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INTRODUCTION |
ADP-ribosylation factors
(ARFs)1 are ~20-kDa guanine
nucleotide-binding proteins, initially identified by their ability to enhance cholera toxin-catalyzed ADP-ribosylation of the GTP-binding protein G
s (1). ARFs are ubiquitous in eukaryotic cells from Giardia to human and are known to play an important
role in intracellular vesicular trafficking (for review, see Ref. 2), as well as to activate phospholipase D (3, 4). Six mammalian ARFs,
identified by cDNA cloning, are grouped into three classes based on
size, amino acid sequence, phylogenetic analysis, and gene structure:
ARFs 1-3 in class I, ARFs 4 and 5 in class II, and ARF6 in class III
(5, 6).
Like other GTPases that regulate many kinds of intracellular processes,
ARFs are active and associate with membranes when GTP is bound, whereas
inactive ARF·GDP is cytosolic. Replacement of GDP by GTP is
accelerated by ARF GEPs or guanine nucleotide-exchange proteins,
several of which have been identified (7). These include Gea1 and Gea2
from yeast (8), mammalian B2-1 or cytohesin-1 (9) and cytohesin-2 or
ARNO (10), which are 83% identical in amino acid sequence, and GRP1
(general receptor for
phosphoinositides), a third member of the cytohesin group
(11).
All ARF GEPs of known structure contain Sec7 domains (8-13). Sec7 was
identified in a group of conditionally lethal yeast mutants as a gene
involved in Golgi vesicular trafficking and secretion (14). The Sec7
gene product is a ~230-kDa phosphoprotein that moves between membrane
and cytosolic fractions (15). Its Sec7 domain was demonstrated
relatively recently to function as a brefeldin A (BFA)-inhibited ARF
GEP (16). BFA is a fungal fatty acid metabolite with a monocyclic
lactone ring that blocks protein secretion reversibly and causes
apparent collapse of Golgi membranes into the endoplasmic reticulum
(17, 18). These effects result from BFA inhibition of GEP-catalyzed ARF
activation (GTP binding) (19, 20).
The two major types of ARF GEPs differ in size and susceptibility to
inhibition by BFA. Their Sec7 domains contain the determinants of GEP
activity as well as its BFA sensitivity. The larger ~200-kDa GEPs
(e.g. Sec7, Gea1, and Gea2 from yeast) are inhibited by BFA, whereas those of the cytohesin family are BFA-insensitive, ~50-kDa proteins. Morinaga et al. (21, 22) reported the purification and cloning of a BFA-inhibited ARF GEP from bovine brain, which was
referred to as p200. We describe here the cloning from a human brain
library of two GEP cDNAs. One, with a deduced amino acid sequence
(1,849 residues) 99.5% identical to that of p200, probably represents
the human form of p200. The other, encoding a 202-kDa protein 74%
identical in sequence, is a new BFA-inhibited human GEP. We propose
that because of their BFA sensitivity and size relative to the
cytohesins, these mammalian ARF GEPs be named BIG1 (for
BFA-inhibited GEP) and BIG2, respectively.
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EXPERIMENTAL PROCEDURES |
Materials--
[
-32P]dCTP,
[
-32P]dATP, and [35S]GTP
S were
purchased from NEN Life Science Products. All restriction enzymes and
Taq polymerase used to prepare BIG1-In and BIG2-In were
purchased from Roche Molecular Biochemicals (formerly Boehringer
Mannheim). Sources of other materials are noted below or have been
reported (22).
DNA Sequencing--
All plasmids were purified using Qiagen
Plasmid Midi kits (Qiagen, Chatsworth, CA). An Applied Biosystems Inc.
373 DNA sequenator was used for sequencing. 500 ng of plasmid DNA, 3.2 pmol of primer, and 8 µl of Terminator Ready Reaction Mix
(Perkin-Elmer) were used for cycle sequencing (25 cycles of 96 °C
for 30 s, 50 °C for 15 s, and 60 °C for 4 min) in total
volume of 20 µl.
