(Received for publication, June 1, 1995; and in revised form, September 25, 1995)
From the
The human and rat homologues of a novel Ras-related GTPase with
unique structural features were cloned by polymerase chain reaction
amplification and cDNA library screening. Their deduced amino acid
sequences are highly homologous (97% identical amino acids; 88.3%
identical nucleotides within the coding region) and comprise all six of
the conserved motifs presumably involved in GTP binding. Because the
sequences exhibit some similarity with members of the ADP-ribosylation
factor (ARF) family (33% identity with ADP-ribosylation factor 1
(ARF1), 39% identity with ARF-like 3), the protein was designated ARP
(ARF-related protein). In contrast to all other members of the ARF
family, ARP lacks the myristoylation site at position 2 and comprises
an insertion of 8 amino acids in the region between PM1 and PM2. mRNA
was found in most rat tissues examined (skeletal muscle, fat, liver,
kidney, spleen, testis, adrenals, ovary, thymus, intestine, and lung).
Western blot analysis with antiserum against recombinant ARP showed a
25-kDa protein in membranes from rat liver, testis, and kidney. Thus,
the protein appears to be posttranslationally modified for membrane
anchoring. Unlike ARF, the ARP immunoreactivity was detected in plasma
membranes but not in cytosol of fractionated 3T3-L1 cells. Recombinant
ARP exhibited specific and saturable GTPS (guanosine
5`-3-O-(thio)triphosphate) binding and, unlike ARF isotypes,
GTPase activity in the absence of tissue extracts or phospholipids.
Thus, the structural and functional characteristics of ARP indicate
that it represents a novel subtype of GTPases, presumably exerting a
unique function and possibly involved in plasma membrane-related
signaling events.
ADP-ribosylation factors (ARF) ()represent a
subfamily of Ras-homologous GTPases (Kahn and Gilman, 1984; Bobak et al., 1989; Kahn et al., 1991) presumably involved
in basic cellular functions, e.g. regulation of phospholipase
D (Brown et al., 1993; Kahn et al., 1993; Cockcroft et al., 1994), exocrine secretion (Zeuzem et al.,
1992b), vesicle traffic from ER to Golgi (Donaldson et al.,
1991; Serafini et al., 1991; Kahn et al., 1992a), and
endocytosis (D'Souza-Schorey et al., 1995). To date, six
mammalian isotypes of ARF (ARF1-ARF6), three isotypes from yeast,
and three from Drosophila have been identified (Lee et
al., 1994a, 1994b; Kahn et al., 1995). All isoforms
exhibit a high degree of overall structural similarity (65-96%
identical amino acids) and share motifs determining common functional
characteristics, e.g. the N-terminal myristoylation site. In
addition, several genes encoding proteins with homology to the ARF
isoforms have been cloned and designated ARF-like genes
(Tamkun et al., 1991; Clark et al., 1993;
Schürmann et al., 1994; Cavenagh et
al., 1994). The products of these genes appear to lack
ADP-ribosylation enhancing activity, and show different characteristics
of GTP-binding (dependence on magnesium and phospholipids) than the ARF
isoforms (Cavenagh et al., 1994). Although some of these
proteins e.g. ARL4 (Schürmann et
al., 1994) exhibit tissue and/or differentiation-specific
expression, little is so far known about their cellular function. In
the present paper, we describe the identification of a new GTPase which
exhibits a remote similarity with other members of the extended ARF
family. Since it differed in important characteristics from other ARF
and ARF-like proteins, e.g. in the lack of a myristoylation
motif, in a constitutive GTPase activity, and in its subcellular
distribution, the protein was designated ARF-related protein (ARP).
Figure 1: Nucleotide and deduced amino acid sequence of rat ARP. The sequence was composed with an incomplete cDNA clone isolated from a rat heart library and with two independent PCR clones (bp 1-194) isolated by the RACE procedure. A complete cDNA clone starting at nucleotide 67 was later isolated from a rat brain library; this clone contained an insertion of 6 bp (underlined) in the untranslated 5` region. Amino acids are given in one-letter code above the respective codons. The conserved consensus motifs of GTP binding are boxed.
