(Received for publication, August 7, 1996, and in revised form, December 19, 1996)
From the Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
ADP-ribosylation factors (ARFs) have been
implicated in vesicle transport in the Golgi complex. Employing yeast
two-hybrid screening of an HL60 cDNA library using a constitutively
active mutant of ARF3 (ARF3·Q71L), as a probe, we have identified a
cDNA encoding a novel protein with a calculated molecular mass of
38.6 kDa, which we have named arfaptin 1. The mRNA of arfaptin 1 was ubiquitously expressed, and recombinant arfaptin 1 bound
preferentially to class I ARFs, especially ARF1, but only in the
GTP-bound form. The interactions were independent of myristoylation of
ARF. Arfaptin 1 in cytosol was recruited to Golgi membranes by ARF in a
guanosine 5-O-(3-thiotriphosphate)-dependent
and brefeldin A-sensitive manner. When expressed in COS cells, arfaptin
1 was localized to the Golgi complex. The yeast two-hybrid system
yielded another clone, which encoded a putative protein, which we have
named arfaptin 2. This consisted of the same number of amino acids as
arfaptin 1 and was 60% identical to it. Arfaptin 2 was also
ubiquitously expressed and bound to the GTP-, but not GDP-liganded form
of class I ARFs, especially ARF1. These results suggest that arfaptins 1 and 2 may be direct target proteins of class 1 ARFs. Arfaptin 1 may
be involved in Golgi function along with ARF1.
ADP-ribosylation factors (ARFs),1 which were originally identified and purified by their ability to enhance the ADP-ribosyltransferase activity of cholera toxin, comprise a distinct subfamily of Ras-related small GTP-binding proteins and have been found in all eukaryotic cells from yeast to human (1). To date, at least six mammalian ARF genes have been cloned. They can be divided into three classes based on deduced amino acid sequence, protein size, phylogenetic analysis, and gene structure: class I, including ARF1, -2, and -3; class II, including ARF4 and -5; and class III, including ARF6 (2, 3). ARFs have recently been recognized as regulators of intracellular vesicular transport. They are essential molecules for coated vesicle formation in the Golgi complex (4, 5) and have also been implicated in vesicle transport between endoplasmic reticulum and Golgi (6, 7) and in nuclear vesicle fusion (8). More recently, ARFs have been shown to activate phospholipase D (9, 10).
Like other members of the Ras superfamily, ARF proteins transmit signals to downstream effectors in a cyclical and guanine nucleotide-dependent manner. Conformational differences between their GDP- and GTP-bound forms determine their interaction with regulatory proteins, namely a guanine nucleotide exchange protein (GEP) and a GTPase-activating protein (GAP). Class I ARF-directed GEP has been purified (11), and ARF1-directed GAP has also been purified and cloned (12, 13). In contrast, the downstream effectors of ARF remain to be clarified. An ARF-responsive phospholipase D has been purified to a high degree from porcine brain (14). More recently, a form of mammalian phospholipase D has been cloned, and the recombinant enzyme expressed in Sf9 cells has been shown to be activated by recombinant ARF1 (15). Although this does not prove direct interaction between ARF and phospholipase D, it strongly suggests that it is a downstream effector. Nevertheless, it is still unclear if phospholipase D mediates ARF signals to initiate coated vesicle formation.
A point mutation of ARF1 at Gln71 with Leu or Ile (Q71L or Q71I) slows the rate of GTP hydrolysis and therefore makes the mutant constitutively active (16). In an attempt to identify a direct downstream effector of ARF, we have employed the Q71L mutant of ARF3, which is 96% identical to ARF1, to screen a cDNA library using the yeast two-hybrid system. We have identified two novel proteins, termed arfaptins2 1 and 2, which interact with class I ARFs only in their GTP-bound conformation. Arfaptin 1 was shown to be recruited to Golgi membranes by GTP-bound ARF.
The yeast strain used in the two-hybrid screening and interaction assay was HF7c (MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3, 112, gal4-542, gal80-538, LYS2::GAL1-HIS3, URA3::(GAL4 17-mers)3-CYC1-lacZ). HL60 cells were purchased from ATCC and maintained as described (17). COS7 cells were purchased from ATCC and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin at 37 °C in a humidified atmosphere of 10% CO2 and 90% air.
Plasmids and cDNA LibraryThe ARF cDNAs used in the
two-hybrid screening and interaction assays were polymerase chain
reaction-amplified from human ARF3 cDNA as a template (kindly
provided by J. Moss, National Institutes of Health). Full-length wild
type ARF3 cDNA was generated using 5 (sense) oligonucleotide
primer A (5
-CG GAA TTC ATG GGC AAT ATC TTT GGA AAC CTT CTC) and 3
(antisense) oligonucleotide primer B (3
-CG GGA TCC TCA CTT CTT GTT TTT
GAG CTG ATT GGC C). Substitution of Gln71 with Leu (Q71L)
was introduced by synthesizing the N-terminal half (corresponding to
amino acids 1-71) and C-terminal half (corresponding to amino acids
71-181). The N-terminal half was generated using 5
primer A and
mutagenic 3
primer C (3
-G CTC
ACC ACC CAC ATC CCA CAC
TGT AAA GCT; Leu71 is underlined). The C-terminal half was
generated using mutagenic 5
primer D (5
-GCT
GAC AAG
ATT CGA CCC CTC TGG AGA CA; Leu71 is underlined) and 3
primer B. The fragments were digested with XbaI and ligated
to produce ARF3·Q71L. Double mutant ARF3·G2A·Q71L was also
generated using N-terminal (amino acids 1-71) and C-terminal (amino
acids 71-181) fragments. The N-terminal half was synthesized using
mutagenic 5
primer E (5
-CG GAA TTC ATG
AAT ATC TTT GGA AAC CTT CTC; Ala2 is underlined) and 3
primer C. The
C-terminal half was the same as described above. The fragments were
digested with XbaI and ligated. Wild type and mutated ARFs
were subcloned into the EcoRI/BamHI sites of
pGBT9 yeast/Escherichia coli shuttle vector (Clontech) to
generate fusions with the GAL4 DNA binding domain (pGBT9-ARF3, pGBT9-ARF3·Q71L, and pGBT9-ARF3·G2A·Q71L). All constructs were confirmed by DNA sequencing.