Cloning of the cDNAs from Human Brain--
Probes for
screening a human frontal cortex
-ZAP cDNA library (1.2 × 106 plaque-forming units) (Stratagene) were prepared by PCR
with bovine BIG1 cDNA (22) as template. Sequences were: nucleotides 306-762 (probe 1), 2086-2728 (probe 2), and 5207-5541 (probe 3). Prehybridization overnight at 42 °C in 10% dextran sulfate, 1% SDS, 50 mM Tris, pH 7.4, 0.5 M NaCl, and 30%
formamide was followed by hybridization with 50 ng of radiolabeled
probe (2 × 105 cpm/ml) in 100 ml of the same buffer
overnight at 42 °C. Filters were then washed with 2 × SSC and
0.1% SDS at room temperature for 10 min twice and 1 × SSC and
0.1% SDS at 50 °C for 10 min. Two positive plaques were obtained
with probe 1. The first contained bases 1-2487 of the BIG2 coding
region named BIG2-N. (Base numbers are relative to A of ATG, equal to
1.) The other contained bases 1-420 of BIG1 (named BIG1-N). One plaque
obtained with probe 2 contained bases 1738-4657 of BIG1 (named
BIG1-C), and one with probe 3 (BIG2-C) contained bases 3514-5860 of BIG2.
The sequence between BIG2-N and BIG2-C was obtained by a nested PCR.
Primers for the first PCR (30 cycles of 94 °C for 1 min, 50 °C
for 1 min, and 72 °C for 2 min) were BIG2-N bases 2359-2385 (forward) and 3655-3631 in BIG2-C (reverse), with 1 µl of human adrenal gland QUICK-Clone cDNA (CLONTECH) as
template (total volume 100 µl). A sample (10 µl) of this PCR
mixture was used as template in a nested PCR (30 cycles of 94 °C for
1 min, 52 °C for 1 min, and 72 °C for 2 min) with forward primer
In-F (bases 2374-2402) and reverse primer In-R (bases 3593-3570). The
single 1.2-kb product was subcloned in pCR2.1 (Invitrogen) to produce
BIG2-In. Sequencing of the individual fragments generated a composite BIG2.
The sequence between bases 421 and 1737 in BIG1 was obtained by an
analogous procedure. Primers for the first PCR (30 cycles of 94 °C
for 1 min, 55 °C for 1 min, and 72 °C for 2 min) were bases
275-299 in BIG1-N (forward) and 1886-1860 in BIG1-C (reverse). A
sample (2 µl) of this PCR product was used as template in a nested
PCR (30 cycles of 94 °C for 1 min, 52 °C for 1 min, and 72 °C
for 2 min) with bases 300-329 (forward) and 1836-1811 (reverse) as
primers. The 1.5-kb product was subcloned in pCR2.1 (named BIG1-In).
Sequence of the 3'-terminus of the BIG1 cDNA was obtained by a
rapid amplification of cDNA ends procedure using 2.5 units of
Pfu DNA polymerase (Stratagene) and a Marathon Ready
cDNA kit (CLONTECH), which included the DNA
template and two reverse primers with adapters (AP-1 and AP-2).
Gene-specific primers in BIG1-C were bases 4468-4491 (G-1, forward)
and bases 4587-4611 (G-2, forward). In the first PCR (total volume 50 µl), 1 µl of template with G-1 and AP-1 was used for 30 cycles of
94 °C for 1 min, 68 °C for 1 min, and 72 °C for 3 min. 1 µl
of this PCR mixture was used as template in the second PCR (total
volume 50 µl) with G-2 and AP-2 for 30 cycles of 94 °C for 1 min,
65 °C for 1 min, and 72 °C for 3 min. The PCR product, which was
purified using a QIAEX II gel extraction kit (Qiagen), was subcloned in
pCR-Blunt (Invitrogen) and sequenced. Sequences of these cDNAs
yielded a composite BIG1 sequence.
