Arl1(G2A, Q71L)-EGFP (Fig. 9G-I).
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DISCUSSION |
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The functional importance of Arl1 was established by the expression of two different mutant versions of Arl1. The Golgi apparatus (marked by GT and GS28) disassembled when Arl1(T31N), which was expected to be restricted to the GDP-bound status, was expressed. Since the rate-limiting step in Arl1 Golgi recruitment is the activation step mediated by guanine nucleotide exchange factors, the likely explanation for the observed effect of Arl1(T31N) is that it competes with endogenous Arl1 for the limiting amount of guanine nucleotide exchange factors, resulting in an inhibition of activation and Golgi recruitment of endogenous Arl1. The failure to recruit Arl1 onto the Golgi may also lead to defective Golgi association of its putative effectors, which, together with Arl1, play a key role in maintaining the normal structure and function of the Golgi apparatus. On the other hand, expression of Arl1(Q71L), expected to be restricted to the GTP-bound activated form, led to an expansion of the Golgi apparatus. The ordered stacks of the Golgi were transformed into an extensive vesicular-tubular network upon expression at high levels. Although the molecular mechanism remains to be established, it is plausible that the uncontrolled Golgi association of Arl1(Q71L) leads to a massive recruitment of its effectors onto the Golgi and an alteration of the normal structure of the stacked cisternae of the Golgi apparatus. As the ordered stacked cisternae are believed to facilitate efficient traffic through this compartment and to govern effective sorting on both the cis as well on the trans side of the Golgi, the transformation of Golgi into an extensive vesicular-tubular network may interfere with the normal function and lead to an inhibition of protein transport through the compartment and/or to inhibition of export from the Golgi. In support of this interpretation, our experiments showed that transport of VSV-G from the ER to the cell surface was arrested at the level of the expanded structure upon overexpression of Arl1(Q71L). The accumulation of VSV-G protein in this vesicular-tubular network indeed indicates that transport through or export from the altered Golgi is blocked. On the basis of these results, we propose that one of the mechanisms for the cell to regulate the size and the stacked cisternae structure of the Golgi apparatus is by controlling the activation and Golgi recruitment of Arl1 and its effectors.
The massive recruitment of COPI and AP-1 onto the expanded Golgi apparatus upon overexpression of Arl1(Q71L) is of interest because a similar effect was observed upon expression of ARF1(Q71L) (Dascher and Balch, 1994; Zhang et al., 1994). As COPI and AP-1 are redistributed from the Golgi to the cytosol within two minutes of Brefeldin A treatment, whereas Golgi association of Arl1 remains intact after five minutes of Brefeldin A treatment (Lowe et al., 1996), it is unlikely that Golgi recruitment of COPI and AP-1 are directly regulated by Arl1. How do we explain the remarkable similarity of the effects observed for Arl1(Q71L) as compared to those observed for ARF1(Q71L) with regards to Golgi recruitment of COPI and AP-1? One possibility is that Arl1 and ARF1 share some common effectors in addition to effectors that are unique to each of them. Upon expression of the activated form of Arl1 and its Golgi recruitment, its effectors are subsequently recruited onto the Golgi membrane. As it is known that effectors for ARF1 have a strong positive feedback on ARF1 GDP-GTP exchange and activation (Zhu et al., 2000), some of the effectors recruited by Arl1(Q71L) will effect positive regulation of ARF1, resulting in activation and Golgi recruitment of ARF1 followed by its unique set of effectors.
POR1/Arfaptin2 is an effector of ARF1/3 (Kanoh et al., 1997) as well as activated Rac1 (Van Aelst et al., 1996). Our yeast two-hybrid screening using Arl1(Q711) as the bait retrieved POR1/Arfaptin-2. Using the yeast two-hybrid assay, it was shown that POR1/Arfaptin-2 interacted with GTP- but not GDP-bound forms of both Arl1 and ARF1. In addition, POR1/Arfaptin-2 could be retained by an immobilized, activated form of Arl1 and ARF1 but not by their respective GDP-bound forms. However, the activated form of Arl1 did not interact with GGA-1, suggesting that GGA-1 is a specific effector for ARF1. These results suggest that POR1/Arfaptin-2 is a common effector shared by Arl1, ARF1, ARF3 and Rac1. POR1/arfaptin-2 potently increase the affinity of ARF1 and ARF3 for GTP in vitro. The stoichiometry of GTP binding to ARF can be increased from about 0.05 mol/mol to almost 0.5 mol/mol of recombinant ARF. The observed effects of Arl1(Q71L) on massive COPI and AP-1 Golgi association could be potentially mediated by POR1/arfaptin-2 or other effectors that lead to activation and Golgi recruitment of endogenous ARF1/3. In support of this hypothesis, expression of Arl1(Q71L) also resulted in massive recruitment of ARFs onto the Golgi in a Brefeldin-A-resistant manner. Although it might be due to an indirect effect of Golgi disassembly, the observed dissociation of AP-1 from the perinuclear Golgi upon expression of Arl1(T31N) could result from indirect inactivation of Golgi-localized ARFs. Our results thus highlight a potentially important mechanism for cross-talk among different small GTPases in regulating membrane traffic.
During the preparation of this manuscript, a report from Kahns laboratory (Van Valkenburgh et al., 2001) appeared. It was different from our results in that they show that the Golgi localization of HA-tagged Arl1 is quickly disrupted within three minutes of Brefeldin A treatment, and HA-tagged Arl1(Q71L) is only partially resistant to Brefeldin A treatment, such that it also dissociates from Golgi within five minutes. The discrepancy is possiblly due to the tag that we think may interfere with their Arl1 function, as we observed that the C-terminal fusion of EGFP to Arl1 mutants makes them less potent than non-tagged versions in affecting Golgi structure and function. In their study, Van Valkenburgh et al. also uncovered an interaction of Arl1 with POR1/arfaptin-2 as well as with other proteins such as Golgin-245 and SCOCO, highlighting the point that Arl1 and ARF1 may have some common effectors in addition to effectors that are unique to each of them. Our unique establishment of Arl1 enrichment in the vesicular-tubular structures in the trans-Golgi, the demonstration that Golgi recruitment of Arl1 depends on both activation by guanine nucleotide exchange as well as on N-terminal myristoylation, and our investigation of the effects of various mutant forms of Arl1 on subcellular distribution of COPI, AP-1, and ARFs, and on the Golgi structure and function underscore the importance of regulation of Golgi structure and function by Arl1.
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ACKNOWLEDGMENTS |
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