A cDNA fragment of rat ARP was used for screening of a human liver cDNA library, and two clones with identical size (1.6 kb) were isolated. Fig. 2depicts the nucleotide sequence, which harbors an open reading frame of 603 codons. Its deduced amino acid sequence is highly homologous to that of rat ARP (Fig. 3A, 195 (97%) identical and 6 differing amino acids, 5 of them conservative substitutions), indicating that the cDNA indeed represented the human homologue. Within the coding region, the nucleotide sequences of rat and human ARP were 88% identical. In the untranslated 3`-region, the overall homology was 71%, reaching even 91% over a stretch of 45 bp (underlined sequence of nucleotides 738-783 in the human cDNA).
Figure 2: Nucleotide and deduced amino acid sequence of human ARP as determined with a full-length cDNA clone isolated from a human liver cDNA library. Amino acids are given in one-letter code above the respective codons. A stretch of 45 nucleotides in the untranslated 3`-region that is highly homologous (91% identical nucleotides) to the rat sequence is underlined.
Figure 3:
Sequence alignments of ARP and other
Ras-related GTPases. Panel A, alignment of the amino acid
sequences of human and rat ARP. The alignment of the deduced amino acid
sequences was performed with the PALIGN program (open gap cost 7, unit
gap cost 1). Identical amino acid residues were highlighted by vertical lines (). Panel B, alignment of rat
ARP with rat ARL3: The alignment was performed as in panel A.
Amino acids identical with residues that are conserved in all other
members of the ARF family are outlined by asterisks. The
conserved motifs of GTP-binding (PM1-PM3, G1-G3) are
depicted above the sequences. Panel C, dendrogram of an
alignment of rat ARP with other members of the Ras superfamily. The
alignment was performed with the CLUSTAL program (gap penalty 5, open
gap cost 10, unit gap cost 10). Accession numbers of the compared
isoforms are as follows: rat Ha-Ras, P20171; canine Rho1, P24406; rat
Rab1, P05711; rat Rab2, P05712; rat Rab4A, X06890, human Ran, P17080;
human ARF1, P10947; human ARF6, P26438; rat ARL1, X76920; rat ARL3,
X76921; rat ARL4, X77235; mouse SarA, P36536; yeast CIN4,
L36669.
It should be noted that the sequence of ARP exhibited several differences to other members of the ARF family. Most strikingly, it lacks the conserved myristoylation site, a glycine in position 2. Furthermore, the loop region between PM1 and PM2 is longer than that in other ARFs because of an insertion of 8 additional amino acids. In addition, the C terminus is longer than that of other ARFs. A multiple alignment of the amino acid sequence of ARP with prototypes of the other Ras-homologous GTPases confirmed that ARP is a distant relative of the ARF family. As is illustrated by the dendogram of the alignment (Fig. 3C), ARP is located on a branch of the ARF family, its similarity to the other ARFs being higher than that of the GTP-binding proteins SarA and CIN4, but considerably lower than all previously identified ARF-like proteins.
Figure 4:
Expression of rat ARP in E. coli (A) or COS-7-cells (B). A, full-length
or truncated ARP-cDNA were subcloned into the pGEX expression vector
and transformed into E. coli DH5, and fusion proteins
were generated as described. The proteins were separated on SDS-PAGE
and stained with Coomassie Blue. GST, preparation of GST
obtained with bland vector; GST-ARP, fusion protein eluted
from the affinity column with glutathione; ARP, recombinant
ARP, which was isolated from the affinity column by thrombin cleavage; GST-ARP
21, fusion protein generated with the incomplete
cDNA clone and eluted with GST. B, the full-length ARP-cDNA
was subcloned into the mammalian expression vector pCMV and COS-7 cells
were transfected as described. Cells were homogenized, and a
particulate fraction of total membranes was isolated. Proteins were
separated by SDS-PAGE and transferred on to nitrocellulose membranes.
Immunochemical detection was performed with antiserum raised against
GST-ARP
21.
The cDNA of ARP was subcloned into the mammalian expression vector
pCMV, and COS-7 cells were transiently transfected with the construct.
Cells were homogenized, and a pellet of total membranes was assayed for
ARP immunoreactivity. Indeed, as is illustrated in Fig. 4(panel B), a 25-kDa protein was detected with
serum against GST-ARP21 in cells transfected with the construct;
it was not present in cells transfected with blank vector. The protein
was not detected with serum that was blocked with recombinant ARP
generated by thrombin cleavage of GST-ARP (not shown).