For the preparation of the HL60 cDNA library (custom made by Clontech), total RNA was isolated from undifferentiated HL60 cells by the acid guanidium thiocyanate-phenol-chloroform extraction method (18), and poly(A)+ RNA was purified through an oligo(dT)-cellulose column (19). Double-stranded cDNA was made using both oligo(dT) and random primers and introduced into pGAD10 yeast/E. coli shuttle vector using EcoRI-NotI-SalI adaptor to generate fusions with GAL4 activation domain.
Two-hybrid ScreeningTwo-hybrid screening was performed using a Matchmaker Two-Hybrid System (Clontech) according to the instructions provided by the manufacturer. The yeast reporter strain HF7c was transformed sequentially with pGBT9-ARF3·Q71L and then with the HL60 cDNA library using a lithium acetate-based method. The double transformants were grown on SD agar medium lacking Trp, Leu, and His for 5 days at 30 °C before positive colonies were picked, restreaked onto triple minus plates, and assayed for the LacZ phenotype. Plasmids containing GAL4-activation domain library fusion were isolated from HIS3 and LacZ positive colonies and used for further investigations.
For the -galactosidase filter assay, colonies of yeast transformants
were spread in small patches onto Whatman No. 1 filter papers and
permeabilized in liquid nitrogen. Each filter was placed on another
filter paper that had been presoaked in Z buffer (60 mM
Na2HPO4, 40 mM
NaH2PO4, 10 mM KCI, 1 mM MgCl2, 37.5 mM
-mercaptoethanol) containing 0.33 mg/ml
5-bromo-4-chloro-3-indolyl-
-D-galactoside and incubated
at 30 °C until color developed.
HL60 poly(A)+ RNA (2 µg) isolated as described above was separated by a denaturing formaldehyde 1% agarose gel, transferred to a nitrocellulose membrane (Durarose-UV, Stratagene), and hybridized with a 32P-labeled probe using a standard procedure (19). To examine the expression in various human tissues, a Northern blot filter with 2 µg of poly(A)+ RNA from different human tissues (Clontech) was hybridized with a 32P-labeled probe according to the manufacturer's recommendations. A 1.2-kb cDNA insert of arfaptin 1 and a 1.6-kb cDNA insert of arfaptin 2 were 32P-labeled using a random primer DNA labeling kit (Bio-Rad) and used as probes.
Recombinant Arfaptin 1 and Arfaptin 2Recombinant arfaptin
1 and arfaptin 2 were produced as glutathione S-transferase
(GST) fusion proteins. For GST-arfaptin 1, a short 5-fragment from
nucleotide 130 to 180 (internal SpeI site) with a
SmaI site at the 5
-end was synthesized by annealing sense
and antisense oligonucleotides in 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM dithiothreitol,
and 0.1 mM EDTA at 37 °C for 1 h. The fragment was
ligated to a SpeI/EcoRI fragment (nucleotides 177-1234) at the SpeI site. The resulting
SmaI/EcoRI fragment was subcloned into the same
sites of GST fusion vector pGEX-2T (Pharmacia Biotech Inc.) to generate
pGEX-arfaptin 1. For GST-arfaptin 2, a short 5
-fragment stretching
from the predicted translation initiation codon (nucleotide 68) to the
internal BamHI site (nucleotide 82) with an EcoRI
site at the 5
-end was synthesized by annealing sense and antisense
oligonucleotides as described above and ligated to a
BamHI/NotI fragment (nucleotides 78-1654) at the
BamHI site. The resulting EcoRI/NotI
fragment was subcloned into the same sites of pGEX-4T vector
(Pharmacia) to generate pGEX-arfaptin 2.
E. coli strain BL21 was transformed with pGEX-arfaptin 1 or
pGEX-arfaptin 2. Transformed cells were grown at 37 °C to
A600 = 0.8, and protein expression was induced
with 0.1 mM
isopropyl-1-thio--D-galactopyranoside for 3 h at
27 °C. The cells were resuspended in phosphate-buffered saline
containing 0.5 mM EGTA, 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and
10 µg/ml antipain and lysed by sonication. The lysate was incubated
with glutathione-Sepharose beads (Pharmacia) for 30 min at room
temperature. The beads were then washed with phosphate-buffered saline,
and the GST fusion protein was eluted by 10 mM glutathione
in 50 mM Tris-HCl (pH 8.0). Recombinant arfaptin 1 without
the GST moiety was obtained by treating GST-arfaptin 1-immobilized
glutathione-Sepharose beads with thrombin (4 µg/ml) in the buffer
consisting of 50 mM Tris-HCl (pH 8.0), 150 mM
NaCl, 2.5 mM CaCl2, and 14.2 mM
-mercaptoethanol for 40 min at room temperature. The supernatant was
later subjected to benzamidine-Sepharose to remove thrombin.
