(Received for publication, October 26, 1994)
From the
ADP-ribosylation factors (ARFs), initially described as
activators of cholera toxin ADP-ribosyltransferase activity, regulate
intracellular vesicular membrane trafficking and stimulate a
phospholipase D (PLD) isoform. ARF-like (ARL) proteins are structurally
related to ARFs but do not activate cholera toxin and have relatively
little effect on PLD. A new human ARL gene termed hARL1, which shares
57% amino acid identity with hARF1, was identified using a polymerase
chain reaction-based cloning method. To determine whether different
structural elements are responsible for the activation of the A subunit
of cholera toxin and PLD, chimeric proteins were constructed by
switching the amino-terminal 73 amino acids of ARF1 and ARL1. The
recombinant rL73/F protein, in which the amino-terminal 73 amino acids
of ARL1 replaced those of ARF1, activated the A subunit of cholera
toxin, whereas the rF73/L protein, in which the NH-terminal
73 amino acids of ARF1 replaced those of ARL1, was inactive. The two
chimeric proteins had quite opposite effects on PLD activity. rF73/L
activated PLD as effectively as rARF1, whereas rL73/F protein activated
PLD only slightly. It appears that the amino-terminal region of ARF1 is
not critical for its action as a GTP-dependent activator of cholera
toxin, whereas it is necessary for activation of the putative effector
enzyme, PLD.
Cholera toxin, a secretory product of Vibrio cholerae that is responsible (in large part) for the devastating diarrheal
syndrome characteristic of cholera, exerts its effect on intestinal
cells via the ADP-ribosylation of the subunit of a heterotrimeric
guanine nucleotide-binding (G) protein, i.e. G
,
the stimulatory G protein of adenylyl cyclase, that serves as a
signal-transducing element from surface receptors to intracellular
effectors(1, 2, 3) . The
ADP-ribosyltransferase activity of the A subunit of cholera toxin (CTA) (
)is stimulated by a family of
20-kDa guanine
nucleotide-binding proteins known as ADP-ribosylation factors or
ARFs(4, 5, 6) . Sequence comparisons of the
ARF proteins and other GTP-binding proteins revealed that ARF proteins
have a similar degree of relatedness to the Ras superfamily and the
heterotrimeric G protein
subunits(7) . The family of ARFs
includes the ARF-like proteins (ARLs), which share 30-60% amino
acid identity with
ARFs(8, 9, 10, 11) . Recombinant
ARLs bind and hydrolyze GTP but do not function as cofactors for CTA
activation, suggesting that ARLs are not functional homologues of
ARFs(8) . Under physiological conditions, ARFs participate in
vesicular transport through the Golgi in mammalian cells (12) and are present on both nonclathrin- (13) and
clathrin-coated vesicles (14, 15) that are accumulated
in the presence of GTP
S.
ARF proteins were recently reported to activate phospholipase D (PLD), an enzyme that cleaves phosphatidylcholine (PC) to produce phosphatidic acid (PA) and choline(16, 17) . PA can serve as an effector in several physiological processes including DNA synthesis, cell proliferation, and secretory responses. PA may also be metabolized by PA phosphohydrolase to diacylglycerol, a well characterized activator of protein kinase C. Brown et al.(16) and Cockcroft et al.(17) purified a cytosolic component from bovine brain that markedly enhanced PLD activity in membranes in a GTP-dependent manner. This cytosolic factor was identified as two small GTP-binding proteins, ARF1 and ARF3. Recent work of Massenburg et al.(18) has shown that all three classes of ARFs can activate PLD. The effects of ARL proteins on PLD have not been reported. To determine whether different structural elements are responsible for the two ARF activities, chimeric proteins were constructed by switching the amino-terminal amino acid sequences of ARF1 and ARL1. We report here the identification of different ARF domains involved in the activation of cholera toxin and phospholipase D.
Figure 1: Comparison of deduced amino acid sequences of hARF1 and hARL1. The human ARL1 cDNA and the predicted protein sequences may be accessed in GenBank using accession no. L28997. Arrow indicates the position of ligation of the two parts of the chimeric proteins, rF73/L and rL73/F.
To define the
domains responsible for CTA and PLD activation by ARF, two chimeric
recombinant proteins were constructed by switching the amino-terminal
73 amino acids of ARF1 and ARL1 (Fig. 1). The first 73 amino
acids contain a region similar to the effector loop found in Ras (7, 20) plus the two conserved nucleotide-binding
sequences GXXXXGK and DXXGQ. The rL73/F chimera
contains the NH-terminal 73 amino acids of ARL1 (40% of the
ARL1 sequence) linked to the COOH-terminal 108 residues of ARF1 (60% of
the ARF1 sequence). The rF73/L chimera encodes the
NH
-terminal 73 amino acids of ARF1 linked to the
COOH-terminal 108 residues of ARL1. The chimeras, expressed as
nonmyristoylated proteins, were purified from E. coli for use
in biochemical studies. (Although it is not known whether ARL proteins
are myristoylated in vivo, it is known that myristoylation is
not absolutely required for the activation of CTA (18, 21) or PLD (16, 17, 18) by rARF1.)
Figure 2:
Stimulation of ADP-ribosyltransferase
activity of cholera toxin by rARF1, rF73/L, and rL73/F. Activation of
CTA by rARF1 (A), rL73/F (B), and rF73/L (C) was evaluated in the NAD:agmatine ADP-ribosyltransferase
assay (25) in 0.003% SDS with 0.1-4.0 µg of ARF.