Figure 5:
Specific binding of GTPS, nucleotide
exchange, and GTPase activity of recombinant rat ARP. Panel A,
magnesium dependence of GTP
S binding. Samples of 5 µg of
recombinant ARP were incubated with tracer GTP
S and the indicated
magnesium concentrations, and bound tracer was separated by filtration
on nitrocellulose membranes after 60 min. Panel B, time course
of GTP
S binding. Samples of 5 µg of recombinant ARP (open
circles) or 10 µg of GST-ARP
21 (filled circles)
were incubated with tracer GTP
S and 10 mM magnesium, and
bound tracer was separated by filtration on nitrocellulose membranes
after the indicated times. Nonspecific binding as determined with
samples containing 100 µM unlabeled GTP
S was less
than 300 cpm/sample. Binding to GST was determined in separate series
and was not distinguishable from nonspecific binding. Panel C,
nucleotide exchange and GTPase activity of ARP. Samples of 1.25 µg
of recombinant ARP were loaded with [
-
P]GTP (circles) or [
S]GTP
S (triangles) for 60 min, and the decrease in bound tracer in
the presence (filled symbols) or absence (open
symbols) of 1 mM GTP or GTP
S was assayed at the
indicated time points. All assays were performed in the presence of 10
mM magnesium chloride without added phospholipids. Panel
D, analysis of the nucleotides bound to recombinant ARP. ARP was
loaded with [
-
P]GTP, and bound nucleotides
were isolated after the indicated times and separated by
TLC.
Unlike the
recombinant ARP, the truncated fusion protein (GST-ARP21) bound
only minute amounts of tracer GTP during a 180 min incubation period.
However, full GTP binding was observed after incubation with tracer for
24 h. Furthermore, HPLC analysis of the native recombinant proteins
indicated that both ARP and GST-ARP
21 were loaded with GTP/GDP in
a ratio of 1:3 after isolation from the E. coli.
Panel
C of Fig. 5illustrates experiments designed to assess the
nucleotide exchange and the GTPase activity of recombinant ARP. ARP was
loaded for 60 min with [S]GTP
S (triangles) or [
-
P]GTP (circles), and the nucleotide exchange or hydrolysis,
respectively, was assayed as the decreases in bound radioactivity. Fig. 5(panel C) illustrates the time course of these
experiments. In the absence of added unlabeled nucleotide, the
GTP
S binding was stable for 60 min, whereas a slow exchange was
visible when 1 mM unlabeled GTP
S (filled
triangles) was added. A marked release of radioactivity from the
recombinant ARP was observed (kinetic constant 0.093
min
), when the protein was loaded with
[
-
P]GTP (open circles), probably
reflecting the release of phosphate from the GTP. Since it was
previously reported that preparations of recombinant proteins may
contain bacterial GTPases (Welsh et al., 1994), we ran
parallel samples containing excess GTP (filled circles) in
order to inhibit these potential contaminants. However, the addition
failed to inhibit the decrease in tracer binding to the recombinant
ARP. Moreover, the decrease in bound GTP and the increase in bound GDP
could be demonstrated directly by TLC analysis of the bound nucleotides (Fig. 5, panel D). Thus, it is concluded that the
release of radioactivity from the bound
[
-
P]GTP indeed reflects the intrinsic
GTPase activity of ARP.
Figure 6: Northern blot analysis of ARP mRNA in different rat tissues. Total RNA from the indicated tissues was hybridized with a probe generated from a cDNA fragment comprising the coding region of ARP. B, total brain; H, heart; M, skeletal muscle; A, fat cells; Li, liver; K, kidney; S, spleen; T, testes; Ad, adrenal gland; O, ovary; Th, thymus; I, intestine; Lu, lung. As judged from ethidium bromide staining (data not shown) the amounts of RNA in each lane were essentially identical with the exception of kidney (partial degradation) and brain (lower amounts).