Construction of Full-length BIG2 cDNA in pAcHLT-C--
For
insertion of BIG2 into the baculovirus transfer vector pAcHLT-C
(Pharmingen), restriction sites for NdeI and NotI
were introduced, respectively, before the initiation and after the termination codons of BIG2. To insert the NdeI site, the
forward primer was
5'-CATATGTATGCAGGAGAGCCAGACCAAG-3' including the
NdeI site (italics) and BIG2 initiation codon (underlined)
with reverse primer (bases 298-274) and 500 ng of BIG2-N as template
in a PCR (100 µl) with 5 units of Taq polymerase for 30 cycles of 94 °C for 30 s, 65 °C for 30 s, and 72 °C
for 30 s. The 305-bp PCR product was subcloned into pCR-2.1
vector. The DNA excised from the purified plasmid with
HindIII (site in pCR 2.1 vector) and NheI (base
219 of BIG2) was ligated to BIG2-N, which had been digested with the same enzymes, and named BIG2-Nde-Nhe.
To ligate BIG2-Nde-Nhe and BIG2-In, BIG2-In (bases 2374-3593) was the
DNA template with primers In-F and In-R and 2.5 units of Pfu
DNA polymerase (total volume 100 µl) for the first PCR (30 cycles of
94 °C for 1 min, 65 °C for 1 min, and 72 °C for 3 min). The
1.2-kb product (1.2-kb PCR) was purified using a QIAEXII gel extraction
kit and eluted in 40 µl of distilled water. 1 µl of 1.2-kb PCR
product and 500 ng of BIG2-N were used as templates in the second PCR
(30 cycles of 94 °C for 1 min, 65 °C for 1 min, and 72 °C for
4 min) with Pfu DNA polymerase, forward primer (bases 1826-1848), and reverse primer In-R (total volume 100 µl). The 1.7-kb PCR (bases 1836-3593), which was purified using a QIAEXII gel
extraction kit, was subcloned into pCR-Blunt vector. The plasmid DNA
was digested with AatII (base 1849 of BIG2) and
SpeI (site in pCR-Blunt vector). The excised DNA and the
BIG2-Nde-Nhe, which had been digested with AatII and
XbaI, were ligated and subcloned (named BIG2-Nde-Xba).
To insert an NotI site after the termination codon, primers
were BIG2 bases 3516-3540 (forward) and
5'-GCGGCCGCTACCACACTGGTG-3' (reverse), which has
an NotI site (italics) and the termination codon of BIG2
(underlined), with Pfu DNA polymerase (2.5 units) and 500 ng
of BIG2-C as template in a volume of 100 µl for 30 cycles of 94 °C
for 1 min, 65 °C for 1 min, and 72 °C for 4 min. The 1.8-kb PCR
product was purified using a QIAEXII Gel Extraction kit and subcloned
into pCR-Blunt vector (named BIG2-Not).
BIG2-C (500 ng) and 1 µl of 1.2-kb PCR were used as templates for PCR
(30 cycles of 94 °C/1 min, 60 °C/1 min, 72 °C/4 min) with
primers In-F and bases 4249-4222 (reverse) in a total volume of 100 µl to obtain bases 2374-4249 (1.9 kb). The 1.9-kb PCR product was
subcloned into pCR-Blunt vector (named BIG2-Nsi-ClaI) based on
subsequent digestion with Nsi and ClaI. A 1.7-kbp
DNA excised from BIG2-Not with ClaI and NotI was
ligated to BIG2-Nsi-ClaI, which had been digested with ClaI
(base 3791) and NotI (named BIG2-Nsi-Not).
To construct the full-length BIG2 DNA, the 2.4-kb DNA, excised from
BIG2-Nsi-Not with NsiI (base 2986 of BIG2) and
NotI, was ligated to BIG2-Nde-Xba which had been digested
with NsiI and NotI. The subcloned plasmid DNA was
named BIG2-Full.