Human ARF3 cDNA was polymerase chain
reaction-amplified with the NcoI site upstream of the
initiator methionine codon and the HindIII site downstream
of the termination codon and subcloned into pKK233-2 expression vector
(Clontech) to generate pKK233-2-ARF3. E. coli strain JM109
was transformed with pKK233-2-ARF3, and protein expression was induced
with 2 mM
isopropyl-1-thio--D-galactopyranoside for 5 h at
37 °C. Recombinant ARF3 was purified by successive column
chromatography on DEAE-Sephacel and then Sephadex 75 as described by
Weiss et al. (20) with slight modifications.
For production of myristoylated ARF3, JM109 bacteria were
co-transformed with pKK233-2-ARF3 and pBB131 (yeast
N-myristoyltransferase, kindly provided by Dr. J. I. Gordon,
Washington University; see Ref. 21) and selected for both ampicillin
and kanamycin resistance. Transformed cells were grown at 37 °C to
A600 = 0.6, and protein expression was induced
with 1 mM
isopropyl-1-thio--D-galactopyranoside in the presence of
200 µM sodium myristate for 3 h at 27 °C. The lower temperature (27 °C) was employed to increase the efficiency of
N-myristoylation (22). The myristoylated ARF3 was partially purified (~20%) employing the same procedure as described for nonmyristoylated ARF3. Efficiency of myristoylation was approximately 50% as judged by the change in mobility on SDS-PAGE.
Recombinant nonmyristoylated human ARF1, ARF5, and ARF6 were kindly provided by W. Patton and J. Moss (National Institutes of Health).
AntiseraRecombinant arfaptin 1 obtained by thrombin cleavage of GST-arfaptin 1 was used as an immunogen to raise polyclonal antisera in rabbits. In Western blotting, anti-arfaptin 1 was used at a 1:2000 dilution. Anti-sARFII, which recognizes ARF1 and ARF3 equally (23), was a kind gift of Dr. J. Moss and was used at a 1:1000 dilution in Western blotting.
ImmunoprecipitationImmunoprecipitation was performed according to standard procedures (24). Briefly, HL60 cells (4 × 107 cells) were lysed in 1 ml of Nonidet P-40 lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml antipain), and the lysate was incubated with 1-3 µl of anti-arfaptin 1 antiserum for 1.5 h at 4 °C. The immune complex formed was purified using Protein A-Sepharose (Pharmacia).
In Vitro Binding StudiesNucleotide-free ARF was prepared
by dialysis of recombinant ARF against 7 M urea as
described (20) and incubated at 0.5 µM with or without
guanosine 5-O-(3-thiotriphosphate) (GTP
S) (5 µM) in a reaction mixture consisting of 25 mM
Hepes (pH 7.4), 100 mM NaCl, 25 mM KCl, 2.5 mM MgCl2, 1 mM EDTA, 0.1% Triton
X-100, 1 mM dithiothreitol, and 0.3 mg/ml bovine serum
albumin at 30 °C for 30 min. Triton X-100 was included to increase
GTP
S loading and to stabilize the GTP
S-ARF formed (25). GDP-bound
ARF was prepared by incubating ARF (not treated with urea) with GDP (5 µM) in the same reaction mixture. GST-, GST-arfaptin 1-, and GST-arfaptin 2-immobilized glutathione-Sepharose beads prepared as
described above were equilibrated with washing buffer A (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol).
Fifty µl of nucleotide-preloaded ARF mixture was then incubated with 25 µl of GST-, GST-arfaptin 1-, or GST-arfaptin 2-immobilized beads
for 20 min at 4 °C with gentle rocking. The beads were then collected by pulse centrifugation in a microcentrifuge, washed four
times with 0.5 ml of washing buffer A, and resuspended in SDS-sample
buffer. ARF associated with the beads was detected by 14% SDS-PAGE
followed by immunoblotting using anti-sARFII antibody.
To determine the affinity of arfaptin 1 for ARF-GTPS, ARF3 was
loaded with 10 µM [35S]GTP
S (4 µCi/nmol) as described above. The concentration of ARF3-[35S]GTP
S was determined by a nitrocellulose
filter binding assay, based on the specific activity of
[35S]GTP
S. GST-arfaptin 1 was then incubated with
ARF-[35S]GTP
S at different concentrations at 30 °C
for 30 min. Anti-arfaptin 1 antiserum and protein A-Sepharose beads
were then added, and the mixture was incubated at 4 °C for 60 min.
The beads were then washed three times in washing buffer A and counted
to determine ARF-[35S]GTP
S binding. The data were
analyzed by Eadie-Hofstee plot to yield Kd
values.
When the relative affinity of each ARF isozyme to arfaptin 1 or
arfaptin 2 was examined, nucleotide-free ARF was preloaded with 5 µM [35S]GTPS (4 µCi/nmol) as described
above. Thereafter, aliquots were determined for
[35S]GTP
S binding by the nitrocellulose filter assay.
