Phospholipid- and GTP-dependent Activation of Cholera Toxin and Phospholipase D by Human ADP-ribosylation Factor-like Protein 1 (HARL1)*

Jin-Xin HongDagger , Fang-Jen S. Lee§, Walter A. PattonDagger , Ching Yi Lin§, Joel MossDagger , and Martha VaughanDagger

From the Dagger  Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 and the § Institute of Molecular Medicine, School of Medicine, National Taiwan University, Taipei 100, Taiwan, Republic of China

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins named for their ability to activate cholera toxin (CT) ADP-ribosyltransferase activity, have a critical role in vesicular transport and activate a phospholipase D (PLD) isoform. Although ARF-like (ARL) proteins are very similar in sequence to ARFs, they were initially believed not to activate CT or PLD. mRNA for human ARL1 (hARL1), which is 57% identical in amino acid sequence to hARF1, is present in all tissues, with the highest amounts in kidney and pancreas and barely detectable amounts in brain. Relative amounts of hARL1 protein were similar to mRNA levels. Purified hARL1 (rARL1) synthesized in Escherichia coli had less activity toward PLD than did rARF1, although PLD activation by both proteins was guanosine guanosine 5'-(gamma -thio)triphosphate (GTPgamma S)-dependent. ARL1 stimulation of CT-catalyzed ADP-ribosylation was considerably less than that by rARF1 and was phospholipid dependent. GTPgamma S-binding by rARL1 was also phospholipid- and detergent-dependent, and in assays containing phosphatidylserine, was greater than that by rARF1. In vitro, the activities of rARL1 and rARF1 are similar. Rather than being a member of a separate subfamily, hARL1, which activates PLD and CT in a phospholipiddependent manner, appears to be part of a continuum of ARF family proteins.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

ADP-ribosylation factors (ARFs),1 a family of 20-kDa GTP-binding proteins, were discovered as activators of cholera toxin (CTA)-catalyzed ADP-ribosylation of Gsalpha (1) and subsequently shown to serve as allosteric activators of the toxin catalytic unit (2). ARFs are known to function in vesicular transport and membrane trafficking (3) and, more recently, were found to activate phospholipase D (PLD), an enzyme that cleaves phosphatidylcholine (PC) to produce phosphatidic acid and choline (4, 5). ARFs require phospholipid and/or detergent for high affinity GTP binding and optimal enhancement of CTA activity (6, 7). There are six known mammalian ARF proteins, which can be divided into three classes based on deduced amino acid sequence, protein size, and gene structure (8, 9). Members of all three classes can associate with specific intracellular membranes, consistent with roles in vesicular transport, and can also activate PLD (10), processes that could be intimately related.

Although the ARF-like proteins (ARLs) have deduced amino acid sequences very similar to ARFs (11-15), they appeared initially not to activate cholera toxin and failed to rescue the lethal Saccharomyces cerevisiae mutant (11) in which the arf1 and arf2 genes are no longer functional. Like ARFs, ARLs bind GTP; ARLs, however, hydrolyze bound GTP, whereas ARFs do not (16). Some ARLs exhibit tissue-, cell- and/or differentiation stage-specific expression (12-15). ARL1 reportedly localized to the Golgi in normal rat kidney cells (17) and in S. cerevisiae (18), suggesting a role in vesicular trafficking. Unlike the combination of ARFs 1 and 2, the yARL1 gene knock out construct was not lethal. Moreover, yARL1 did not rescue the arf1- arf2- construct and thus may not have a role in constitutive secretion, but rather in regulated secretion.

We report here that the hARL1 protein is abundant in most human tissues, except brain (adult and fetal). Human ARL1, which is more similar in amino acid sequence to hARF1 than to other human ARLs, can, under certain conditions, activate PLD and enhance CTA-catalyzed ADP-ribosyltransferase activity.

    EXPERIMENTAL PROCEDURES
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Results & Discussion
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Materials-- Brain phosphatidylethanolamine (PE) and L-alpha -phosphatidylcholine (PC) were purchased from Avanti Polar Lipids (Alabaster, AL); L-alpha -phosphatidyl-D-myoinositol 4,5-bisphosphate (PIP2), cardiolipin (CL), and L-alpha -phosphatidylserine (PS) from Sigma, choline-[methyl-3H]DPPC (50 Ci/mmol) from NEN Life Science Products; and [adenosine-14C]NAD (280 mCi/mmol) from Amersham Pharmacia Biotech. ARF-activated PLD was highly purified as published (19). rARF1 and rARL1 proteins were synthesized in Escherichia coli and purified as described (20, 21). Sources of other reagents have been published (10, 20).

