From the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322-3050
Received for publication, March 15, 2001, and in revised form, April 6, 2001
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ABSTRACT |
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Despite the 40-60% identity between
ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins, distinct
functional roles have been inferred from findings that ARLs lack the
biochemical or genetic activities characteristic of ARFs. The potential
for functional overlap between ARFs and ARLs was examined by comparing effects of expression on intact cells and the ability to bind effectors. Expression of [Q71L]ARL1 in mammalian cells led to altered
Golgi structure similar to, but less dramatic than, that reported
previously for [Q71L]ARF1 (1). Two previously identified partners of
ARFs, MKLP1 and Arfaptin2/POR1, also bind ARL1 but not ARL2 or ARL3.
Two-hybrid screens of human cDNA libraries with dominant active
mutants of human ARL1, ARL2, and ARL3 identified eight different but
overlapping sets of binding partners. Specific interactions between
ARL1 and two binding proteins, SCOCO and Golgin-245, are defined and
characterized in more detail. Like ARFs and ARL1, the binding of SCOCO
to Golgi membranes is rapidly reversed by brefeldin A, suggesting the
presence of a brefeldin A-sensitive ARL1 exchange factor. These data
reveal a complex network of interactions between GTPases in the ARF
family and their effectors and reveal a potential for cross-talk not
demonstrated previously.
ADP-ribosylation factors
(ARFs)1 are highly conserved,
ubiquitous, 21-kDa GTP-binding proteins with roles in multiple steps of
membrane traffic and other cellular processes (for review, see Refs.
2-6). Within the ARF family are three subgroups that have been defined
by sequence and functional relatedness (7). The ARFs share >60%
sequence identity and share activities as cofactors in the
ADP-ribosylation of G More than 10 ARLs have been identified in humans, and three in S. cerevisiae (15, 18-21). Although ARFs have been purified repeatedly by laboratories using different biochemical assays, no ARL
has ever been purified or cloned based on an activity. The lack of ARL
activity in ARF assays has led to the conclusion that ARLs have
distinct biochemical activities and thus cellular functions, despite
their similarity in sequence and structure (15).2 The lack of functional
overlap between ARFs and ARLs is evident from the findings that ARLs in
yeast cannot rescue the synthetic lethality of the
arf1 In contrast to the many activities and functions assigned to ARFs, none
has yet been assigned to any ARL from any species. The number of
binding partners for mammalian ARF proteins has increased dramatically
in recent years in large part because of the use of two-hybrid screens
that have identified seven new ARF effectors: Arfaptin1 (25),
Arfaptin2/POR1 (independently isolated by two groups (25, 26) and named
Arfaptin 2 and POR1, respectively), MKLP1 (27), Arfophilin (28), and
the three GGAs (29-31). The use of carboxyl-terminal fusion proteins
and dominant activated ARF mutants has enhanced the ability to select
for binding partners that interact preferentially with the activated
form of the GTPase (27, 29, 32). Two proteins, "binder of ARL2"
(BART (33)) and the Role(s) for ARF in Golgi functions were evident when expression of the
persistently activated mutant allele [Q71L]ARF1 in cells led to
expansion and vesiculation of the Golgi compartment (1). Because ARL1
was reported also to bind Golgi membranes in intact cells, we used the
same procedures to test for a functional role of ARL1 at the Golgi. We
were surprised by the similarities between effects of activated ARF1
and ARL1 on Golgi morphology, and this led us to reexamine the extent
of functional overlap between ARF and ARL proteins.
Data Collection--
Every experiment reported herein was
repeated at least twice and in most cases additional times with
essentially the same results.
Cell Culture--
Growth media were prepared, and maintenance of
yeast strains was performed as described in Sherman et al.
(35). Transformation of yeast was performed using the method of
Schiestl and Gietz (36). Plasmids were rescued from yeast as described
(37) followed by further purification on Qiagen minipreparation
columns. DNA was transformed into E. coli strain DH5 Yeast Two-hybrid Screens--
Yeast strains Y190 (MATa
gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3, -112 URA3::GAL
Screening of human cDNA libraries was performed as described
previously (38) with the modifications described in Boman et al. (27, 29). Briefly, the dominant activating mutations in each
human ARL protein were used as bait to screen human fetal brain or B
cell libraries in Y190 by selection for growth on selective plates
containing 25 mM 3-aminotriazole (3-AT). Positives were then assayed for Purification of Recombinant Proteins--
The open reading
frames of human ARL1 and [ GTP GAP Assay--
ARL1 GAP activity was assayed by a modification
of the method of Randazzo and Kahn (41) using 0.18 µM
purified, recombinant [ Gel Overlay Assay--
Direct interaction between human ARL1, 2, or 3 and BART, PDE Affinity Chromatography--
10 µM purified
recombinant [ Immunohistochemistry--
NRK cells were fixed in 3.7%
formaldehyde and permeabilized in 0.2% saponin with 10% goat serum,
as described in Zhang et al. (1). Cells were visualized either on an
Olympus BX60 fluorescent microscope or a Bio-Rad 1024 laser scanning
confocal microscope coupled to a Zeiss Axioskop. For confocal
microscopy, a Z-series of images was collected with 5-µm steps and
processed using IMAGE J software from NIH IMAGE.
SCOCO Antibody Generation and Affinity
Purification--
Recombinant SCOCO-(His)6 was purified as
described above and used as antigen in rabbits, after conjugation to
keyhole limpet hemocyanin through both the NH2 terminus
(using glutaraldehyde) and the COOH terminus (using carbodiimide).
Antibodies were affinity purified by sequential protein G-Sepharose and
affinity chromatography. 6.5 mg of untagged, recombinant SCOCO was
covalently attached to 1-ml Affi-Gel 15 beads, according to the
manufacturer's directions. Serum from rabbit R97679 was enriched for
immunoglobulins on a 1-ml protein G column (Amersham Pharmacia Biotech)
and eluted with 0.1 M glycine HCl, pH, 2.7. The eluted
antibodies were exchanged into 0.1 M MOPS, pH 7.2, and
applied to the Affi-Gel 15-SCOCO column. The column was washed with 10 ml of phosphate-buffered saline containing 1 M NaCl.
Antibodies were eluted with 10 ml of 0.1 M glycine-HCl, pH
2.4, collecting 1-ml fractions into 100 µl of 1 M
Tris-HCl, pH 9, to neutralize the glycine buffer. Fractions containing
protein were pooled, and the buffer was exchanged for phosphate-buffered saline.
Interferon-inducible ARL1 Cell Lines--
COOH-terminal,
myc-epitope tagged constructs of ARL1 or [Q71L]ARL1 were subcloned
into pSS2-2 for expression in mammalian cells under regulation by the
interferon-inducible Mx1 promoter, as described in Zhang et
al. (Table I and Ref. 1). Each of these or the parental vector was
cotransfected with pSV2-neo (ATCC), at a 10:1 ratio of DNA, into NRK
cells using Fugene 6 (Invitrogen) reagent, following the
manufacturer's directions. 48 h after transfection, the cells
were split and diluted into medium containing 400 µg/ml Geneticin
disulfate (G418; Sigma). G418-resistant clones were isolated and later
cloned by limited dilution. Cell lines carrying stably integrated
plasmids directing expression of ARL1-myc or [Q71L]ARL1-myc with no
ARL1 5'-untranslated sequence were NRK-HV1-9 and NRK-HV2-20,
respectively. Protein expression was induced with 1,000 units/ml
We redesigned the pSS2-2 constructs to include 50 bp of ARL1 5'-UTR, to
decrease transcriptional initiation from internal methionines. We
also incorporated the gene conferring resistance to neomycin into the
pSS2-2 vector to facilitate selections and included the hemagglutinin
(HA) epitope at the COOH terminus of the ARL1 (Table I). Stably
transfected cell lines were obtained and cloned as described above.