GTP
S-bound ARF was stable on ice for at least 5 h. Each mixture
was adjusted to contain the same amount of
[35S]GTP
S-ARF and used for the interaction experiment
described above. After washing, the beads were mixed with 10 ml of
Ready Safe liquid scintillation mixture (Beckman), and the
radioactivity was counted. The stoichiometry of
[35S]GTP
S binding for ARF1, ARF3, ARF5, and ARF6 was
0.26, 0.28, 0.08, and 0.21, respectively (means of the three
determinations).
Highly concentrated
cytosol was prepared from HL60 cells as described by Malhotra et
al. (26) except that the cells were homogenized by sonication.
Prior to use, the cytosol was stored at 80 °C and thawed and
centrifuged at 100,000 × g for 1 h at 4 °C to
remove any aggregated proteins. Golgi-enriched membrane fractions were
prepared from rat liver by sucrose gradient centrifugation according to
Malhotra et al. (26). Membranes were collected at the 0.5 M sucrose/1 M sucrose interface and stored at
80 °C. Prior to use, membranes were washed with 10 mM
Tris-HCl (pH 7.4).
Golgi membranes were incubated with HL60 cytosol or comparable amounts
of gel-filtered cytosol under conditions previously defined (27) with
slight modifications. Briefly, Golgi membranes (7.5 µg of protein)
and a saturating concentration of cytosol protein (300 µg) were
incubated with or without GTPS (25 µM) at 37 °C for
10 min in the reaction mixture (100 µl) consisting of 25 mM Hepes-KOH (pH 7.0), 125 mM KCl, 2.5 mM MgCl2, 1 mM dithiothreitol, 0.2 M sucrose, 1 mM ATP, 5 mM creatine
phosphate, and 10 units/ml creatine kinase. When the effect of
brefeldin A was examined, Golgi membranes were incubated with brefeldin
A for 10 min at 37 °C prior to the addition of cytosol and GTP
S.
After incubation, 90 µl of the mixture was layered on 330 µl of
25% (w/v) sucrose in washing buffer B, consisting of 25 mM
Hepes-KOH (pH 7.0), 125 mM KCI, 2.5 mM
MgCl2, and 1 mM dithiothreitol, and Golgi
membranes were collected as pellets by centrifugation for 30 min at
14,000 rpm at 4 °C in a microcentrifuge as described (28). The
sucrose cushion greatly reduced the amounts of ARF and arfaptin 1 that were nonspecifically precipitated by centrifugation when Golgi membranes were omitted from the reaction mixture. The pellet was washed
with the washing buffer B containing 0.2 M sucrose and resuspended in SDS-sample buffer. ARF and arfaptin 1 associated with
the membranes were separated on 14% SDS-PAGE and detected by
immunoblotting using anti-sARFII and anti-arfaptin 1, respectively. In
some experiments, the immunoblots were analyzed by densitometry (Apple
Macintosh, One Scanner).
An epitope sequence corresponding to
FLAG octapeptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys), which is
specifically recognized by anti-FLAG M2 monoclonal antibody (Eastman
Kodak Co.), was introduced at the N terminus of arfaptin 1. A
5-fragment from nucleotides 131-180 was synthesized with a
HindIII site upstream and the FLAG encoding sequence
downstream of the translation initiation codon by annealing sense and
antisense oligonucleotides as described above. This fragment was
ligated to the SpeI/EcoRI fragment (base pairs
177-1234) at the SpeI site. The resulting
HindIII/EcoRI fragment was subcloned into the
same sites of the mammalian expression vector pcDNA3 (Invitrogen)
to generate pcDNA3-FLAG·arfaptin 1.
COS7 cells grown to 50-80% confluency in 100-mm dishes were transiently transfected by electroporation at 220 V and 960 microfarads using 15 µg of pcDNA3-FLAG·arfaptin 1 and grown on glass coverslips. Two days after transfection, cells were fixed using 3.7% formaldehyde and then incubated with mouse anti-FLAG M2 monoclonal antibody (20 µg/ml) followed by Texas red-conjugated horse anti-mouse IgG (1:150 dilution, Vector) according to standard procedures (29). Expression of exogenous arfaptin 1 was detected by fluorescent microscopy. Transfection efficiency was 10-20%.
Miscellaneous ProceduresSDS-PAGE and Western blotting were
performed as described (17) except that an ECL kit (Amersham Corp.) was
used for detection. Protein concentrations were determined using the
Coomassie Plus protein assay reagent (Pierce) with bovine serum albumin
as a standard. DNA sequencing was performed using a Sequenase II DNA sequencing kit (U.S. Biochemical Corp.). HL60 cytosol and crude membrane fractions used in Fig. 2 were prepared as follows. HL60 cells
were homogenized by sonication in a buffer consisting of 50 mM Hepes (pH 7.2), 100 mM KCl, 5 mM
NaCl, 0.5 mM EGTA, 3.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and
10 µg/ml antipain. After unbroken cells and nuclei were removed by
centrifugation at 500 × g for 10 min, crude membranes
and cytosol were obtained by centrifugation at 100,000 × g for 90 min. Membranes were washed once with the sonication
buffer.