BIG2 Synthesis and Purification--
BIG2-Full, excised with
NdeI and NotI, was ligated to the baculovirus
transfer vector pAcHLT-C with the His6 sequence encoded at
its NH2 terminus. A sample (2 µg) and 0.5 µg of
BaculoGold DNA (Pharmingen) were mixed with 2 × 106
Sf9 cells in 3 ml of TNM-FH Insect medium (Pharmingen). After incubation at 27 °C for 5 days, a sample (10 µl) of the cell
supernatant (Sup 1) was added to 1 × 105 Sf9
cells in 3 ml of medium followed by incubation for 5 days at 27 °C
before the supernatant was collected (Sup 2). Sf9 cells (2 × 107) were added to 1 ml of Sup 2 and 30 ml of TNM-FH.
After 5 days at 27 °C, cells were harvested by centrifugation,
suspended in 1 ml of ice-cold 10 mM sodium phosphate (pH
8.0) and 100 mM NaCl containing 8 µg of benzamidine
hydrochloride, 0.5 mM AEBSF, 5 µg of phenanthroline, 5 µg of aprotinin, and 5 µg of leupeptin, placed on ice for 45 min,
and finally lysed by freezing and thawing twice. Clear lysate was
separated from cellular debris by centrifugation (20,000 × g, 15 min). The lysate (1 ml) was mixed with 0.3 ml of
nickel-nitrilotriacetic acid agarose (Qiagen). After 1 h at 4 °C, the affinity matrix was washed five times with 1-ml portions of 5 mM imidazole and 50 mM sodium phosphate
(pH 8.0) in 300 mM NaCl, 10% glycerol, and 0.5 mM AEBSF. Bound protein was eluted with 100 mM
imidazole and 50 mM sodium phosphate (pH 6.0) in the same
solution of 300 mM NaCl, 10% glycerol, and 0.5 mM AEBSF. The eluted protein was dialyzed against 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM NaN3, 1 mM dithiothreitol, 0.25 M sucrose, 5 mM MgCl2, 0.5 mM AEBSF, and 30 mM NaCl overnight at
4 °C.
Northern Blot--
Prehybridization and hybridization with a
human Multiple Tissue Northern (MTN) (CLONTECH)
were carried out according to the manufacturer's instructions. For
prehybridization, ExpressHyb solution (CLONTECH)
was used at 60 °C for 30 min. DNA probes, a 382-bp PCR product
representing bases 121-502, in the 3'-untranslated region of BIG2 and
a 467-bp PCR product corresponding to bases 715-1181 in the coding
region of BIG1 were labeled with [
-32P]dCTP using a
random primed DNA labeling kit (Roche Molecular Biochemicals). After
hybridization, membranes were washed at room temperature with 2 × SSC and 0.05% SDS for 30 min, 2 × SSC and 0.1% SDS at 50 °C
for 20 min, and 1 × SSC and 0.1% SDS at 50 °C for 10 min. For
hybridization with glyceraldehyde-3-phosphate dehydrogenase mRNA,
20 pmol of oligonucleotide (ACACAAATTCGAAGCTAAATAAAGCCGAGAGCTGGTAGT) was labeled with [
-32P]dATP using 10 units of
polynucleotide kinase (Roche Molecular Biochemicals). 1.5 × 107 cpm of this or other labeled probe was used in 10 ml of
ExpressHyb solution. Membranes were washed at room temperature in
2 × SSC and 0.05% SDS for 40 min 2 × SSC and 0.1% SDS at
42 °C for 15 min. Autoradiography film was exposed overnight at
80 °C.
Chromosomal Localization--
The NIGMS human/rodent somatic
cell hybrid panel 2, version 3, containing DNA samples from human
IMR-91 cells (NAIMR91), Chinese hamster RJK88 cells, and mouse 3T6
cells, was purchased from the Corielle Institute for Medical Research.