Antisera were
raised in rabbits against the fusion protein GST-ARP21 and were
tested in preliminary experiments with COS-7 cells transfected with the
ARP cDNA. As is illustrated in Fig. 4and Fig. 7, the
immune serum reacted with a 25-kDa protein in membranes from
transfected COS-7 cells, testis, liver, and kidney. This signal
corresponded with that of recombinant ARP (right lanes in Fig. 7), and was blocked by preincubation of the serum with
recombinant ARP (not shown here; see also Fig. 8). Other
nonspecific bands, i.e. the additional band at 10 kDa, were
not blocked by recombinant ARP. By longer exposure (not shown), a weak
signal was detected in membranes from adipocytes; no immunoreactivity
was present in heart. By a rough comparison with the immunoreactivity
of recombinant ARP, the amounts of ARP present in the membranes of
kidney and testis can be estimated to approximately 5 ng/15 µg of
membrane protein. The apparent molecular mass of the specific
immunoreactivity correlates reasonably well with the calculated value
(23 kDa), indicating that ARP is considerably bigger than all
previously identified members of the ARF family. As anticipated, the
ARP immunoreactivity in membranes from rat tissues migrated at a
smaller apparent molecular weight than recombinant ARP, which, due to
the cloning strategy, has an extended N terminus. A second antiserum
(not shown) detected more nonspecific bands but reacted with the same
rARP-inhibitable immunoreactivity at 25 kDa.
Figure 7: Immunochemical detection of ARP in membranes from rat tissues. Samples of membrane proteins (15 µg/lane) from adipocytes, heart, testis, kidney, liver, and brain were separated by SDS-PAGE (12% gels), transferred on to nitrocellulose membranes, and probed with specific antiserum. Note that the apparent molecular mass of recombinant ARP is approximately 2 kDa higher because of an extension of the N terminus due to the cloning strategy.
Figure 8: Immunochemical detection of ARP in membrane fractions from 3T3-L1 cells. Differentiated 3T3-L1 cells were fractionated as described. Membrane fractions were separated by SDS-PAGE and transferred on to nitrocellulose membranes. Parallel blots of the same membrane preparation were probed with ARP antiserum (upper left), blocked antiserum (upper right), and antisera against Ha-Ras and the glucose transporter GLUT4 (lower panel) as markers of plasma membranes or intracellular microsomes, respectively. The reproduction of the blot probed with Ha-Ras antiserum was cut for rearrangement of the lanes in the same order as the other panels.
In order to assess the subcellular distribution of ARP, 3T3-L1 cells were homogenized and fractionated by differential centrifugation. With this previously established procedure (Weiland et al., 1990; Ziehm et al., 1993), fractions of plasma membranes (PM), ER-enriched high density microsomes, Golgi-enriched low density microsomes, and cytosol were isolated. ARP immunoreactivity (Fig. 8) was detected mainly in the plasma membrane fraction of 3T3-L1 cells; only traces were present in high-density microsomes and no immunoreactivity was detected in the cytosol. The signals were blocked by preincubation of the antiserum with recombinant ARP (Fig. 8, upper right). These data indicate that ARP is exclusively associated with membranes and does not occur in a soluble, cytoplasmic form. In low density microsomes, a 25-kDa band was visible, but this immunoreactivity appeared not inhibited by blocking of the serum with recombinant ARP (upper right). However, since the antiserum reacted with several nonspecific bands in the low density microsomes, we cannot fully exclude that ARP is also present in these microsomes. As is illustrated in the lower panel of Fig. 8, the purity of the membrane fractions was assessed with Western blots of Ha-Ras (marker for plasma membranes) and glucose transporter GLUT4 (marker for low density microsomes). The pattern of the subcellular distribution of these markers and of ARP indicates that the ARP immunoreactivity in the plasma membrane fraction reflects a specific association rather than a cross-contamination of membrane fractions.
On the basis of structural and functional criteria (Kahn et al., 1992b), the novel GTP-binding protein ARP is a Ras-related GTPase with remote similarity to the family of ADP-ribosylation factors. It contains all six motifs (PM1-PM3, G1-G3) involved in nucleotide binding (Valencia et al., 1991), and a recombinant protein isolated from E. coli indeed binds and hydrolyses GTP. Its closest relatives are members of the ARF family (ARL3, 39% identity; ARF1, 33%), whereas the homology to other Ras-related G-proteins is considerably lower (Ras, 17.5% identical amino acids; Rab, 21.9%; Ran, 20.9%; Rho, 24.4%). Furthermore, ARP harbors motifs and residues that are typical for the ARF family: The PM1 motif GLDNAGKTT, the PM3 motif WDXGGQ, the conserved tryptophan in position 86 between the PM3 and G2 motifs, and the G2 motif ANKQD (instead of GNKQD as in all other Ras-related proteins). However, the sequence of ARP differs from that of other members of the ARF family by a lack of the myristoylation site (glycine 2), a striking insertion of 8 amino acids between PM1 and PM2, and an extended C terminus that is highly charged but lacks the lysine residues present in all other ARF isoforms. Also, as is discussed below, the new GTPase exhibited striking functional differences (GTPase activity, subcellular distribution) to the other members of the ARF family. In order to emphasize these differences, we decided to designate the protein ARF-related protein (ARP).