To identify molecules that act as downstream effectors
of ARF, we employed the yeast two-hybrid system and screened an HL60 cDNA library using a constitutively active mutant of ARF3
(ARF3·Q71L) (16, 29) as a target. When approximately 2 × 106 double transformants were screened and selected for
histidine prototrophy, three positive colonies were obtained. These
colonies were assayed for activation of second reporter gene
LacZ by -galactosidase filter assay, and all were
revealed to be positive. Partial sequencing and restriction mapping of
the cDNA inserts of these library clones revealed that two clones
(clones 1 and 3) had the same cDNA insert (1.2 kb), while another
clone (number 2) had a insert (1.6 kb) distinct from clones 1 and 3. To
eliminate false positives, further genetic assays were conducted (Table
I). Neither clone 1 nor clone 2 by itself was capable of
activating
-galactosidase activity, indicating that they do not
contain a latent transcriptional activator. The GAL4-binding domain
alone or human lamin C, which is a protein unrelated to ARF, did not
interact with either clone 1 or 2. These observations rule out the
possibility that clone 1 and clone 2 nonspecifically interact with
other proteins.
|
ARFs are modified by myristoylation at Gly2 through the action of an N-myristoyltransferase in vivo (30). However, ARF3 expressed as a fusion protein with a GAL4-binding domain cannot be modified, because N-myristoyltransferase can only act at the N terminus (21). Therefore, it is presumed that the interaction of ARF3·Q71L with clone 1 or clone 2 in yeast is independent of myristoylation. This was confirmed by studies with the double mutant ARF3·G2A·Q71L, which has an additional mutation at Gly2 to abolish myristoylation, which showed that this also interacted with clones 1 and 2 with apparently the same affinity as ARF3·Q71L (Table I). Interestingly, the two-hybrid assay indicated that neither clone 1 nor 2 interacted with wild-type ARF3, suggesting that both clones interact only with the active, GTP-bound form of ARF (Table I).
Clone 1 was revealed to contain a full-length open reading frame
encoding a protein of 341 amino acids with a calculated molecular mass
of 38,596 Da (Fig. 1). The translation initiation codon
(nucleotides 131-133) was surrounded by a consensus Kozak sequence
(31). Furthermore, an antibody raised against a recombinant protein encoded by this open reading frame immunoprecipitated a protein from
HL60 cell lysate whose migration on SDS-PAGE was similar to that
of the recombinant protein (~44 kDa) (Fig. 2). Because the recombinant protein was obtained by thrombin cleavage of the GST
fusion protein, it had four additional amino acids at the N terminus.
This could partly explain the slight difference in migration. We have
named the protein encoded by clone 1 arfaptin 1.2 When
cytosol and crude membranes from HL60 cells were subjected to
immunoblotting, arfaptin 1 was detected predominantly in cytosol (Fig.
2).
Interestingly, sequence analysis of clone 2 revealed the presence of a
potential open reading frame whose size was exactly the same as that of
arfaptin 1. The nucleotide sequence of this open reading frame was 57%
identical to that of arfaptin 1, and the deduced amino acid sequence
was 60% identical and 81% homologous when consensus substitutions
were included. The homology was pronounced at the C-terminal half
of the molecule (Fig. 3). Thus we have tentatively
named the protein encoded by the putative open reading frame of clone 2 arfaptin 2.
The predicted amino acid sequences of arfaptins 1 and 2 were used to search a protein data base (the BLAST network service at the National Center for Biotechnology Information (NCBI)). However, no homologous proteins of known function were found.3 The highest identity score was found with a Caenorhabditis elegans hypothetical 35-kDa protein of unknown function (CEF54C8.7, EMBL/GenBankTM accession number Z22178[GenBank]). The primary structure analysis indicated that arfaptin 1 and arfaptin 2 were hydrophilic proteins with a few minor hydrophobic regions. Both proteins had several potential phosphorylation sites for protein kinase C,4 protein kinase A, and casein kinase II. Interestingly, arfaptin 2 had a periodic repetition of five leucine residues at every seventh position (leucine zipper) (residues 150, 157, 164, 171, and 178 in Fig. 3). Such a motif, with a highly positively charged region immediately adjacent to its N-terminal side, forms a DNA-binding domain (33). Arfaptin 2, however, lacks such a positively charged region. Arfaptin 1 also has a leucine zipper motif, but this is interrupted at residue 157 by replacement with methionine (Fig. 3).
Northern blot analysis indicated that arfaptins 1 and 2 were
ubiquitously expressed as mRNAs of approximately 3.4 and 2.1 kb,
respectively, in various human tissues as well as HL60 cells, ruling
out the possibility that they are cancer cell-specific products. Both
arfaptins 1 and 2 were expressed at a relatively high level in liver,
pancreas, and placenta. In addition, arfaptin 1 was expressed at a
relatively high level in skeletal muscle and heart (Fig.
4).