Expression and Purification of Recombinant Proteins-- The open reading frame of human ARL1 was obtained by polymerase chain reaction using primers that incorporated NdeI and BamHI sites at the initiating methionine and 6 base pairs downstream of the stop codon, respectively. For the His-tagged hARL1 fusion protein, the polymerase chain reaction product was inserted into vector pET15b (Novagen), yielding pET15bhARL1. For the nonfusion protein, the polymerase chain reaction product was inserted into vector pT7 (20), yielding pT7hARL1. BL21(DE3) cells transformed with the above constructs were grown to a density of A600 ~ 1.0 in LB broth containing ampicillin (100 µg/ml), at which time protein synthesis was induced with 1 mM isopropyl-1-thio-beta -D-galactopyranoside. After 3 h, cells were harvested by centrifugation, washed once in 20 mM Tris, pH 7.4, 1 mM EDTA, and stored at -80 °C. For large scale protein production, one liter of LB broth containing ampicillin was inoculated with 5 ml of an overnight culture, followed by shaking at 37 °C. When the culture reached A600 of ~ 0.6-0.8, protein production was induced with 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside. After 3 h, bacteria were collected by centrifugation and stored at -20 °C. Cell pellets were suspended in 10 ml of phosphate-buffered saline, pH 7.4, containing lysozyme (0.5 mg/ml) and disrupted by sonification. The lysate was centrifuged for 30 min at 100,000 × g and the supernatant recovered. His-tagged protein was isolated on Ni2+-nitrilotriacetic acid resin (Qiagen, Chatsworth, CA) by standard methods; nonfusion protein was isolated by gel permeation chromatography (20). Purity was assessed by Coomassie Blue staining of SDS-polyacrylamide gels, and protein was quantified by Coomassie Blue dye-binding assays (Bio-Rad).

PLD Assays-- DPPC hydrolysis was assayed as described previously (10) with slight modifications. Briefly, 50 µl of mixed lipid vesicles (PC/PE/PIP2, molar ratio 16:1.0:1.4) with choline-[methyl-3H]DPPC to yield ~300,000 cpm/assay were added to 2 µl of PLD (~0.5 µg) in a total of 300 µl containing 42 mM Hepes, pH 7.5, 2.5 mM EGTA, 67 mM KCl, 2.5 mM MgCl2, 300 nM free calcium, and 10 mM DTT with GTPgamma S and other additions as indicated. Assays were incubated at 37 °C for 1 h before addition of 1 ml of CHCl3/CH3OH/concentrated HCl (50:50:0.5, v/v/v), and 0.35 ml of 1 M HCl, 5 mM EGTA. After centrifugation (5 min, 3000 rpm, 4 °C, Sorvall RC3B, H2000B swinging bucket rotor), 3H in 575 µl of the aqueous phase was quantified by liquid scintillation counting.

GTPgamma S Binding Assay-- Binding by rARL1 and rARF1 was assayed in total volume of 150 µl containing 1 µM [35S]GTPgamma S (1.25 µCi), 150 µM agmatine, 10 mM DTT, 200 µM NAD, and indicated lipids. After 1 h at 30 °C and addition of 2 ml of washing buffer (50 mM potassium phosphate, pH 7.5, 1 mM DTT, 5 mM MgCl2), proteins were collected on nitrocellulose filters and washed (five times) with 2 ml of buffer; radioactivity was quantified by liquid scintillation counting of dry filters.

CTA-catalyzed ADP-ribosyltransferase Activity-- Assays (total volume: 150 µl) contained 1 µg of CTA, 10 mM agmatine, 20 mM MgCl2, 200 µM [adenosine-14C]NAD (0.05 µCi), and 10 mM DTT with lipid, detergent, GTPgamma S, and other additions (as indicated), in the same buffer used in the PLD assay. Assays were terminated after 1 h at 30 °C, and ADP-ribosylagmatine was isolated for radioassay (22).

Northern Blotting-- Filters with mRNA from human tissues (CLONTECH) were hybridized as described (23) with a 3'-end-32P-labeled, 45-base oligonucleotide probe (5'-CCG AGT TCC AAA CAG ACT GGA AAA TAT ACT TGA GAA AAA GCC ACC-3') complementary to the 5'-end of hARL1. The probe (specific activity: ~6 × 108 cpm/mg) was prepared by incubation with [alpha -32P]dATP and terminal deoxynucleotidyltransferase, followed by purification on a NAP-5 column (Amersham Pharmacia Biotech, Uppsala, Sweden). Blots were also hybridized with a random-primed, 32P-radiolabeled human actin probe as an internal control. Bands were quantified on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) for calculation of ARL/actin ratios.

