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'-(
-thio)triphosphate (GTP
S)-dependent. ARL1 stimulation of CT-catalyzed
ADP-ribosylation was considerably less than that by rARF1 and was
phospholipid dependent. GTP
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.
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INTRODUCTION |
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 Gs
(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.
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EXPERIMENTAL PROCEDURES |
Materials--
Brain phosphatidylethanolamine (PE) and
L-
-phosphatidylcholine (PC) were purchased from Avanti
Polar Lipids (Alabaster, AL); L-
-phosphatidyl-D-myoinositol
4,5-bisphosphate (PIP2), cardiolipin (CL), and
L-
-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-
-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-
-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 GTP
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.
GTP
S Binding Assay--
Binding by rARL1 and rARF1 was
assayed in total volume of 150 µl containing 1 µM
[35S]GTP
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, GTP
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
[
-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-
-tubulin antiserum.
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RESULTS AND DISCUSSION |
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 -actin probe was performed as a control
for sample loading. The ratio of the sum of intensities of all hARL1
bands and -actin bands is at the bottom of each lane.
Data shown in Figs. 1-5 are representative of those from two separate
experiments.
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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- -tubulin
antiserum. Positions of protein standards (kDa) are to the
left.
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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), r
13ARF1, and rpka14ARF1 (20) that were isolated in an active
form with GTP bound, rARL1 required dialysis against 7 M
urea before GTP
S-dependent CTA activation could be
detected (Table II). As shown in Table
III, GTP
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 GTP
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 GTP 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 GTP 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
( ), rARL1 ( ), or ovalbumin ( ), 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 GTP 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
GTP S binding to rARL1 and rARF1
Samples of rARF1 or rARL1 (3 µg), 1 µM GTP 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.
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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
GTP
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 GTP S, 10 mM DTT, and 2 µl of PLD (~0.5 µg) were performed with
the indicated concentration of rARF1 ( ) and rARL1 ( ). The
experiment was repeated three times.
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Fig. 5.
Effects of GTP S on PLD activation by rARL1
and rARF1. Duplicate assays containing PLD (2 µl), 10 mM DTT, the indicated concentration of GTP S, and 5 µM of rARL1 ( ) or rARL1 ( ) were incubated at
37 °C for 1 h. The experiment was repeated twice.
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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 GTP
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.
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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.
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.