Arfaptins 1 and 2 Bind Directly to the GTP-bound Form of Class 1 ARFs in Vitro
As described above, the two-hybrid interaction
assay suggested that arfaptins 1 and 2 bind ARF3 only in its GTP-bound
conformation and that the binding was independent of myristoylation of
ARF. To confirm this biochemically, in vitro interactions of
nonmyristoylated ARF3 with arfaptins 1 and 2 were examined using
recombinant proteins (Fig. 5). Since recombinant ARF
binds GTP or GTPS with very low stoichiometry due to its tightly
bound GDP, we utilized nucleotide-free ARF to prepare GTP
S-bound
ARF. Under the conditions employed, the stoichiometry of GTP
S
binding of ARF3 was 0.28. When GST-arfaptin 1-immobilized
glutathione-Sepharose beads were incubated with GTP
S-ARF3, GDP-ARF3,
or nucleotide-free ARF3, only GTP
S-ARF was associated with the
beads, while very little nucleotide-free ARF and no GDP-ARF were
detected. In addition, GTP
S-ARF3 did not bind to GST-immobilized
beads, indicating that the binding of GTP
S-ARF3 to the beads was
through arfaptin 1. The binding, however, was not quantitative (see
below). These results indicated that arfaptin 1 was capable of
interacting directly with nonmyristoylated GTP-ARF3 but not GDP-ARF3,
consistent with the results of the two-hybrid interaction assay.
Qualitatively similar results were obtained with arfaptin 2 and ARF3
(Fig. 5). The affinity of arfaptin 1 for GTP
S-ARF3 was measured as
described under "Materials and Methods." The mean
Kd from two experiments was 1.4 × 10
7 M.
The binding of arfaptins 1 and 2 to other ARF isoforms was examined
using recombinant nonmyristoylated ARFs preloaded with [35S]GTPS (Table II). ARF1, which is
96% identical to ARF3 in amino acid sequence, was used as another
representative of class I ARF, and ARF5 and ARF6 were used as
representatives of class II and III, respectively. After preloading of
nucleotide-free ARF with [35S]GTP
S, equal amounts of
each GTP
S-bound ARF were incubated with GST-arfaptin 1- or
GST-arfaptin 2-immobilized beads. Both arfaptins 1 and 2 bound ARF1
with the highest affinity. On the other hand, they bound much less ARF5
and very little ARF6. It was confirmed that both arfaptins 1 and 2 bound ARF1 only in its GTP
S-bound form by employing the same
experiments described for ARF3 in Fig. 5 (not shown). These results
indicate that arfaptins 1 and 2 interact preferentially with class I
ARFs, especially ARF1.
|
Among the subfamilies of Ras
low molecular weight GTP-binding proteins, ARF is the clearest example
of a GTPase whose GTP binding and hydrolysis cycle appears to be
strictly coupled to a membrane-cytosol localization cycle. Inactive
GDP-bound ARF is cytosolic, whereas active GTP-bound ARF is associated
with membranes, especially with the Golgi apparatus (24). As described above, arfaptin 1 is present in the cytosolic fraction and specifically binds GTP-bound ARF. Therefore, we examined the possibility that arfaptin 1 was translocated from cytosol to Golgi membranes in association with GTP-bound ARF (Fig. 6). Golgi-enriched
membranes from rat liver were incubated with HL60 cytosol in the
presence or absence of GTPS and then the membranes were collected by
centrifugation. In the presence of GTP
S, arfaptin 1 and ARF were
detected in the pellet (lane 3), whereas in the absence of
GTP
S very little arfaptin 1 or ARF was detected (lane 1).
When Golgi membranes were omitted from the reaction mixture, arfaptin 1 and ARF were barely detectable in the pellet (lane 2),
ruling out the possibility that arfaptin 1 and ARF were precipitated
due to nonspecific aggregation. Brefeldin A has been shown to disrupt
Golgi membranes and inhibit the binding of ARF to Golgi membranes (34).
When Golgi membranes were treated with brefeldin A (40 µg/ml) prior
to the addition of cytosol and GTP
S, translocation of arfaptin 1 and
ARF was inhibited (lane 4), suggesting that the association
of arfaptin 1 with Golgi membranes depends on ARF. When the results of
three experiments were analyzed by densitometry, the inhibition of
arfaptin translocation (32 ± 2%) was similar to that of ARF
(38 ± 7%).
To confirm that translocation of arfaptin 1 is
ARF-dependent, we utilized ARF-depleted cytosol (Fig.
7B). To obtain this, HL60 cytosol was
subjected to Sephacryl S-200 gel filtration column. Since arfaptin 1 was present in a high molecular weight complex, it could easily be
separated from ARF (Fig. 7A). When Golgi membranes were
incubated with ARF-depleted cytosol (fractions 22-28) alone, only a
trace amount of arfaptin 1 was translocated to membranes even in the
presence of GTPS. This was probably caused by the tiny amount of
aggregated ARF that was co-eluted with arfaptin 1. On the other hand,
when fractions containing native ARF (fractions 35-40 in Fig.
7A) were included in the above mixture,
GTP
S-dependent translocation of arfaptin 1 to the Golgi
membranes was restored, and this was well correlated with translocation
of ARF. When partially purified recombinant myristoylated ARF3 was used
instead of native ARF fraction, the same results were observed. These
results indicate that translocation of arfaptin 1 to Golgi membranes
requires GTP-bound ARF. It should be noted that although HL60 cytosol
contained both ARF1 (lower band of doublet, Fig.
7A) and ARF3 (upper band of doublet,
Fig. 7A), translocation of native ARF3 was much less than
that of native ARF1. This differential interaction of ARF1 and ARF3
with Golgi membranes is consistent with the previous observations (23).
Thus, it appears that translocation of arfaptin 1 to Golgi membranes is
mediated mainly by ARF1.