Cell lines carrying the stably integrated plasmids directing expression
of ARL1-HA or [Q71L]ARL1-HA, including ARL1 Antibody Generation--
Recombinant human
ARL1-(His)6 was purified as described above and used as
antigen in rabbits. The antibodies were characterized, and the lower
limit of sensitivity to purified recombinant antigen was determined to
be 5 ng. The antibodies were specific to human ARL1, as they did not
recognize 100 ng of purified recombinant human ARF1-6, ARL2, or ARL3.
Western blot analysis (50 µg of total protein) using these antibodies
did not detect endogenous ARL1, although a variety of rat tissues (rat
and human ARL1 are 99% identical) were surveyed, including brain,
heart, skeletal muscle, liver, lung, and kidney. Western blots using
these antibodies also failed to detect endogenous ARL1 in NRK (rat) or
SF295 (human) cells (50 µg of total protein loaded), suggesting that
the amount of endogenous ARL1 present in these tissues and cell lines
is < 0.01% of total cellular protein or at least 10-fold less
than ARF1.
Northern Blot Analysis--
Northern blotting was performed with
the use of the Multiple Human Tissue I Blot
(CLONTECH), according to manufacturer's
directions. The SCOCO open reading frame was used as the probe (~250
bp in length) and was labeled by incorporating
[ Electron Microscopy--
After fixation in 1% glutaraldehyde,
samples were poststained with 1% osmium tetroxide, dehydrated through
a graded series of ethanol, and subsequently embedded in Embed
812 (Electron Microscopy Sciences). Thin sections were stained
with uranyl acetate and lead citrate. Electron microscopy was
performed on a Philips CM-10 transmission electron microscope at 60 kV.
Guanine Nucleotide Exchange Factor (GEF) Assays--
Binding of
GTP Expression of [Q71L]ARL1-altered Golgi Structures--
A role
for ARFs in Golgi function was supported by the finding that the
expression of dominant active [Q71L]ARF1 in NRK cells led to
expansion and vesiculation of the Golgi compartment (1). Because ARL1,
like ARF, localizes to Golgi membranes (21), we used the same
procedures to test for a functional role of ARL1 at the Golgi. To
enable specific detection of ARL1 in cells, we engineered an epitope
(myc) tag at the COOH terminus of both the wild type and [Q71L]ARL1
proteins. Stable NRK cell lines were obtained which were capable of
expressing ARL1-myc or [Q71L]ARL1-myc upon induction with interferon,
as described under "Materials and Methods." Localization of each
protein to the Golgi was confirmed by indirect immunofluorescence using
the monoclonal myc antibody (data not shown). However, the Golgi
staining in cells expressing [Q71L]ARL1-myc appeared more diffuse
than that seen with the wild type protein. The change in Golgi staining
induced by [Q71L]ARL1 was subtle and was not accompanied by the
progressive development of large perinuclear vesicles which often
surrounded the nucleus, as seen previously for [Q71L]ARF1 (1). When
viewed by electron microscopy, it was evident that cells expressing
[Q71L]ARL1 contained a Golgi with an engorged lumen (Fig.
1), similar to early time points in the
expression of [Q71L]ARF1 and particularly at the ends of stacks. In
contrast, the Golgi in cells expressing comparable levels of wild type
ARL1 were indistinguishable from those seen in either uninduced or
induced control cells. No other differences were seen in these cell
lines. Golgi were clearly identified by viewing serial sections and
seeing the complete expanded phenotype (shown in Fig. 1) as well as
partial expansion in which normal Golgi stacks were contiguous with
expanded ones.
The level of expression of [Q71L]ARL1 in these lines was similar to
that of [Q71L]ARF1 in the analogous lines. It should be noted that in
NRK cells endogenous ARF1 is expressed to A Subset of ARF Effectors Also Binds to ARL1--
We next asked
whether a set of known ARF-binding proteins could also bind to any of
three ARLs. Toward this end, we applied the yeast two-hybrid system
(38) to test binding of LTA, Arfaptin2/POR1, GGA1, 2, and 3, and MKLP1
to each of three human ARL proteins, ARL1, ARL2, and ARL3. The results
are summarized in Table II. Two proteins,
Arfaptin2/POR1 and MKLP1, each interacted as well with [Q71L]ARL1 as
with any ARF protein (similar activities with activated human ARF1,
ARF3, ARF4, ARF5, or ARF6). Binding to ARL1 was predicted to be
GTP-dependent because neither Arfaptin2/POR1 nor MKLP1
bound to wild type ARL1. None of the GGAs or LTA bound ARL1 or
[Q71L]ARL1, although each interacted with all five activated ARFs.
The wild type and activated mutants of ARL2 and ARL3 lacked binding
activity to any of the ARF effectors tested. Thus, ARL1 was the only
one of these three ARLs which bound to a subset of ARF binding partners
in two-hybrid assays.
ARL1 and Arfaptin2/POR1 Bind Directly--
Several ARF-binding
proteins have recently been shown to increase the stoichiometry of GTP
binding to ARFs (32). This property extends to Arfaptin2/POR1, which
can increase the equilibrium binding of GTP Identification and Characterization of ARL1-3 Binding
Partners--
We initiated a series of yeast two-hybrid based screens
to identify novel ARL-binding proteins. Human ARL1, ARL2, and ARL3 were
each engineered as wild type or dominant activated mutants, fused at
their carboxyl termini with the binding domain of GAL4. Human fetal
brain and B cell cDNA libraries were screened with the activated
mutant form of each human ARL, as described previously (27, 29) and
under "Materials and Methods." As predicted from the direct tests
described above, the [Q71L]ARL1 screens led to the cloning of
fragments of Arfaptin2/POR1 twice and MKLP1 once, and these cDNAs
were not pulled from either of the other ARL screens ([Q70L]ARL2 or
[Q71L]ARL3).
Screens of human cDNA libraries with the activating mutants of
ARL1-3 led to the identification of six binding partners that were not
identified by previous screens with activated ARF proteins, and each
failed to bind ARFs (wild type or activated mutants) when tested
individually in two-hybrid assays. These proteins include two
previously identified ARL-binding partners: the delta subunit of
rod-specific cGMP phosphodiesterase 6 (PDE PDE
The open reading frame of HRG4 is 720 bp in length, encoding a protein
of 240 residues with a predicted mass of 27 kDa. Sequence homology
between PDE BART Binds ARL2 and ARL3--
BART is a 19-kDa protein, shown
previously to bind ARL2·GTP with high affinity (Kd
Golgin-245 and RanBP2
Because ARL1, like ARFs, binds activating guanine nucleotides to only
low stoichiometry in vitro, we designed an additional screen, using the NH2-terminal truncation mutation,
[
Because rat ARL1 and murine Golgin-245 have been found independently to
localize to the Golgi (21, 49, 56), we wanted to confirm the
colocalization of the human orthologs in the same cell and test their
interdependence of Golgi membrane binding. Murine and human Golgin-245
are only 66% identical overall, although the GRIP domains are 97%
identical. We expressed an epitope (myc)-tagged GRIP domain of human
Golgin-245 in NRK cells that had previously been stably transfected
with plasmid-directing expression of ARL1-HA and determined the
location of each protein by indirect immunofluorescence. Extensive
overlap between the staining of ARL1 and the GRIP domain of Golgin-245
at the Golgi was apparent (Fig. 3).