Taq DNA polymerase (5 units) was used to prepare all DNA
probes for chromosomal localizations. To construct the BIG1 probe, PCR
R-1 was carried out with 2 µl of human/rodent hybrid cell genomic DNA
as template, the forward primer 5'-CATTTACATCCCTCTGCTCTTT-3' (which
starts 24 bp downstream of the termination codon), and reverse primer
5'-TTTCTTCTTCTCTCCCCACTCC-3' (which starts 425 bp downstream of the
termination codon) for 30 cycles of 94 °C for 30 s, 55 °C
for 30 s, and 72 °C for 30 s (total volume 20 µl). An
intron from BIG2 was amplified from 200 ng of human genomic DNA
(CLONTECH), with forward primer CL2-A (bases
2359-2385) and reverse primer (bases 2591-2568), in a volume of 100 µl for 30 cycles of 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 2 min. The single PCR product was sequenced to verify the
presence of the 622-bp intron at position 2533 in the coding region and
ligated into pCR2.1. Human/rodent hybrid cell genomic DNA (2 µl) was
used as template in PCR R-2 (total volume 20 µl) with forward primer
CL2-A and reverse primer 5'-ACTCCTAAATCCTCCCAACC-3' (bases 533-514 of
the intron) for 30 cycles of 94 °C for 1 min, 55 °C for 1 min,
and 72 °C for 1 min.
Assay for GEP Activity--
GEP stimulation of
[35S]GTP
S binding to ARF was assayed using a rapid
filtration procedure. Partially purified ARF (predominantly ARFs 1 and
3) was prepared from bovine brain cytosol (21). Assays, which were
incubated for 20 min at 25 °C, contained 4 µM
[35S]GTP
S (5 × 106 cpm), 20 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 3 mM MgCl2, 1 mM EDTA, 50 µg of
bovine serum albumin, and 10 µg of phosphatidylserine with ARF
substrate and GEP preparation as indicated (final volume, 50 µl).
Assays were terminated by transfer to nitrocellulose filters (Millipore
Corp.) followed by six washes, each with 2 ml of ice-cold buffer
containing 20 mM Tris-HCl (pH 8.0), 100 mM
NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 3 mM MgCl2. Filters were dissolved in
scintillation fluid for radioassay. Data presented are means ± S.E. of values from triplicate determinations.
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RESULTS |
Human BIG1 and BIG2 cDNAs--
BIG2 was initially purified as
part of a macromolecular complex of ~670 kDa from bovine brain. After
separation of complex components by SDS-polyacrylamide gel
electrophoresis, proteins of ~200 (p200) and 190 kDa exhibited
BFA-inhibited ARF GEP activity (22). Sequences of peptides from p200
were used as the basis for cloning the bovine cDNA for BIG1.
Cloning of human BIG1 and BIG2 cDNAs is described under
"Experimental Procedures." The cDNA for human BIG2 encodes a
protein of 1,785 amino acids with a calculated molecular weight of
202,000. Both human and bovine BIG1 are somewhat larger with 1,849 amino acids and molecular weights of 209,000.
Sequences of nine peptides produced by tryptic digestion of the p190
that had been purified from bovine brain (22) are included in the
deduced amino acid sequence of BIG2 (Table
I). Seven are identical. In peptide 2 (19 amino acids), a phenylalanine in the human clone is tyrosine in the
bovine, and in peptide 8 (9 amino acids), an alanine in the bovine
sequence is proline in the human. Both differences are considered
conservative. Peptides 2 and 3 are from the Sec7 domains.
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Table I
Amino acid sequence of nine peptides from BIG2
Shown is the deduced sequence from the human brain BIG2 cDNA. Below
peptides 2 and 8 are shown only those amino acids that differ in the
tryptic peptides from purified bovine brain BIG2 (22).
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The deduced amino acid sequence of human BIG2 is aligned with those of
human and bovine BIG1 in Fig. 1. BIG2,
which is overall 74% identical to human BIG1, is 90% identical in the
190 amino acids of the Sec7 domain (Fig.
2). The BIG2 Sec7 domain is only ~50%
identical to the Sec7 domains of the BFA-insensitive cytohesins B2-1,
ARNO, and GRP1 (which are themselves 85-90% identical), slightly less
so to those from yeast Sec7 and EMB30, and only 34% identical to the
Sec7 sequence from yeast Gea1 (Table II). Outside of the Sec7 domain, BIG1 and BIG2 have other large regions of
>70% identity, much greater overall similarity than, for example, to
the yeast Sec7 itself (Fig. 3).

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Fig. 1.