A striking feature of ARP is the lack of
a known lipid modification signal in its sequence. In analogy to other
Ras-related GTPases, and on the basis of the Western blot analysis,
which demonstrated exclusive association of ARP to membranes, it is
reasonable to assume that the protein is co-translationally or
post-translationally modified for membrane anchoring. Other GTPases
have C-terminal motifs for farnesylation (e.g. Ras) or fatty
acid acylation (e.g. Rab), or are myristoylated at glycine 2
(all so far known relatives of ARF). None of these motifs are apparent
in ARP. Thus, the nature of membrane attachment remains to be
determined. At present, we consider the possibility that cysteine 186
is a site of palmitoylation, in analogy to G-protein -subunits or
Ras (Casey, 1995). In spite of the lack of further data, it appears
reasonable to assume that targeting and membrane anchoring of ARP is
fundamentally different from that of ARP isotypes.
A second striking
difference between ARP and other members of the ARF family is its high
GTPase activity. ARF and ARF-like proteins do not hydrolyze GTP in the
absence of tissue extracts containing GTPase activating factors and
phospholipids (Randazzo and Kahn, 1994; Makler et al., 1995).
In contrast, a marked decrease in bound tracer from ARP preloaded with
[-
P]GTP was detected in the absence of any
added tissue extracts or phospholipids. This activity does not appear
to be due to bacterial contamination of the recombinant protein, since
it was not inhibitable by excess unlabeled GTP. Furthermore, other
recombinant GTPases, e.g. ARL4, which we isolated by the same
procedure were essentially devoid of GTPase activity. (
)It
should also be noted that the binding of GTP
S to ARP proceeded
relatively slowly, equilibrium being reached after 30-60 min.
Thus, ARP appears to require the presence of an exchange factor in
addition to a GTPase-activating protein in order to function as a fast
GTP-dependent switch. However, it has to be taken into account that the
GTP-binding assays were conducted in an artificial in vitro system, and with protein that certainly lacked post-translational
modification. Recently, it has been shown that myristoylation of ARF1
accelerates GDP exchange and markedly decreases dissociation of GTP
(Franco et al., 1995).
The truncated ARP (GST-ARP21),
which we had prepared with the incomplete cDNA clone bound GTP in a
specific manner but with a very low association rate. Since the native
GST-ARP
21 is loaded with unlabeled GTP/GDP when isolated from E. coli, the finding indicates that the truncation had
markedly reduced the nucleotide exchange rate and suggests that the N
terminus of ARP is essential for nucleotide exchange. Previous
experiments with ARF constructs comprising N-terminal modifications
have suggested that the N terminus of ARF1 is essential for its GTPase
activity, and might therefore interact with ARF GTPase-activating
protein (Randazzo et al., 1994). ARP
21, however, seems to
have significant GTPase activity, since the native recombinant protein
was isolated in its GDP-bound form. It should be noted that truncated
ARP lacks a basic residue (Lys-15), which is conserved in all members
of the ARF family and corresponds with Lys-15 in ARF1. Furthermore,
almost all other previously described Ras-homologous GTPases harbor a
basic residue corresponding with lysine 5 in Ras in their N terminus.
Thus, it is tempting to speculate that it is the lack of a basic
residue preceding the PM1 motif that causes the marked reduction in the
nucleotide exchange rate in GST-ARP
21.
The subcellular distribution of ARP in 3T3-L1 cells is remarkable in that it was predominantly associated with the plasma membrane fraction of 3T3-L1 cells. This subcellular distribution resembles that of Ha-Ras, which we used as a marker for the plasma membrane fraction. It thereby differs strikingly from that of ARF, which has been found in the cytosol and in Golgi-derived vesicles of other cells (Randazzo et al., 1994; Serafini et al., 1991). Thus, since some structural elements, the targeting, and the membrane anchoring of ARP are fundamentally different from that of ARF isoforms, ARP appears to exert a unique, yet unknown, function. From its subcellular distribution we derive the speculative working hypothesis that it is involved in plasma membrane-related signaling events.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X78603 [GenBank]and X91504[GenBank].