One hallmark of ARF binding to Golgi membranes is its sensitivity to
brefeldin A, an inhibitor of ARF GEFs. We tested the sensitivity of
ARL1 to brefeldin A and found that, like ARFs, and as described
previously (21), the binding of ARL1 to the Golgi is lost rapidly upon
exposure to brefeldin A (see Fig. 8). This effect clearly precedes the
changes in Golgi morphology and integrity which result from long term
exposure to brefeldin A and the eventual fusion of Golgi and
endoplasmic reticulum elements (data not shown). Although ARL1
localization to the Golgi was disrupted by treatment with brefeldin A
within 3 min (see Fig. 8 and Ref. 21), the GRIP domain of Golgin-245
remained localized at the Golgi even after 5 min of brefeldin A
treatment (data not shown). Thus the GRIP domain can bind to the Golgi
independently of ARL1, and its association is insensitive to brefeldin A.
The ARL1-independent binding of the GRIP domain of human Golgin-245 to
Golgi membranes suggested that instead of being recruited to membranes
by the GTPase it may be acting as a docking site for activated ARL1.
Mutation of a tyrosine residue ([Y2032A]Golgin-245), conserved in all
GRIP domains, results in the loss of Golgi binding by this domain (56,
57). The relationship between binding of the GRIP domain to Golgi and
ARL1 was investigated further by testing for effects of the homologous
mutation in human Golgin-245 on the localization of the expressed
protein in mammalian cells and on the binding of [Q71L]ARL1 in
two-hybrid assays. The GRIP domain of human [Y2032A]Golgin-245
(residues 2025-2083) was expressed in NRK cells using the same method
described above for the nonmutated domain. Mutation of the single
tyrosine residue was sufficient to prevent the binding to Golgi
membranes because the mutant protein was seen located diffusely
throughout the cytosol (data not shown), consistent with previously
published observations made with the murine GRIP domain (56, 57). The
expression of [Y2032A]Golgin-245(2025-2083) in NRK cells had no
effect on the localization of ARL1-HA (data not shown). When tested in
two-hybrid assays, the mutated domain was also unable to bind
[Q71L]ARL1 (Table II). Thus, it appears that this tyrosine in the
GRIP domain is involved in the binding to both Golgi membranes and ARL1.
Like ARF proteins, ARL1 has no detectable intrinsic GTPase activity
(58) and is predicted to depend upon interaction with an ARL1 GAP for
hydrolysis of bound GTP. The GRIP domain of Golgin-245 was assayed and
found to lack any ARL1 GAP activity. The addition of a 10-fold molar
excess of the purified Golgin-245 GRIP domain did not alter the rate of
GTP hydrolysis by ARL1 when assayed under the same conditions in which
ARF GAP activity can be readily detected ("Materials and Methods"
and data not shown). This same assay has been used recently to detect
and purify an ARL2 GAP activity.3
SCOCO--
The full-length human homolog of SCOCO (mouse accession
number NP062682) was identified from the yeast two-hybrid B cell
library as a specific binder of [Q71L]ARL1. The entire human SCOCO
protein is 82 amino acids in length, 100% identical at the amino acid level to the mouse protein and predicted by the COILS (59) program to
fold predominantly into a coiled-coil structure (Fig.
4). Northern blot analyses of human tissues
with a cDNA probe from human SCOCO revealed a single
~2.2-kilobase message, present in all tissues tested, except
perhaps lung (Fig. 5). SCOCO mRNA was
most abundant in brain, heart, and skeletal muscle.
Homology searches of sequence data bases revealed only three proteins
with high homology to SCOCO, one in humans and two in yeast (S. cerevisiae). Human Golgin-95, yeast IMH1, and yeast VPS30 have
26%/51%, 22%/58%, and 30%/64% identity/homology over a stretch
of
Human SCOCO was expressed as a soluble protein in bacteria and purified
to >95% homogeneity, taking advantage of a hexahistidine sequence
added to the COOH terminus. Recombinant human SCOCO migrated in
SDS-polyacrylamide gels with the mobility expected of a 9-kDa protein,
but its mobility in nondenaturing gel filtration medium was faster than
predicted, more consistent with that of a protein of
Binding of guanine nucleotides to purified ARL1 was similar to ARFs,
with low stoichiometry that is partially relieved by added lipids or
detergents. To assist in the biochemical characterization of ARL1 and
its binding partners, we expressed an NH2-terminal truncation mutant of ARL1, [
The cellular location of SCOCO and its relationship to ARL1 were next
analyzed in cultured mammalian cells. Rabbit polyclonal antibodies were
raised against human SCOCO-(His)6 and affinity purified, as
described under "Materials and Methods." These antibodies were
sufficiently sensitive and specific (see "Materials and Methods") to allow detection of endogenous SCOCO in NRK cells. A punctate, perinuclear staining pattern, very similar to that seen for markers of
the Golgi, was observed (Fig. 7), along with
punctate staining in cytosol, and some staining at the plasma membrane.
Double labeling with SCOCO and either
Because the activating mutations of ARF and ARLs block the
actions of GAPs and lead to increased abundance of the GTP-bound proteins in cells, these mutations confer a limited degree of resistance to brefeldin A, as described in Zhang et al. (1) for
[Q71L]ARF1. The ability of [Q71L]ARL1 to provide such resistance to
the actions of brefeldin A on itself or SCOCO were examined in NRK
cells expressing comparable levels of ARL1 or the activated mutant. As
a control, comparisons were also made between these cells and those
expressing wild type or [Q71L]ARF1 (1). The binding of endogenous or
overexpressed ARF1 and its effector, COP-I, were clearly diminished
within 1 min of exposure to 10 µM brefeldin A and were
essentially absent by 3 min (Ref. 1 and data not shown). Expression of
[Q71L]ARF1 delayed this response such that it took 5 min or longer
for complete dissociation of the signal from Golgi membranes (1). We
observed a very similar level of resistance to brefeldin A provided to
SCOCO by expression of [Q71L]ARL1 (Fig. 8).
Both [Q71L]ARL1 and SCOCO were still present on Golgi membranes after
3 min of brefeldin A treatment (Fig. 8). The activating mutation only
delayed the release of the GTPase and binding partner from the Golgi,
it did not prevent it. By 5 min of exposure to the drug, both
[Q71L]ARL1 and SCOCO appeared fully dissociated from Golgi membranes
(Fig. 8). The expansion and vesiculation of the Golgi which resulted
from expression of [Q71L]ARF1 also resulted in a dilution of the
staining of SCOCO which makes comparisons technically more demanding.
However, we saw no indication of protection of SCOCO localization to
Golgi membranes when such cells were treated with brefeldin A (data not
shown).
ARF GEFs each contain a common structural motif, termed the SEC7
domain, which binds to both ARFs and brefeldin A (for review, see Ref.
65). We assayed for ARL1 GEF activity of the brefeldin A-sensitive SEC7
domain of the SEC7 protein, using ARL1 and ARF3 as substrates in the
absence or presence of the SEC7 domain. As seen in Fig.
9, the SEC7 domain increased the binding of
GTP Despite previous evidence of clear functional distinctions between
the actions of ARF and ARL proteins, our studies have revealed a number
of functional similarities between human ARL1 and ARF1-6. This
apparent overlap in functions with ARFs did not extend to either human
ARL2 or ARL3. Similarities included changes in Golgi morphology with
expression of the dominant activated mutant, brefeldin A-sensitive
localization to the Golgi, and a subset of shared binding partners. The
extent to which different ARF or ARL proteins shared common effectors
was investigated further, using two-hybrid technology, to reveal both
specificity and extensive overlap between binders of ARL1, ARL2, and
ARL3, but only ARL1 was found to share binding partners with ARFs.
Thus, the functional overlap between ARF and ARLs may be limited to
ARL1. Two-hybrid screens of human cDNA libraries identified five
novel and specific binding partners of ARL1, none of which bound ARFs.