Deduced amino acid sequences of BIG1 and
BIG2. The sequence of human BIG2 is aligned with those of human
(h) and bovine (b) BIG1 (22). Dots
denote identical amino acids. Conservative differences from BIG2 are in
capital letters and nonconservative in lowercase.
Hyphens indicate gaps; Sec7 domains are in
boldface. Conservative replacements are A, G, P, S, T/I, L,
M, V/H, K, R/F, W, Y/D, E, N, Q. Alignment was generated using
GeneWorks 2.5.1.
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Fig. 2.
Alignment of deduced amino acid sequences of
Sec7 domains of ARF GEPs. The amino acid sequences are presented
as in Fig. 1 for hBIG2, hBIG1, ySec7 (14), EMB30 (12), yGea1 (8), hB2-1
(9), hARNO (10), and mGRP1 (11). Alignments were produced by GeneWorks
2.5.1 and adjusted by inspection.
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Table II
Percentage identity and similarity of amino acid sequences of Sec7
domains of nine proteins with ARF GEP activity
Percentage identities are shown above and similarities below the
diagonal. A, G. P, S, T/I, L, M, V/H, K, R/F, W, Y/D, E, N, Q
were considered as conservative differences for calculation of
similarity. References for sequences are in the legend for Fig. 2.
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Fig. 3.
Percentage identity of amino acid sequences
of hBIG2 and hBIG1 or ySec7. Residue numbers follow the name of
each protein with arrows indicating Sec7 domains. The
percentage of identical amino acids is recorded in each segment of the
diagram and the range indicated with shading from >90%
identity (black) to <20% (white). With the
exception of the Sec7 domains, divisions were arranged to maximize
identity of hBIG2 with hBIG1 (above) or ySec7 (below), thereby
accounting for the differences in segmentation of the two proteins.
Alignments were produced by GeneWorks 2.5.1. The Clustal program and
the following parameters were used: cost to open a gap = 5, cost
to lengthen a gap = 5, gap penalty = 3, number of top
diagonals = 5, window size = 5, PAM matrix = Identity,
k-tuple size = 1, consensus cutoff = 50%.
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Tissue Distribution of BIG1 and BIG2 mRNA--
On Northern
blots with poly(A)+ RNA from six human tissues (Fig.
4), a single 7.5-kb band hybridized with
BIG1 cDNA; a 9.4-kb transcript hybridized with the BIG2 probe. When
quantified by densitometry and expressed as a fraction of the density
of the corresponding glyceraldehyde-3-phosphate dehydrogenase mRNA,
BIG1 mRNA in placenta and lung was considerably more abundant than it was in heart, brain, kidney, or pancreas. The amounts of BIG2 mRNA were relatively similar in all of those tissues.

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Fig. 4.
BIG1 and BIG2 mRNA in human tissues.
Positions of size markers (in kb) are on the right. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
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Chromosomal Localization of BIG1 and BIG2--
The BIG1 cDNA
hybridized clearly with a single band (400 bp) from chromosome 8 (data
not shown) and with a band of the same size in the lane containing
NAIMR91 DNA (positive control). The BIG2 probe hybridized with a 700-bp
band from chromosome 20, which was also present in the total human DNA
(data not shown). No hybridization with DNA from other chromosomes was detected.
Function of Recombinant BIG2 Synthesized in Sf9
Cells--
To demonstrate the GEP activity of BIG2, it was synthesized
as a His6 fusion protein in Sf9 cells. After
purification on nickel-nitrilotriacetic acid agarose, a single protein
band corresponding to a molecule of about 200 kDa was detected on
SDS-polyacrylamide gel electrophoresis (Fig.
5, inset). At 25 °C, the
purified BIG2 accelerated GTP
S binding by rARF1 in a
concentration-dependent manner (Fig. 5). The basal rate of
binding was essentially constant for at least 30 min, although the
initial rate of BIG2-stimulated binding was apparently beginning to
decline by 5 min (Fig. 6). BIG2 itself did not bind GTP
S under these conditions (data not shown).

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Fig. 5.