Polyclonal Antibody Production-- Purified recombinant His-tagged hARL1, prepared as described above, was further purified by SDS-PAGE. A band corresponding to the pure protein was excised from the gel for immunization of rabbits as described (24). The preparation of antibodies against hARL5 has been described (18).

Western Blotting-- Two µg each of purified nonfusion and/or His tag fusion ARLs (rARL1, rARL2, rARL3, mARL4, rARL5) and ARFs (rARF1, rARF5, and rARF6) were subjected to SDS-PAGE under reducing conditions, before transfer to polyvinylidene difluoride membranes in 25 mM Tris base, 192 mM glycine, 20% methanol. After incubation with 3% gelatin in Tris-buffered saline, membranes were incubated with anti-hARL1 polyclonal antibodies, followed by donkey anti-rabbit horseradish peroxidase conjugate (Amersham Pharmacia Biotech) and detection with the ECL chemiluminescent system (Amersham Pharmacia Biotech), and exposure to Hyper-film-MP. A human adult tissue blot (6.5-100 kDa, CLONTECH) was incubated with rabbit antibodies against rARL1; reactivity was detected with ECL. As a control, primary and secondary antibodies were removed from the blot using the multiple tissue Western blot-stripping protocol and the blot reacted with rabbit anti-alpha -tubulin antiserum.

    RESULTS AND DISCUSSION
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Results & Discussion
References

Using a hARL1-specific oligonucleotide probe for Northern blotting, hARL1 mRNA was detected in most human tissues (lung, heart, liver, placenta, kidney, pancreas, and skeletal muscle) with the largest amount in pancreas and kidney; less ARL1 mRNA was present in adult or fetal brain (Fig. 1). The hARL1-specific oligonucleotide probe hybridized with three distinct bands of 3.2, 1.7, and 1.2 kb in RNA from all tissues except brain, where only the 3.2-kb species was found. In additional blotting experiments, a different sequence-specific probe yielded identical results for hARL1 (data not shown). The level of rat ARL1 mRNA was reported to be reduced after differentiation of 3T3-L1 cells and only a single 2.2-kb mRNA was detected (14). In contrast, Lowe et al. (17) reported that the major rat ARL1 mRNA had a size of 1.8 kb and was present in all tissues tested, albeit at relatively low levels in lung and spleen. The discrepancy in size between human and rat message is probably due to species differences. In addition to those shown for rat ARL1 (17), multiple mRNA species were also noted for human ARL3 (13) and rat ARL5 (15).


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Fig. 1.   hARL1 mRNA in human tissues. RNA blots (MTN blots, CLONTECH) containing poly(A)+ RNA (2 µg) from various adult human tissues (upper panels) and fetal human tissues (lower panels) were hybridized with a 3'-end-32P-labeled 45-base oligonucleotide probe (5'-CCG AGT TCC AAA CAG ACT GGA AAA TAT ACT TGA GAA AAA GCC ACC-3') complementary to a 5'-end region of hARL1. Arrows indicate 1.2-, 1.7-, and 3.2-kb hARL1 mRNAs. Hybridization with a human beta -actin probe was performed as a control for sample loading. The ratio of the sum of intensities of all hARL1 bands and beta -actin bands is at the bottom of each lane. Data shown in Figs. 1-5 are representative of those from two separate experiments.

To determine the tissue distribution of hARL protein, we prepared polyclonal antibodies against 6-His hARL1, which reacted on Western blots with preparations of hARL1 synthesized in E. coli (Fig. 2, A and B). (His-tagged proteins were purified by Ni2+/affinity chromatography and nonfusion proteins by size-exclusion chromatography.) The antibodies against hARL1 did not react detectably with other human ARLs or human ARFs 1, 5, or 6 (Fig. 2, A and B). By Western blotting, amounts of hARL1 protein were highest in kidney, liver, heart, and lung (Fig. 2C), as were mRNA levels. The nature of the lower molecular weight immunoreactive species in heart and skeletal muscle is unknown, but it was also seen when the same blot was stripped and incubated with antibodies against His-tagged hARL5. Differences in protein size may relate to tissue-specific, differential protein processing.