The presence of GRIP domains at the COOH termini of two of these
reveals this domain to be an ARL1 binding structure. The distinct
specificities in binding proteins for ARFs and each ARL suggest that
signaling by members of the ARF family involves complex networks of
multiple overlapping effectors.
Expression of [Q71L]ARL1 led to an engorgement of the Golgi
apparatus, most evident at the ends of stacks. This expansion was
similar in appearance to that seen with [Q71L]ARF1 (1) but was less
dramatic. We saw no evidence of expansion of the endoplasmic reticulum
lumen. The effect on Golgi morphology was specific to the activating
mutant, [Q71L]ARL1, and was not observed when similar levels of wild
type ARL1 were expressed. The effects of [Q71L]ARF1 on Golgi and
endoplasmic reticulum morphologies were seen with less than 2-fold
overexpression of ARF1 (1). Although the amounts of [Q71L]ARL1
expressed in NRK cells were comparable to those of [Q71L]ARF1 the
fold increase was much more because endogenous levels of ARL1 are at
least 10-fold lower than those of ARF1. This relatively high level of
[Q71L]ARL1 expression was required for effects on Golgi morphology in
that the phenotype was lost at lower levels of expression, resulting
from the addition of Similarities between ARL1 and ARF extended to the ability
of the activated form of each protein to become partially resistant to
the actions of brefeldin A (Fig. 8 and Ref. 1). One of three likely
explanations may account for these observations: 1) there exists at
least one ARL1-specific GEF that is brefeldin A-sensitive; 2) one or
more of the brefeldin A-sensitive ARF GEFs can also bind and activate
ARL1; or 3) the binding of ARL1 to Golgi membranes is dependent on the
activation of one or more ARFs. The finding that at least one SEC7
domain, active as a brefeldin A-sensitive GEF for human ARFs, is
inactive when human ARL1 is the substrate, tempers support for
explanation 2 above (Fig. 9 and Ref. 66) but does not exclude it. The
similarities in the time course of responses of ARF1 and its effector
(COP-I) and ARL1 and its binding partner (SCOCO) to brefeldin A
similarly run counter to explanation 3 (Fig. 8 and Ref. 1). We
therefore favor the possibility that there exist one or more brefeldin
A-sensitive ARL1 GEF, although more definitive tests of these three
models are required. The identification of such an ARL1 GEF would
provide further evidence that at least some of the brefeldin
A-sensitive activities in cells, currently ascribed to ARFs, may
actually result from the actions of ARL1.
Ultimately, the functional description of the actions of a
regulatory GTPase will be determined by the molecules with which it
specifically interacts. Current descriptions of specific GTPases, e.g. Ras, can include as many as a dozen distinct downstream
binding partners (for review, see Ref. 68). The identity and
specificity of these regulated protein interactions will define the
signaling pathways that may be activated in response to the activation
of each GTPase. Toward this end we and others have identified more than
10 direct, GTP-dependent, ARF-binding partners (10, 11, 14,
25-31, 64, 69-71). Biochemical and genetic evidence indicate a lack
of functional overlap between ARF and ARL actions. It was surprising,
therefore, to discover that two-hybrid assays revealed ARL1 interacting
with two ARF-binding proteins, Arfaptin2/POR1 and MKLP1, in a specific
and GTP-dependent fashion. The presence of an extensive
coiled-coil domain in MKLP1 likely contributes to its inability to be
expressed as a recombinant protein, precluding biochemical analyses. We
focused instead on Arfaptin2/POR1 and have confirmed its direct binding
to ARL1, using the fact that GTP Screens of three different human cDNA libraries (B
cell, kidney, and fetal brain) led to the isolation of seven different cDNAs, encoding proteins predicted to bind ARL1. Two of these were
Arfaptin2/POR1 and MKLP1. Cross-reactivity between ARF and ARL1
partners prompted tests for specificity between these novel partners
with both ARFs and ARL1-3 (Fig. 10).
PDE
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S by bacterial toxins (cholera and
Escherichia coli heat-labile toxin) (8, 9), activators of
phospholipase D (10, 11), suppression of the lethal double mutation
arf1
arf2
in
the yeast Saccharomyces cerevisiae (12, 13), and activator of phosphatidylinositol 4-phosphate 5-kinase (14). The ARF-like (ARL) proteins are 40-60% identical to each other or to any ARF and
are essentially devoid of the activities described for ARFs (12, 15,
16). A third group of proteins, including yeast SAR1 and CIN4, is
included in the ARF family although the proteins share only 25-35%
sequence identity and have clearly distinct activities in cells (5,
17).
arf2
mutations (12), whereas any of the six mammalian ARFs (13, 23) or ARFs
from other organisms (e.g. Drosophila (24), or Giardia (23)) can suppress the lethality of
arf1
arf2
in
yeast. Similarly, deletion of the Drosophila arl1 gene
causes zygotic lethality despite the presence of the full complement of
ARF genes in flies (15).
subunit of cGMP phosphodiesterase 6 (PDE
(34)), have been cloned based on their GTP-dependent
interaction with either ARL2 or ARL3, respectively. To date, however,
no clear biological significance has been attributed to these interactions.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
prior to plasmid preparation. Normal rat kidney (NRK) cells were
obtained from American Type Culture Collection (ATCC; Rockville, MD)
and grown and passaged in RPMI 1640 medium (Life Technologies, Inc.)
containing 10% fetal bovine serum (Life Technologies, Inc.) at
37 °C in a humidified atmosphere containing 10%
CO2.
lacZ) and Y187 (MAT
gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3,-112 met-
URA3::GAL
lacZ), plasmids pAS2 and pACT2,
and the human B cell library in pACT were the gifts of Steven J. Elledge (Baylor University). This system utilizes the GAL4 binding and
activation domains and allows for two independent read-outs for a
positive interaction in the two-hybrid system: histidine auxotrophy and
-galactosidase expression (38). The human fetal brain cDNA
library, in pACT2, was purchased from
CLONTECH. Plasmid pBG4D was a gift
from Rob Brazas and allowed the expression of proteins fused through
their carboxyl termini to the GAL4 binding domain. The open reading
frame of each ARL was cloned in-frame into pBG4D via BamHI
and NotI sites. Mutations were generated using Stratagene's
QuikChange site-directed mutagenesis kit. All DNA generated by
polymerase chain reaction was sequenced both to confirm the presence of
designed mutations and prevent the introduction of others.
-galactosidase activity, using the nitrocellulose filter binding assay of Breeden and Lasmyth (39). Filters were incubated at 30 °C for up to 3 h. A strong interaction was
defined as the development of a dark blue color within 15 min; a weak interaction required the full 3 h for development of a pale blue color. Further tests for specificity of interactions included: 1)
counterselection for the loss of the ARL plasmid on cycloheximide plates resulted in loss of activity in the
-galactosidase assay; 2)
cells carrying each potential positive were mated to unrelated partners
(we used CDK2, lamin, and p53, each fused to the GAL4 activation
domain) and assayed for
-galactosidase activity; and 3) rescued
library plasmids yielded growth on 3-AT plates and
-galactosidase
activity after transformation into the original ARL-bearing yeast
strain. In most cases, positives were counterscreened against the wild
type protein, to help identify those proteins that interacted
preferentially with the GTP-bound form of the GTPase. Table
I lists each of the plasmids used in the
studies described below.
Plasmids used in this study
17]ARL1 were subcloned into pET20b
(Novagen) at the NdeI and NotI sites (Table I).