Effect of His6 BIG2 on
[35S]GTP S binding by rARF1.
rARF1 (0.5 µg, 25 pmol) was incubated with the indicated amount of
BIG2 and 2 µM [35S]GTP S for 20 min at
25 °C. [35S]GTP S bound in the absence of BIG2 has
been subtracted. Data are means ± S.E. of values from triplicate
assays. This experiment was replicated twice. Inset,
silver-stained 8% gel after SDS-PAGE. Lane 1, total soluble
protein (10 µg) from Sf9 cells containing the
His6-BIG2; lane 2, His6-BIG2 (2 µg) after purification with Ni-NTA agarose; lane 3,
standard proteins with size (kDa) on the right.
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Fig. 6.
Effect of BIG2 on
[35S]GTP S binding by
rARF1. rARF1 (0.5 µg) was incubated with 2 µM
[35S]GTP S in 50 µl at 25 °C for the indicated
time without ( ) or with ( ) 0.25 µg of BIG2. Data are the
means ± S.E. of values from triplicate assays. Error
bars smaller than symbols are not shown. This
experiment was replicated twice.
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BFA at a concentration of 60 µg/ml (214 µM) inhibited
BIG2 activity ~70%, and 20 µg/ml caused ~25% inhibition (Fig.
7). B17, a structural analog of BFA which
does not interfere with Golgi function, did not inhibit BIG2 activity
(Table III).

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Fig. 7.
Effect of BFA on BIG2 stimulation of
GTP S binding by rARF1. rARF1 (0.5 µg),
2 µM [35S]GTP S, and 250 µg of BIG2
were incubated with the indicated concentrations of BFA in 50 µl at
25 °C for 20 min. GTP S bound is shown as percentage of that bound
in the absence of BFA. Data are the means ± S.E. of values from
triplicate assays. This experiment was replicated twice.
IC50 = 41.6 ± 8.5 µg/ml (mean ± S.E.,
n = 6).
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Table III
Effects of BFA and B17 on BIG2 stimulation of GTP S binding by
rARF1
Samples containing 0.5 µg of rARF1, 2 µM
[35S]GTP S, and 0.25 µg of BIG2 were incubated without or
with 3 µg of BFA or B17 for 20 min at 25 °C. Data are means ± S.E. of values from triplicate assays.
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As shown in Table IV, BIG2 was similarly
effective in enhancing GTP
S binding by rARF1, rARF5, and rARF6. It
activated the native ARF (chiefly ARF1 and ARF3) more effectively than
it did any of the recombinant nonmyristoylated ARFs.
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Table IV
Effect of BIG2 on [35S]GTP S binding by native ARF and
recombinant class I, class II, and class III ARFs
Samples (0.5 µg) of mixed native ARF, rARF1 (class I), rARF5 (class
II), or rARF6 (class III) with 2 µM
[35S]GTP S without or with 0.25 µg of BIG2 were incubated
at 25 °C for 20 min. Data are means ± S.E. of values from
triplicate assays.
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DISCUSSION |
BIG1 and BIG2, when initially identified as parts of a
~>600-kDa complex that was purified on the basis of its
BFA-inhibited GEP activity, were referred to as p200 and p190 (21).
Both are considerably larger molecules than the ~50-kDa
BFA-insensitive cytohesins (cytohesin-1, ARNO, GRP1). We suggest,
therefore, that they be named, respectively,
BFA-inhibited GEP1 and 2, i.e. BIG1 and BIG2. A cDNA for the latter protein, which
has now been cloned from a human brain library, is described here.
Sequences of nine tryptic peptides from the bovine p190 protein (21)
are almost identical (106 of a total of 108 amino acids) to that
deduced from the human BIG2 clone.