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Fig. 2.   Reactivity of anti-hARL1 polyclonal antibodies with ARFs and ARLs detection of hARL1 protein in human tissues. A, recombinant nonfusion ARFs as well as nonfusion and 6-His ARLs (2 µg each) (B) were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membranes, reacted with anti-hARL1 polyclonal antibodies, developed, and exposed to Hyper-film-MP. Positions of protein standards (kDa) are noted in A. C, a multiple human tissue blot (MTN blot, 6.5-100-kDa proteins, purchased from CLONTECH) was incubated with a rabbit polyclonal antibody against hARL1. The chemiluminescent signal was imaged on Hyper-film-MP with a 5-min exposure. hARL1 was detected, as indicated by the arrows. Primary and secondary antibodies and luminol substrate were removed from the blot using the multiple tissue Western blot-stripping protocol, before it was reacted with a rabbit anti-alpha -tubulin antiserum. Positions of protein standards (kDa) are to the left.

Activation of CTA by ARF requires phospholipid or detergent (7). In the presence of PS or PC under PLD assay conditions, hARL1 activated CTA, albeit much less effectively than did hARF1 (Table I and Fig. 3). Activation of CTA by hARL1 was, for the most part, not GTP-dependent (Table II). Similar to preparations of rARF6 (25), rDelta 13ARF1, and rpka14ARF1 (20) that were isolated in an active form with GTP bound, rARL1 required dialysis against M urea before GTPgamma S-dependent CTA activation could be detected (Table II). As shown in Table III, GTPgamma S binding by hARL1 in vitro, like that by hARF1, was modified by phospholipids and detergent. In the presence of CL, binding by hARF1 was greater than by hARL1, but hARL1 bound more GTPgamma S than hARF1 in the presence of PS. In all cases, only a small fraction of the recombinant proteins exhibited high affinity binding.

                              
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Table I
Effect of lipids and detergent on hARL1 stimulation of CTA activity
Assays without or with hARF1 or hARL1 (5 µg), CTA (1 µg), 200 µM [adenosine-14C]NAD, 10 mM agmatine, 200 µM GTPgamma S, and phospholipid or detergent were incubated at 30 °C for 1 h. Data are means of values from duplicate assays that agreed within 10% and are representative of two separate experiments.


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Fig. 3.   Activation of CTA by rARL1 and rARF1. Duplicate assays containing 200 µM GTPgamma S, 253 µM PS, 1 µg of CTA, 200 µM [adenosine-14C]NAD (0.05 µCi), 10 mM agmatine, the PLD buffer system described under "Experimental Procedures" with the indicated amounts of rARF1 (bullet ), rARL1 (open circle ), or ovalbumin (black-triangle), were incubated at 30 °C for 1 h. For Figs. 3-5, points are means of values from duplicate assays ± one-half the range.

                              
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Table II
Effect of nucleotides on rARL1 stimulation of CTA activity
Assays contained 6 µg of the indicated protein (with or without 200 µM GTPgamma S or GDP) and were carried out as described under "Experimental Procedures." rARL1-D1 and rARL1-D2 were two separate preparations of rARL1 dialyzed against 7 M urea in KEND buffer (10 mM potassium phosphate, pH 7.4, 1 mM EDTA, 100 mM NaCl, 1 mM dithiothreitol) (to remove bound nucleotide) for 24 and 48 h, respectively followed by several changes of buffer without urea (20). Data are means of values from duplicate assays that agreed within 10% and are representative of two separate experiments.

                              
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Table III
GTPgamma S binding to rARL1 and rARF1
Samples of rARF1 or rARL1 (3 µg), 1 µM GTPgamma S, and the indicated lipid or detergent were incubated at 30 °C for 1 h. Data are means of values from duplicate assays that agreed within 10% and are representative of two separate experiments.

Initially, hARL1 was thought not to activate PLD significantly (21, 26). Activation of a highly purified PLD by hARL1 was less than that by hARF1 (Fig. 4), but was GTPgamma S-dependent (Fig. 5). Activation of PLD by hARL, along with the prior observation of rat ARL1 localization to the Golgi, could be consistent with a role in vesicular trafficking similar to that of ARFs.


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Fig. 4.   Effect of rARL1 on PLD activity. Duplicate assays containing 200 µM GTPgamma S, 10 mM DTT, and 2 µl of PLD (~0.5 µg) were performed with the indicated concentration of rARF1 (bullet ) and rARL1 (open circle ). The experiment was repeated three times.


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Fig. 5.   Effects of GTPgamma S on PLD activation by rARL1 and rARF1. Duplicate assays containing PLD (2 µl), 10 mM DTT, the indicated concentration of GTPgamma S, and 5 µM of rARL1 (bullet ) or rARL1 (open circle ) were incubated at 37 °C for 1 h. The experiment was repeated twice.