The open reading frames of SCOCO and the GRIP domain of Golgin-245 (amino acids 2025-2083) were cloned into pET20b (via NdeI
and NotI restriction sites) and pET14b (via NdeI
and BamHI restriction sites), respectively (Table I). The
expression of (His)6-tagged protein was induced for 3 h in mid-log phase BL21(DE3) cells with 1 mM isopropyl
-D-thiogalactopyranoside at 37 °C. Cells were lysed with a French pressure cell, and the recombinant protein was
purified using a 1-ml HiTrap chelating (Amersham Pharmacia Biotech)
column charged with nickel chloride and developed with a 0-500
mM imidazole gradient according to the manufacturer's instructions. Fractions that contained (His)6-tagged
protein, as determined by SDS-polyacrylamide gel
electrophoresis, were pooled, and the buffer was exchanged into 25 mM Tris, pH 7.4, 100 mM NaCl, 2 mM
MgCl2, and 1 mM dithiothreitol. Human ARL2, ARL3, and ARF1 cDNA were all cloned into pET3C via NdeI
and BamHI restriction sites (Table I). They were each
expressed and purified as described previously (18, 33). POR1 fused to
the maltose-binding protein (a gift from Linda Van Aelst) was purified
as described (26).
S Binding Assay--
The GTP
S binding assay was
performed at 30 °C in 20 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM dithiothreitol, 1 mM
EDTA, 0.5 mM MgCl2, 50 µg/ml BSA, and 10 µM [
-35S]GTP
S (1,000 cpm/pmol),
as described previously (40).
17]ARL1-(His)6 as the substrate
with or without the addition of 1.8 µM GRIP domain of
Golgin-245 or SCOCO. Purified, recombinant human ARF3 and ARF GAP (42)
served as positive controls for the assay.
, or HRG4 was assayed by gel overlay, as described
in Sharer and Kahn (33). Briefly, SDS-solubilized protein lysates were prepared from BL21(DE3) cells containing either the PDE
open reading
frame in pET15b (the gift of Ahmed Zahraoui, Compartimentation et
Dynamique Cellulaires, Institut Curie, Paris, France), HRG4 open
reading frame in pET14b at NdeI and BamHI
restriction sites, or an empty vector control (Table I). 25 µg of
total protein was resolved on a 15% polyacrylamide gel before
electrophoretic transfer to 0.2-µm nitrocellulose membrane (Bio-Rad).
Proteins adsorbed on the filter were renatured in 10 mM
MOPS, pH 7.1, 100 mM potassium acetate, 0.25% Tween 20, 5 mM magnesium acetate, 0.5% BSA, 5 mM
dithiothreitol, and incubated with 2 µg of recombinant [
17]ARL1-(His)6, ARL2, or ARL3 prebound to 20 µCi of
[
-32P]GTP. The filter was washed three times with
binding buffer (20 mM MOPS, pH 7.1, 100 mM
potassium acetate, 0.1% Triton X-100, 5 mM magnesium
acetate, 0.5% BSA, 50 µM GTP, 50 µM GDP,
and 5 mM dithiothreitol), and specific binding was
determined by phosphorimage analysis.
17]ARL1-(His)6 was loaded with either 100 µM GDP or GTP
S for 15 min at 30 °C in the presence of 20 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 0.5 mM MgCl2, and 50 µg/ml BSA. SCOCO was
covalently attached to Affi-Gel 15 beads according to the
manufacturer's directions (Bio-Rad). SCOCO beads were washed twice in
binding buffer containing 10 µM appropriate nucleotide,
just prior to the addition of preloaded [
17]ARL1-(His)6. SCOCO beads and [
17]ARL1-GDP or
-GTP
S were incubated with gentle rocking for 15 min at room
temperature. The beads were washed twice with binding buffer,
containing 20 µM appropriate nucleotide and then mixed
with an equivalent amount of 2 × Laemmli sample buffer. Proteins
were resolved on a 15% polyacrylamide gel, and the presence of
[
17]ARL1-(His)6 was detected using polyclonal rabbit
antiserum raised against ARL1 (R85722-3).
,
-rat interferon (Lee Biomolecular, San Diego) and assayed by
immunoblot analysis using monoclonal 9E10 (mouse
-myc) antibodies.
When lysates were probed with myc antibodies in immunoblots, we
consistently observed the interferon-dependent expression
of a doublet in which the upper band corresponded to the predicted size
of full-length ARL1 (21 kDa) and a second band migrating as a smaller
fragment (
17 kDa). Human ARL1 is myristoylated at its
NH2 terminus (43), so we tested for the incorporation of
[3H]myristic acid into ARL1 by fluorography (44). Only
the upper band was labeled under these conditions. We believe it likely that the smaller protein was produced by initiation from an internal start codon, Met-18, but we cannot exclude the possibility that it was
the result of a proteolytic event that produced a fairly uniform size
product. However, the fact that it lacks myristate means it is unlikely
to be biologically active, as the nonmyristoylated [G2A]ARF1 and
[
17]ARF1 truncation mutants are null alleles in yeast (45,
46).
50 bp of the
5'-untranslated sequence of human ARL1, were NRK-HV820-9 and
NRK-HV810-13, respectively. Cells were induced with 1,000 units/ml
,
-rat interferon and assayed for ARL1 expression in immunoblots,
using the 12CA5 antibody (mouse
-HA). Interferon induction now led
to the production of only the upper, 21-kDa, band which also was found
to incorporate [3H]myristic acid. Thus, these cells
express full-length ARL1-HA that is properly myristoylated, but in
every case the level of expression of this species was 3-5-fold lower
than that expressed in stable cell lines lacking the 5'-UTR of ARL1.
These lines, expressing [Q71L]ARL1, no longer displayed the enlarged
Golgi phenotype.
-32P]dCTP (NEN Life Science Products) using the
random priming kit from Life Technologies, Inc. Hybridizing bands were
detected by exposure to Phosphorscreens.
S to human ARL1-(His)6 was determined as described in
Kahn and Gilman (8). Briefly, GTPases (1 µM) were incubated at 30 °C with or without the SEC7 domain (10 µM) in 20 mM HEPES, pH 7.4, 1 mM
EDTA, 1 mM dithiothreitol, 100 mM NaCl, 4.5 mM MgCl2, 2.5 mM azolectin
vesicles, 100 µg/ml BSA, and 10 µM
[35S]GTP
S (2,500 cpm/pmol). Duplicates of 10-µl
samples were taken and diluted into 2 ml of ice-cold TNMD buffer (25 mM Tris-Cl, pH 7.4, 100 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol),
followed by rapid filtration onto 25-mm BA85 nitrocellulose filters
(Schleicher & Schuell).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of [Q71L]ARL1 causes engorgement
of Golgi in NRK cells. NRK cells, stably transfected with ARL1-myc
(NRK-HV1-9; left panel) or [Q71L]ARL1-myc (NRK-HV2-20;
right panel), under control of the interferon-inducible Mx1
promoter, were fixed and prepared for electron microscopy after 16 h of induction, as described under "Materials and Methods."
NRK-HV1-9 cells were indistinguishable from untransfected NRK or empty
vector transfected NRK cells. Golgi elements are indicated by
arrows.
0.1% of total cell
protein, but we cannot detect endogenous ARL1 (estimated level of
expression <0.01% total cell protein). Thus, the magnitude of the
changes in Golgi structure was considerably less in cells expressing
[Q71L]ARL1 than in cells expressing [Q71L]ARF1 even though the fold
increase in expression was greater. The similarities in phenotype
between cells expressing [Q71L]ARF1 and [Q71L]ARL1 were suggestive
of similarities in function of these proteins at the Golgi.