Arfaptin 1 Is Not a Component of Coatomer
One of the well
characterized functions of ARF is the recruitment of cytosolic coat
proteins to Golgi membranes, which is thought to play an important role
in vesicle trafficking (35). Coat proteins are preassembled in a
cytosolic complex, coatomer, which has an apparent molecular mass of
~800 kDa (36). As described above, arfaptin 1 was present in cytosol
as a high molecular weight complex that was recruited to Golgi
membranes by activated ARF (Fig. 7). These findings raised the
possibility that arfaptin 1 was a constituent of coatomer. To examine
this, HL60 cytosol was fractionated through a Sephacryl S-400 gel
filtration column. Arfaptin 1, however, was clearly separated from
-COP, one of the components of coatomer, since this was eluted at
around 800 kDa, whereas arfaptin 1 was eluted at around 450 kDa (not
shown). These results indicate that arfaptin 1 is not a constituent of coatomer. It is, however, currently uncertain whether arfaptin 1 is
present in such an 800-kDa complex in vivo or if this
complex is a result of nonspecific aggregation during the preparation of HL60 cytosol.
To
investigate the in vivo localization of arfaptin 1, epitope-tagged arfaptin 1 was transiently expressed in COS7 cells and detected by immunostaining (Fig. 8). Indirect
immunofluorescent microscopy showed perinuclear staining of arfaptin 1. Treatment of cells with brefeldin A caused the redistribution of
arfaptin 1 into the cytoplasm, suggesting that arfaptin 1 is localized to the Golgi complex in intact cells.
In the present study, we have identified a novel protein arfaptin 1, whose mRNA is ubiquitously expressed in human tissues. Arfaptin 1 is a hydrophilic protein with a few minor hydrophobic regions and is present in cytosol. The calculated mass is 38,596 Da, and it migrates on SDS-PAGE at approximately 44 kDa. Recombinant arfaptin 1 binds preferentially class I ARFs, especially ARF1, in their GTP-bound state and not at all in their GDP-bound form (Fig. 5 and Table II). Thus, arfaptin 1 is likely to be a direct target protein for class I ARFs. We have also obtained another clone encoding a putative protein, arfaptin 2, which consists of the same number of amino acids as arfaptin 1. The mRNA of arfaptin 2 is also ubiquitously expressed, and the deduced amino acid sequence of arfaptin 2 is 60% identical and 81% homologous to that of arfaptin 1. Furthermore, recombinant arfaptin 2 has very similar characteristics for ARF binding as arfaptin 1; it binds preferentially class I ARFs, especially ARF1, but only in their GTP-bound conformation, implicating arfaptin 2 as another target protein for class I ARFs.
To date, six distinct but highly homologous mammalian ARF proteins have been identified (2, 3). Our data concerning the relative affinity of arfaptin 1 for different ARF isoforms suggest that ARF domains that may be important for the interaction with arfaptin 1. Among the isoforms examined, arfaptin 1 binds ARF1 with the highest affinity and ARF3 with less affinity. It binds much less ARF5 and very little ARF6. The order of affinity for different ARF isoforms is correlated with their structural homology; ARF3, -5, and -6 differ from ARF1 in amino acid sequence by 4, 20, and 32%, respectively (2). It is surprising, however, that the very small difference in the amino acid sequences of ARF1 and ARF3 (7 of 181 amino acids) results in an approximately 3-fold difference in binding affinity for arfaptin 1. The differences between ARF1 and ARF3 are attributed exclusively to their N and C termini; 4 amino acids are different in the N-terminal 13 amino acids and 3 amino acids in the C-terminal 8 amino acids (37). These facts suggest that the N and/or C termini may be important domains of ARF1 for interaction with arfaptin 1.
Several lines of evidence have pointed out the significance of the N terminus of ARF for its functions. A synthetic peptide derived from the N terminus of ARF1 inhibits the cofactor activity of ARF for cholera toxin ADP-ribosyltransferase activity and ARF-dependent intra-Golgi protein transport in vitro (38). Experiments using a N-terminal deletion mutant of ARF1 have also shown that the N terminus of ARF1 is essential for the interaction with GAP and for the cofactor activity (39). More recently, analysis of the GTP-dependent conformational change of ARF1 has led to the proposal that the N terminus of ARF1 is an effector domain (40). Thus the N terminus rather than C terminus of ARF might be more important for interaction with arfaptin 1. In addition to the N terminus itself, modification of the N terminus by myristoylation is also critical for ARF functions, because myristoylation confers an ability to associate with membranes (41) and is critical for the GTP-dependent conformational change (40). Although the present study indicates that arfaptin 1 interaction with ARF does not require myristoylation (Table I), the possibility that the affinity may be increased by myristoylation remains to be examined.
It is reported that different ARF isoforms have different affinities for Golgi membranes and other cellular membranes. Among the isoforms, it has been shown that ARF1 as well as ARF5 associate with Golgi membranes with high affinity and specificity in vitro, while ARF3 associates with the membranes with lower affinity and is distributed to other cellular membranes (23). Localization of ARF1 to the Golgi complex was confirmed in overexpression experiments, whereas ARF6, to which arfaptin 1 does not bind, was localized to the endosomal/plasma membrane system (42). Another study has shown that ARF6 is uniquely localized to the plasma membrane of Chinese hamster ovary cells (43). Therefore, the preferential binding of arfaptin 1 to ARF1 shown by in vitro interaction experiments is in good agreement with the localization of arfaptin 1 in the Golgi complex shown by overexpression in COS cells in vivo (Fig. 8).