Based on protein size, deduced amino acid sequence, phylogenetic analysis, and gene structure, the six mammalian ARFs can be grouped into three classes (8). ARL proteins are structurally related to ARFs, but due to their relative ineffectiveness in the activation of cholera toxin and inability to rescue the lethal arf1- arf2- double deletion mutant in yeast (8), they were not classified as ARFs. Alignment of the amino acid sequence of hARL1 with those of hARFs and other hARLs reveals the greatest degree of identity exists between hARL1 and class II ARFs (Table IV). Sequences of hARF4 and hARF5 (180 amino acids) are 58% identical to hARL1. Deduced amino acid sequences of hARL1 and hARF1 are 57% identical, and the functional consequences of some of these differences have been demonstrated (21). hARL1 is only 35-43% identical to the other four known ARLs. Based on this alignment, it appears that human ARLs may fall into classes in a manner analogous to human ARFs (Fig. 6). ARL1 differs on one hand from ARLs 2 and 3, which are 53% identical and on the other from ARLs 4 and 5, which are 60% identical. ARL2 is, however, only 27 and 29% identical to ARLs 4 and ARL5, respectively, and ARL3 is 37 and 35% identical to ARL4 and ARL5, respectively. We, therefore, propose that there are at least three distinct classes of ARL (I, II, and III); human ARL1 (Class I) appears to be different, from ARLs 2 and 3 (Class II) and ARLs 4 and 5 (Class III). Human ARLs 2 and 3 did not activate PLD, although, under the same assay conditions, they bound significantly more GTPgamma S than did hARL1.2

                              
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Table IV
Percentage identity and similarity of deduced amino acid sequences of ARFs and ARLs
Percentage amino acid identity is shown above the diagonal and percentage similarity below. Accession numbers for the sequences used to generate this table (using the Palign routine of the PC Gene program) are from the GenBank (GB) and SwissProt (SP) databases: hARF1 (Ref. 27, SP-P32889), bARF2 (Ref. 28, SP-P16500), hARF3 (Ref. 27, SP-P16587), hARF4 (Ref. 29, SP-P18085), hARF5 (Ref. 8, SP-P26437), hARL1 (Ref. 21, GB-L28997), hARL2 (Ref. 12, SP-P36404), hARL3 (Ref. 13, GB-U07151), hARL4 (Ref. 30, GB-U73960), hARL5 (Ref. 31, SP-P49703).


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Fig. 6.   Deduced amino acid sequences of hARFs and hARLs. Sequences were aligned using the Clustal routine of PC Gene (Oxford Molecular, Campbell, CA). Residues identical in members of class II or III are shown as consensus sequences (uppercase), others are in lowercase. Dots indicates an amino acid identical to the consensus sequence for that class.

Because its sequence is more similar to those of hARFs than to other hARLs, and in light of its activity toward PLD and CTA in the presence of appropriate lipids, hARL1 may be part of an ARF continuum rather than a member of a separate subfamily and may have physiological functions similar to those of ARFs.

    ACKNOWLEDGEMENTS

We thank Dr. Joong-Soo Han, Dr. Joon-Ki Chung, and Jason Donaldson in the group of Dr. Sue Goo Rhee (Laboratory of Cell Signaling, NHLBI, National Institutes of Health, Bethesda, MD) for providing us with highly purified PLD from rat brain. We also thank Carol Kosh for expert secretarial assistance.

    FOOTNOTES

* This work was supported in part by a grant from the Institute of Biomedical Sciences, Academia Sinica, Republic of China (IMBS-CRC86-TO1) (to F.-J. S. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: 7 Chung Shan South Rd., Taipei, Taiwan, R.O.C. Tel.: 886-2-2397-0800 (ext. 5730); Fax: 886-2-2321-0977; E-mail: fangjen{at}ha.mc.ntu.edu.tw.

1 The abbreviations used are: ARF, ADP-ribosylation factor; rARF, recombinant human ARF; ARL, ARF-like protein; rARL, recombinant human ARL1; mARL, recombinant mouse ARL; GTPgamma S, guanosine 5'-(gamma -thio)triphosphate; PLD, phospholipase D; CT, cholera toxin; CTA, cholera toxin subunit A; CL, cardiolipin; PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; PIP2, phosphatidylinositol 4,5-bisphosphate; DMPC, dimyristoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s).

2 F.-J. S. Lee, J.-X. Hong, W. A. Patton, C. Y. Lin, J. Moss, and M. Vaughan, unpublished data.

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Abstract
Introduction
Procedures
Results & Discussion
References

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