Interactions between GTPases in the ARF family and binding partners
-Galactosidase activities were determined using the filter lift
assay in yeast carrying the designated pairs of proteins, as described
under "Materials and Methods." Activities when coexpressed with the
wild type and activating mutants of each GTPase are shown. ARF1-6 are
shown collectively because no differences were seen when human ARF1,
ARF3, ARF4, ARF5, and ARF6 were assayed independently. Only the GRIP
domain of Golgin-245 (residues 2025-2083) was included for those
proteins, one of which included the point mutation Y2032A. Interactions
were scored by eye as the time and intensity of blue color development
at 30 °C. Activities were defined as a strong (+++; dark blue within
15 min), intermediate (++; blue within 30 min), or weak (+; pale blue
within 3 hs). Lack of any detectable blue color development within
3 h is shown as no interaction (
). Negative controls (see
"Materials and Methods") were included in every assay but are not
shown. Each result was obtained multiple (
3) times. ND, not
determined.
S to ARF3 by 3-8 fold
(Fig. 2 and Ref. 32). Similar effects were
found for Arfaptin2/POR1 on the binding of GTP
S to purified ARL1
(Fig. 2). The addition of Arfaptin2/POR1 led to a > 4-fold
increase in the amount of GTP
S bound to ARL1 at steady state.
Binding reached a maximum of 0.42 mol of GTP
S bound/mol of ARL1,
very similar to the amount of GTP
S bound to ARF1 under the same
conditions (Fig. 2). In the absence of Arfaptin2/POR1, these
preparations of ARL1 and ARF1 bound 0.08 and 0.09 mol of GTP
S/mol of
GTPase, respectively. This increase in GTP binding induced by
Arfaptin2/POR1 was specific because neither the BSA present in the
assay nor other proteins tested (e.g. GGA1; data not shown)
had any effect on nucleotide binding to ARL1. Thus, ARFs and ARL1 share
the ability to bind directly and specifically to Arfaptin2/POR1.
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Fig. 2.
Arfaptin2/POR1 binds to ARL1 and ARF1 and
increases the binding of GTP S. The
binding of 10 µM [
-35S]GTP to 1 µM purified recombinant ARL1-(His)6
(squares) or 1 µM ARF1 (circles)
was determined at 30 °C in the presence (closed symbols)
or absence (open symbols) of 3.6 µM MBP-POR1, as
described under "Materials and Methods." Each time point contains
10 pmol of ARL1-(His)6 or ARF1.
; accession number
AF045999 (47)) and "binder of ARL2" (BART; accession number
AF126062 (33)), as well as four newly identified ARL-binding proteins:
human retinal gene 4 (HRG4; accession number U40998 (48)), Golgin-245
(accession number U31906 (49)), RanBP2
(50), and a short
coiled-coil protein, termed SCOCO (accession number AF330205). These six human proteins bound the three activated
ARLs with different specificities (Table II).
and HRG4 Bind ARLs 1, 2, and 3--
A direct and
GTP-dependent binding of ARL2 and ARL3 to PDE
has been
reported previously (34). Our results extend the likely set of binders
to include ARL1 (Table II). The complete open reading frame of PDE
was cloned once from the human fetal brain library using [Q71L]ARL1
as bait and 21 times using [Q70L]ARL2. Direct tests for specificity
also revealed an interaction between [Q71L]ARL3 and PDE
(data not
shown). The open reading frame of PDE
is 450 bp and encodes a
predicted 17-kDa protein. We expressed the full-length protein but
found it was insoluble in bacterial extracts under a variety of growth
and extraction conditions. When this protein was resolved by
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes it was able to refold at least partially into
a structure that bound ARL2·[
-32P]GTP or
ARL3·[
-32P]GTP in the gel overlay assay (data not
shown). Weak signals, compared with BART in the same assays, suggest
that refolding was inefficient. No binding of
ARL1·[
-32P]GTP was observed, but the stoichiometry
of GTP binding to ARL1 is at least 4-fold below that of the other ARLs.
Thus, although we were able to confirm the binding of ARL2 and ARL3 to
PDE
, we cannot yet confirm that ARL1 also binds. Although originally described as a component of a tetrameric phosphodiesterase complex with
high expression in retinal cells (51), PDE
is actually expressed in
many other tissues (including brain and adrenal gland (52)) and is not
only found associated with the phosphodiesterase complex, implicating a
more general role for the protein in cells and tissues.
and HRG4 is evident; they are 30% identical (40%
homologous) in their COOH-terminal regions (residues 159-240) and 23%
identical overall (53). Like PDE
, HRG4 is expressed in photoreceptor
cells (54), but in contrast to PDE
, little or no HRG4 message has
been detected in other tissues tested (54). Plasmids encoding the
portion of HRG4 from residue 96-240 (HRG4(96-240)) were isolated
seven times from the human B cell library screen with [Q70L]ARL2. The
presence of the HRG4 message in the B cell cDNA library is evidence
that expression is not limited solely to photoreceptor cells.
HRG4(96-240) was later tested directly and shown to interact with both
[Q71L]ARL1 and [Q71L]ARL3 but not ARFs in two-hybrid assays (Table
II). HRG4 (residues 96-240) bound indistinguishably to both wild type
and activated mutant forms of each ARL in two-hybrid assays (Table II)
but failed to bind to any of the ARF constructs.
15 nM (33)). Library screens led to the isolation of
cDNAs encoding BART a total of 33 times using [Q70]ARL2 as bait
and once using [Q71L]ARL3. Neither [Q71L]ARL1, nor any ARFs, bound
BART when tested directly in two-hybrid assays. The gel overlay assay
was used to confirm the direct and GTP-dependent binding
between BART and ARL3 (data not shown).
--
Fragments of Golgin-245 were
isolated 35 times from library screens using [Q71L]ARL1 as bait and
once using [Q71L]ARL3. Golgins are Golgi-associated antigens in a
number of autoimmune diseases. Golgin-245 is a 245-kDa protein that is
predicted to contain an extensive coiled-coil domain (residues 1-2010)
and is peripherally associated with the cis-Golgi (49, 55).
Alignment of Golgin-245 with another golgin, Golgin-97, revealed a
conserved domain, termed GRIP. GRIP domains are found at the COOH
terminus of at least five proteins (Golgin-245, Golgin-97, RanBP2
,
and two uncharacterized open reading frames) and are capable of
directing the association of attached proteins to the Golgi apparatus
(56, 57). All library inserts included the entire COOH-terminal GRIP
domains, and some included very little else. Thus, the GRIP domain of
Golgin-245, which includes the last 60 residues, binds [Q71L]ARL1 and
[Q71L]ARL3 in two-hybrid assays (Table II). Stronger signals were
observed when fragments of Golgin-245 were paired with the activated
mutants of ARL1 or ARL3, compared with either wild type protein, but
preference for the activated proteins was less marked than those seen
with other partners (Table II).
17]ARL1 (46), coupled with the activating mutation, in efforts to
maximize the amount of activated ARL1 in cells. A screen of the human
fetal brain library with [
17,Q71L]ARL1 resulted in the cloning of
each of the binding partners found with [Q71L]ARL1, but in addition we cloned a COOH-terminal fragment of RanBP2
, containing the entire
GRIP domain. The GRIP domain of RanBP2
gave a strong signal when
paired with [Q71L]ARL1 or [
17,Q71L]ARL1 but did not interact with ARL1. Thus, at least two of the five GRIP-containing proteins bind
to ARL1, though with apparent differences in GTP-dependence when tested
as the isolate GRIP domains.
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Fig. 3.
Human ARL1 and the GRIP domain of
human Golgin-245 colocalize in mammalian cells. NRK-HV820-9 cells
were transiently transfected with plasmids containing the COOH-terminal
GRIP domain of Golgin-245 controlled by the constitutive
cytomegalovirus promoter. 16 h after cells were transfected and
protein expression was induced with interferon, the cells were fixed
and prepared for indirect immunofluorescence, as described under
"Materials and Methods." Mouse monoclonal antibody 9E10 ( -myc)
and rabbit polyclonal antibody SC-805 (
-HA) were used to visualize
the expressed ARL1-HA (left panel) or Golgin-245 GRIP domain
(center panel). The merged images are shown on the
right. The extensive overlap in staining is evident in all
cells expressing both proteins.