Accumulating evidence suggests that there is a cyclic localization of
ARF between cytosol and membrane fractions. Inactive GDP-bound ARF
present in the cytosol is activated by GEP in Golgi membranes or
cytosol in a brefeldin A-sensitive manner and is translocated to Golgi
membranes. Subsequently, GTP bound to ARF is hydrolyzed in the presence
of GAP in Golgi membranes, and the resulting GDP-ARF is released to
cytosol (13, 27, 44-47). Our in vitro translocation data
and in vivo transient expression experiments suggest that
the GTP-bound form of ARF serves as a membrane anchor for arfaptin 1 and that arfaptin 1 cycles between cytosol and Golgi membranes
depending on the activity status of ARF. Such a relationship between a
GTP-binding protein and its target, i.e. a GTP-binding
protein that serves as regulatable membrane anchor for its target
protein, has also been observed for other small GTP-binding proteins
and their functional target proteins. Ras, when it is activated,
translocates its cytosolic target Raf-1 protein kinase to the plasma
membrane, where Raf-1 is activated and initiates a phosphorylation
cascade (48). RhoA, which is involved in morphological events involving
the actin cytoskeleton, recruits its target serine/threonine kinase
ROK to peripheral membranes (49). Rab3A and Rab3C have been
suggested to recruit rabphilin-3A, a Rab3A target protein, to synaptic
vesicle membranes (50). Rab5, a potent regulator of endocytic
transport, recruits its target protein rabaptin-5 to early endosomes in
a GTP-dependent manner (51). Therefore, it is possible that
ARF, which belongs to another subfamily of Ras-related proteins, also
has a cytosolic target protein (arfaptin) and recruits it to membrane
fractions.
Arfaptin 2 has a leucine zipper structure in the middle of the molecule (Fig. 3). Arfaptin 1 also has a similar repetition of leucine residues, although one of them is replaced with methionine (Fig. 3). The leucine zipper structure provides hydrophobic faces through which zipper proteins interact to form dimers, which can be homodimers or heterodimers. The leucine zipper structure is often found in transcription factors, since the dimer can interact with DNA through a domain enriched with positively charged amino acids immediately adjacent to the N terminus of the zipper (33). Since arfaptin 1 and arfaptin 2 lack such a domain, they are unlikely to bind DNA, but they may still be able to form homo- or heterodimers. In fact, when HL60 cytosol was separated by gel filtration chromatography, arfaptin 1 was found as a high molecular weight complex (Fig. 6), raising the possibility that it may exist as an oligomer and/or as a complex with other proteins.
A major question of the present study is the physiological function of
arfaptin 1. This is being explored in in vitro studies carried out in collaboration with the group of Dr. J. Moss (National Institutes of Health). These results will be reported elsewhere when
complete, but initial findings are that recombinant arfaptin acts as an
inhibitor of the in vitro action of ARF on phospholipase D
and cholera toxin-catalyzed ADP-ribosyltransferase activity. However,
it does not alter the binding of GTPS or GDP to ARF in the presence
of GEP (11) or alter GTPase activity. Irrespective of these
observations, the fact that arfaptin 1 only interacts with ARF liganded
to GTP
S, but not GDP, suggests that it may be an effector
i.e. a physiological target of ARF whose function is
presently unknown. An additional possibility is that arfaptin 1 is an
adaptor protein that may require a third component before its function
can be observed.
The relative affinity of arfaptin 2 for different ARF isoforms is strikingly similar to that of arfaptin 1 (Table II). Tissue distribution of arfaptin 2 is also similar to that of arfaptin 1 except for muscle (Fig. 4). Furthermore, arfaptin 2 may form a heterodimer with arfaptin 1 as discussed above, raising the possibility that arfaptin 2 might act cooperatively with arfaptin 1. Alternatively, arfaptin 1 may require other protein(s) beside arfaptin 2. As mentioned above, estimation of the size of arfaptin 1 using gel filtration (Fig. 6) suggests that it may associate with other proteins of unknown identity. GTP-bound ARF recruits not only arfaptin 1 to Golgi membranes, but also coat proteins including coatomer (27, 35), p200 protein (52), and AP1 adaptins (53). Recently, ARF1-GAP has also been shown to be recruited to the Golgi complex by ARF (13), suggesting complex interactions among the various molecules. Thus, experimental conditions that are more physiological may be required to explore the function of arfaptin 1, e.g. those involving overexpression or "knockout" of arfaptin 1.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U52521[GenBank] (arfaptin 1) and U52522[GenBank] (arfaptin 2).
We thank S.-C. Tsai, R. Adamik, W. Patton, and J. Moss (National Institutes of Health) for generous gifts of ARF3 cDNA, anti-sARFII antibody, and recombinant ARF1, -5, and -6 and J. I. Gordon (Washington University) for kindly providing pBB131 yeast N-myristoyltransferase expression vector. We also thank H. Usui, A. R. Siddiqi, and A. H. Ross for the preparation of recombinant ARF3 and G. Venkatakrishnan for helpful comments. The assistance of J. Childs in the preparation of the manuscript is also greatly appreciated.