Activity of CTA without added ARF was 2.8 and 2.9 nmol/h, with 100
µM GTPS (closedsymbols) and 100
µM GDP
S (opensymbols),
respectively. Data are means of values of duplicate assays; variance
was less than 10%. The experiments were repeated at least
twice.
Activation of CTA by ARF requires phospholipid
and/or detergent(6) . In the presence of SDS, the maximal
activity of rL73/F was similar to that of rARF1 (Fig. 3A), although the GTPS concentration
required for half-maximal activation of CTA (EC
) by rL73/F
(
200 µM) was nearly 40 times that required with rARF1
(
5 µM). In the presence of DMPC and sodium cholate or
cardiolipin, however, the maximal activity of rL73/F was much higher
than that of rARF1 (Fig. 3, B and C). The
EC
values of GTP
S for rARF1 and rL73/F were about 0.1
and 10 µM, respectively. Under all these conditions,
rF73/L chimera was inactive, resembling wild type rARL1 (data not
shown).
Figure 3:
GTP-dependent stimulation of
ADP-ribosyltransferase activity of cholera toxin by rARF1, rF73/L, and
rL73/F. Assays contained 1 µg of rARF1 (), rL73/F (
), or
rF73/L (
), the indicated concentration of GTP
S, and 0.003%
SDS (A), 3 mM DMPC plus 0.2% sodium cholate (B), or 0.3 mg/ml cardiolipin (C). Data are means of
values of duplicate assays; variance was less than 10%. The experiments
were repeated at least twice.
As the rL73/F chimera, like wild type ARF1, stimulates cholera toxin ADP-ribosyltransferase activity, whereas rF73/L, like wild type ARL1, cannot, the functional domain of ARF that is responsible for cholera toxin activation is apparently not localized in the amino-terminal region (at least not in the first 73 amino acids). This is consistent with the observation that deletion of the amino-terminal 13 amino acids of ARF1 did not affect the ability of ARF to activate CTA(19) , but differs from the previous view that the amino terminus of ARF is critical for its activity based on the ability of a peptide with that sequence to inhibit ARF activation of CTA(23) . We propose that the carboxyl-terminal 60% of ARF contains a structural element that is crucial for its function as a CTA cofactor.
Under the PLD assay
conditions and in the presence of 100 µM GTPS (but
not GDP
S), CTA was activated by rL73/F and rARF1, but not by
rF73/L (data not shown), which is in agreement with the results shown
in Fig. 2. In contrast, rF73/L, which was inactive in the CTA
assay, activated PLD in the presence of 100 µM GTP
S
in a concentration-dependent manner (Fig. 4C). The
maximal activity of rF73/L was nearly equal to that of rARF1 (Fig. 4, A and C). In the same experiment,
rL73/F (which activated CTA) produced only a small degree of activation (Fig. 4B). The activation of PLD was clearly
GTP-dependent (Fig. 4), although there was a fraction of rARF1
activity (as shown also in Fig. 2) that was not, which could be
explained by the presence in the recombinant ARF protein of tightly
bound GTP(22) .
Figure 4:
Activation of PLD by rARF1, rF73/L, and
rL73/F. Assays contained 0.6 µg of partially purified PLD (10
µl, 0.06 mg/ml), and the indicated amount (0.1-8.0 µg) of
rARF1 (A), rL73/F (B), or rF73/L (C) with 100 µM GTPS (closed
symbols) or 100 µM GDP
S (open symbols).
Basal activity of PLD without added ARF was 2.9 and 2.3 pmol/h, with
GTP
S and GDP
S, respectively. Data are means of values of
triplicate assays; variance was less than
10%.
It appears that an ARF domain that is important for PLD activation is located in the amino-terminal portion of the protein (at least the first 73 amino acids), although the specific location of the activating structure or the mechanism of activation is still unclear. The putative Ras effector loop is also located in this region(7, 20) . The slight GTP-dependent activity of rL73/F, which was observed also with rARL1 (data not shown), may be related to the fact that ARL1 and ARF1 are 84% identical in amino acids 37-55 (the region similar to the putative Ras effector loop) and 68% identical in sequences of the first 73 amino acids. The finding that rL73/F is clearly less potent than rARF1 may mean that it does not contain all of the specific sequence necessary for optimal PLD activation (at least not for this form of partially purified PLD), or that it associates less well with membranes (phospholipids).
In any case, it seems that different functional
domains are involved in the activation of CTA and PLD; thus, ARF could
be a rather versatile protein, perhaps subject to control related to
subcellular location and/or the presence of effector molecules. The
role of PLD activation by ARF in vesicle-mediated membrane trafficking
is at present speculative. Activation of PLD by ARF proteins could
generate second messengers that are involved in signaling processes or
products, e.g. phosphatidic acid, that influence membrane
properties and thereby vesicle budding or fusion. The action of PLD, by
altering membrane phospholipid composition, might directly or
indirectly provide binding sites for membrane docking
proteins(16) . Liscovitch et al.(24) have
recently published an appealing hypothesis involving a positive
feedback between PLD activation and PIP biosynthesis in
ARF-regulated vesicle fusion. It is tempting to speculate that
different functions of ARF are involved in this action and in its
operation as a critical compound required for coatomer or AP-1 binding
in vesicle formation. As the physiological role of ARF-stimulated PC
hydrolysis and the mechanism of ARF-effector interaction are still
unknown, elucidation of structure-function relationships in ARFs and
ARLs may lead to a better understanding of the precise roles of ARF and
ARL proteins in signaling and other cellular processes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L28997[GenBank].