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Fig. 4.
Human SCOCO is predicted to form an extended
coiled-coil structure. Panel A, the protein and
cDNA sequences of human SCOCO are shown, using one-letter
abbreviations. Panel B, the probability for coiled-coils
structures, predicted by the COILS program (59), is shown.
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Fig. 5.
SCOCO is widely expressed in human
tissues. Poly(A)+-enriched RNA (2 µg/lane) was
isolated from eight different human tissues, resolved by agarose-gel
electrophoresis, and transferred to nylon membranes. A human SCOCO
cDNA probe was labeled by random priming and hybridized, as
described under "Materials and Methods." A single hybridizing band
was seen in all tissues surveyed except lung. The mobility of size
standards, in kilobases, is shown on the left.
52 residues in the middle of each protein. Interestingly, all three have predicted roles in vesicle traffic at the Golgi; one is
another Golgin (Golgin-95), and one also contains a GRIP domain at the
COOH terminus (IMH1) (56, 57, 60-63).
54 kDa (data
not shown). This observation is consistent with the formation of a
hexameric complex in solution, but it could also be due to the presence
of an extended coiled-coil with one longer axis.
17]ARL1-(His)6, comparable
to the truncation of ARF1 described previously (46).
[
17]ARL1-(His)6 bound guanine nucleotides to higher
stoichiometry than the full-length protein and did so independently of
added lipids or detergents (data not shown). The nucleotide binding
site of [
17]ARL1-(His)6 was filled with either GDP or
GTP
S by prior incubation in saturating concentrations of the
nucleotide and then incubated with SCOCO or BART (serving as a negative
control) that had been covalently attached to Affi-Gel beads, as
described under "Materials and Methods." The beads were collected
by centrifugation, and the amount of ARL1 retained on the beads after
washing was determined by immunoblot analysis. [
17]ARL1·GDP and
[
17]ARL1·GTP
S were specifically retained on the SCOCO
affinity column, with approximately twice as much [
17]ARL1 bound
to the column in the presence of the activating nucleotide, GTP
S, as
in its absence (Fig. 6). No binding to BART
was detected under these same conditions (data not shown). Preferential
binding to the activated conformation of a regulatory GTPase has been
observed previously with effectors and GAPs (25, 26, 29, 64). Purified
SCOCO had no detectable ARL1 GAP activity, under the same conditions
used for ARF GAPs (see "Materials and Methods").
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Fig. 6.
[ 17]ARL1-(His)6 binds
directly to SCOCO in a GTP-dependent manner. Purified
recombinant human SCOCO was covalently attached to Affi-Gel 15 beads,
as described under "Materials and Methods." These beads were then
incubated with [
17]ARL1-(His)6 that had been
equilibrated previously with either GDP or GTP
S for 15 min at room
temperature. Beads were washed twice with binding buffer containing 20 µM appropriate nucleotide, and proteins were then eluted
by boiling in an equivalent amount of 2 × Laemmli sample buffer.
Proteins were resolved on a 15% polyacrylamide gel, and the presence
of [
17]ARL1-(His)6 was detected using polyclonal ARL1
antiserum (R85722-3). Equivalent volumes of
[
17]ARL1-(His)6 that was loaded onto the Affi-Gel
15-SCOCO beads (L), the supernatant from the second wash
(W2), and the eluted protein from the beads (E)
are shown.
-COP or ARF antibodies
revealed extensive overlap in staining at the Golgi membranes (data not
shown). Because our ARL1-specific antisera are not sufficiently
sensitive to detect endogenous ARL1 by indirect immunofluorescence, we
used stably transfected NRK cells expressing inducible human ARL1-HA to
assess the extent of colocalization of ARL1 and SCOCO. Confocal
microscopy of the induced cells revealed extensive overlap in staining
of ARL1-HA and SCOCO (Fig. 7) at the Golgi.
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Fig. 7.
ARL1-HA and endogenous SCOCO
colocalize in mammalian cells. NRK-HV820-9 cells were treated
with interferon to induce expression of ARL1-HA for 16 h prior to
fixing and processing for indirect immunofluorescence using confocal
microscopy. Affinity-purified rabbit SCOCO (left panel) and
monoclonal 12CA5 ( -HA; center panel) antibodies were used
to label the SCOCO and ARL1, respectively. Each panel represents a
flattened stack of 18 images taken in 5-µm steps. The SCOCO and
ARL1-HA images were merged using Image-Pro Plus software, and the
overlap appears as yellow in the panel on the
right.
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Fig. 8.
Binding of SCOCO to Golgi is made
partially resistant to brefeldin A by expression of [Q71L]ARL1.
NRK-HV820-9 (ARL1-HA) and NRK-HV810-13 ([Q71L]ARL1-HA) cells were
treated with 10 µM brefeldin A for 0, 1, 3, or 5 min.
Cells were then fixed in 3.7% formaldehyde and prepared for indirect
immunofluorescence, as described under "Materials and Methods."
Cells were labeled with monoclonal HA and affinity-purified SCOCO
antibodies and visualized using confocal microscopy. This figure
represents a flattened stack of 24 images taken in 5-µm steps. The
retention of perinuclear staining of ARL1 and SCOCO is evident at 3 min
after brefeldin A addition, only in NRK-HV810-13 cells.
S to ARF3 in a linear fashion over time but had no effect on the binding of GTP
S to ARL1 (also see Ref. 66).
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Fig. 9.
A SEC7 domain does not act as a GEF for
ARL1. Specific binding of [35S]GTP S to 1 µM purified recombinant ARL1-(His)6
(triangles) or 1 µM ARF3 (circles)
in the presence (filled symbols) or absence (open
symbols) of the SEC7 domain of SEC7 (10 µM) was
determined for 10-µl aliquots after varying times of incubation at
30 °C, as described under "Materials and Methods."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
50 bp of the human ARL1 5'-UTR. Such a result
might be predicted if [Q71L]ARL1 and [Q71L]ARF1 have similar
affinities for the effector that mediates Golgi vesiculation. We have
also expressed the homologous mutant of human ARL2, [Q70L]ARL2, in
NRK cells and observed no effects on or localization to Golgi
membranes.4 Instead, we have
noted recently that ARL2 is imported into
mitochondria5 and has actions
that are clearly distinct from those of ARFs and ARL1. We conclude that
ARL1 can function at the Golgi in a manner similar to ARF1.
S binding stoichiometry is sensitive
to the addition of binding proteins, first established for ARFs (32).
Indeed, Arfaptin2/POR1 increased the stoichiometry of GTP
S binding
to ARL1 and ARF1 with the same sensitivity, to the same extent, and
with the same kinetics (Fig. 2). With its previous identification as a
partner of RAC, Arfaptin2/POR1 may now be viewed as an
ARF/RAC/ARL1-binding protein.
was described previously as binding ARL2 and ARL3 (34). Results
from two-hybrid assays confirmed those results and extended the binding
specificity to include ARL1 but not ARFs.
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Fig. 10.
Summary of ARF and ARL binding
partners. The interactions detected by two-hybrid technologies
between ARF and ARL binding partners are depicted. The upper
panel marked SHARED indicates those binding
partners that bind to more than one GTPase (ARF1-6 are shown
collectively because no partner has yet been described which does not
bind them all). The lower panel marked
SPECIFIC indicates the binding partners that
appear to interact with only one GTPase. The question mark
following ARFaptin1 and ARFophilin indicates that they have not been
tested for interaction with ARLs 1-3. Not shown in this figure are the
five or more proteins (e.g. COP-I, AP-1, AP-3, PLD1,
phosphatidylinositol 4-phosphate, 5-kinase) that bind directly to ARFs
but do not work in two-hybrid assays because of their oligomeric nature
or tight membrane binding.
Golgins are a group of autoimmune antigens that share a localization at
the Golgi, and though they lack defined primary sequence homology they
typically contain predicted coiled-coil domains (for review, see Ref.
55). Two of the Golgins, Golgin-97 and Golgin-245, share a sequence
motif, termed the GRIP domain, which is also found in three other
proteins, RanBP2 and two proteins predicted from ESTs sequences
(57). GRIP domains are always found at the COOH terminus, are 50-60
residues in length, and are autonomously folding and acting domains
that are sufficient to direct localization of fusion proteins to Golgi
membranes (22, 57). The GRIP domain of Golgin-245 was pulled
from screens of human B cell and fetal brain cDNA libraries using
the activated mutant of ARL1 as bait. Tests of specificity in
two-hybrid assays indicated that this GRIP domain also binds to
activated ARL3 but not ARL2 or any of the ARFs. Although a preference
for the [Q71L]ARL1 mutant was observed, it was less dramatic than was
found for ARFs with their partners (27, 29; Table II). A related screen
of the brain library also led to the cloning of a fragment of
RanBP2
, including the GRIP domain, as a binder of ARL1 which is
highly dependent on the activation state of the GTPase. Thus, we
independently cloned two of the three known GRIP-containing proteins
using activated ARL1 as bait and conclude that GRIP domains are
involved in both binding to ARL1 and Golgi membranes. It is possible
that the use of full-length Golgin-245 or RanBP2
would yield
different results with regard to the activation dependence on ARL1
binding or Golgi binding, but the previous demonstration of GRIP
domains as autonomously folding and acting domains (22, 57) makes it
likely that these activities are retained and biologically relevant in
the full-length proteins. Indeed, one theme that has been repeated
several times (MKLP1, GGA1-3, Golgin-245, SCOCO) is the binding of ARF
family members to domains close to or overlapping predicted
coiled-coils. The presumed binding of RanBP2
to the nuclear pore may
suggest that the GRIP domain and ARL1 binding may have functions
independent of Golgi membranes. Because no full-length sequence for
human Golgin-245 is available and the GRIP domain had already been
shown to bind Golgi membranes and ARL1, we focused on the relationship between these two activities for the GRIP domain of Golgin-245.
Tests of the interdependence between binding of Golgin-245 and ARL1 to
Golgi membranes were initiated. Expression of the GRIP domain of
Golgin-245 in NRK cells revealed colocalization with ARL1 when viewed
by confocal microscopy. A tyrosine residue that is conserved in all
GRIP domains and is required for Golgi binding (22, 57) was mutated in
the GRIP domain of human Golgin-245 and found to be required for both
Golgi and ARL1 binding. This overlap in ARL1 and Golgi binding domains
suggests that the binding of one protein to the Golgi is likely to
require or be promoted by the other. The insensitivity of the GRIP
domain to short term treatments with brefeldin A suggested that it may
represent a docking site for ARL1 to bind Golgi, rather than the
alternative. The large size of both Golgin-245 and RanBP2 (358 kDa)
makes them candidate scaffolding proteins, and the identification of an
ARL1 binding site at their COOH termini likely represents a site of
protein recruitment and assembly, whether on nuclear pores or Golgi membranes.
Screens of several human cDNA libraries resulted in the isolation of the ARL1-specific binding partner, SCOCO. The name was based on the prediction that >75% of the 82 residues in the full-length protein exist in a coiled-coil (Fig. 4). A small subset of coiled-coil-containing proteins have short (50-80 residues) regions of high homology to SCOCO which may serve as Golgi or ARL1 binding domains. Affinity-purified SCOCO antibodies were used to localize endogenous SCOCO to the Golgi and plasma membrane in NRK cells (Figs. 7 and 8). There was extensive overlap in the staining of ARL1 and SCOCO at the Golgi, and each was sensitive to brefeldin A. Sensitivity of a Golgi membrane protein to brefeldin A has previously been taken as evidence for a role for ARF proteins because the drug binds directly to ARF GEFs (65, 67). However, the brefeldin A-induced dissociation of SCOCO from Golgi was slowed by the presence of the activated ARL1 protein (see Fig. 8) but not by activated ARF1. The simplest interpretation of these data is that there exists a brefeldin A-sensitive ARL1 GEF whose inhibition is sufficient to cause the rapid release of both ARL1 and ARL1-dependent binding proteins from the Golgi. Whether such an ARL1 GEF represents a novel protein or is an ARF GEF with substrate specificity that includes ARL1 is currently under investigation. Thus, earlier conclusions that ARFs and ARLs lack functional overlap must now be altered to include ARL1 as a GTPase with the potential to signal through interactions with ARF GEFs and effectors shared by one or more ARFs. Similarly evidence of brefeldin A sensitivity must now be interpreted as evidence for ARF or ARL1 involvement.
Overlap in binding partners is essentially complete among the ARF
proteins, which share >60% sequence identity, and is nearly absent
between ARFs and ARLs, with the notable exception of ARL1. Given that
the sequence relatedness between ARFs and ARLs is the same as between
any two ARLs, we also predicted very limited, if any, overlap in
binding partners between ARLs. However, we found instead a more
extensive set of shared partners than specific ones between ARL1, ARL2,
and ARL3. These interactions, defined originally by two-hybrid assay,
are summarized in Fig. 10. This evidence for greater potential overlap
in signaling between members of the ARF family indicates that more
extensive tests for specificity should be performed in future
characterizations of effectors, GEFs, and GAPs for members of the
ARF/ARL family.
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ACKNOWLEDGEMENTS |
---|
We thank Steve Elledge for the yeast
strains, B cell library, and associated plasmids; Rob Brazas for the
pBG4D vector; Stan Fields for plasmids carrying lamin-AD and Cdk2-AD;
Ahmed Zahraoui for the PDE expression construct; Nava Segev and Sara
Jones for the SEC7 domain expression system; and Linda Van Aelst for
the myelin basic protein-POR1 expression construct. Additionally, we
thank Kristen Thomas for extensive help with the use of the confocal
microscope and Chun-Jiang Zhang for technical advice with GAP assays
and construction of interferon-inducible cell lines. We also thank Judy
Fridovich-Keil for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* 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: Dept. of Biochemistry,
Emory University School of Medicine, 1510 Clifton Rd., Atlanta, GA
30322-3050. Tel.: 404-727-3561; Fax: 404-727-3746; E-mail:
rkahn@emory.edu.
Published, JBC Papers in Press, April 12, 2001, DOI 10.1074/jbc.M102359200
2 J. C. Amor, X. Zhu, J. Horton, X. Cheng, D. Ringe, and R. A. Kahn, manuscript in preparation.
3 J. D. Sharer, J. Shern, and R. A. Kahn, unpublished observation.
4 J. D. Sharer and R. A. Kahn, unpublished observations.
5 J. D. Sharer, J. F. Shern, H. Van Valkenburgh, D. C. Wallace, and R. A. Kahn, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are:
ARF(s), ADP-ribosylation factor(s);
ARL, ARF-like;
BART, binder of ARL2;
PDE,
subunit of cGMP phosphodiesterase 6;
NRK, normal rat
kidney;
GTP
S, guanosine 5'-3-O-(thio)triphosphate;
BSA, bovine serum albumin;
GAP, GTPase-activating protein;
SCOCO, short coiled-coil;
HRG, human retinal gene;
MOPS, 4-morpholinepropanesulfonic acid;
bp, base pairs;
HA, hemagglutinin;
UTR, untranslated region;
GEF, guanine nucleotide exchange
factor.
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