As shown in Fig. 3, the primary structures of BIG1 and BIG2 exhibit a
very high degree of identity, consistent with the notion that they may
be representatives of a distinct branch of the BFA-inhibited, 200-kDa
GEP family. In all family members the Sec7 domain constitutes only a
small fraction of the entire molecule, although there are clearly some
other elements of the structure which influence its GEP activity (16,
24). There are several regions outside of the Sec7 domains with
sequences that are 70-89% identical in BIG1 and BIG2 and very likely
have similar functions, which remain to be determined. The
corresponding regions of other proteins of the GEP family
(e.g. Sec7) presumably enable them to function in somewhat
different pathways, involving specific interactions with a different
assortment of proteins and other molecules. In addition to anchoring,
scaffolding, and/or adapter roles, these proteins may have other
enzymatic activities. The number of recognized ARF GEPs has increased
dramatically in the last 2 years and far exceeds the five or six known
ARFs (7). Clearly, we are just beginning to glimpse the complexity of
organization and operation of the pathways in which they function. It
appears that the multiple GEPs may contribute in a major way to
accomplishing a specificity of ARF action in cells which, at present,
lacks a mechanistic explanation.
The regions of highest identity (90%) in BIG1 and BIG2 are the Sec7
domains, which in the yeast Sec7 protein are responsible for GEP
activity and its BFA inhibition (16). Similarly, the BFA-insensitive
GEP activities of ARNO (10) and cytohesin-1 (23) are properties of
their Sec7 domains. With fewer than 200 amino acids in the Sec7
domains, ~50% of which are identical in the BFA-sensitive and
-insensitive GEPs, the residues responsible for BFA inhibition should
soon be identified. By comparing systematically the activities of
cytohesin-1 and its Sec7 domain with a variety of ARF-related proteins,
it was established that some determinants of the substrate specificity
of cytohesin-1, which is much more restricted than that of its Sec7
domain, must reside elsewhere in the molecule (24). This seems likely
to be true also for the other ~50-kDa BFA-insensitive GEPs, ARNO (10)
and GRP1 (11). Whether or not it applies to the ~200-kDa
BFA-inhibited GEPs remains to be determined.
The two families of GEPs also differ structurally in the presence of
pleckstrin homology (PH) domains in the cytohesin-1 family. PH domains,
which participate in specific protein-phospholipid and protein-protein
interactions, are critical regions in nucleotide-exchange proteins for
many GTPases and other regulatory molecules (25). Whereas cytohesin-1
and ARNO (and their PH domains) preferentially bind
phosphatidylinositol 4,5-bisphosphate (26), GRP1 has a higher affinity
for phosphatidylinositol 3,4,5-trisphosphate (27). Each
phosphoinositide increased the activity of the respective GEP
dramatically, presumably because it concentrates the protein (along
with the ARF substrate) at a membrane surface (26). PH domains have not
been recognized in the larger BFA-sensitive GEPs, although their
activity is also influenced by specific phospholipids.
With the recent report (28) that activated ARF induces binding of
spectrin and ankyrin isoforms to Golgi membranes, it is tempting to
speculate that an ARF GEP could be part of a soluble complex containing
those and perhaps other anchoring or adapter molecules involved in a
spectrin-ankyrin (or analogous) assembly that is important for
structure and function in vesicular trafficking pathways. It was
suggested that phosphatidylinositol 4,5-bisphosphate, which is bound
preferentially by the PH domain of the Golgi-specific
I
*
spectrin, is one site of spectrin attachment to the membrane. A second
is in a repeat sequence near the spectrin NH2 terminus. Because ARF enhancement of spectrin binding was not dependent on its
activation of phospholipase D or recruitment of coat proteins to the
membrane surface, Godi et al. (28) concluded that it resulted from an ARF-induced increase in phosphatidylinositol 4,5-bisphosphate in Golgi membranes.
BIG1 and BIG2 were purified as components of a >600-kDa protein
complex. Through six steps of purification from bovine brain cytosol,
including (NH4)2SO4 fractionation,
chromatography on DEAE-Sephacel, hydroxylapatite, and Mono Q, followed
by precipitation at pH 5.8, the BFA-inhibited GEP activity remained
associated with a supramolecular complex (21). It was finally eluted in a relatively symmetrical peak from a column of Superose 6B at the
position of thyroglobulin (669 kDa). The active fractions contained
several proteins that were separated by SDS-polyacrylamide gel
electrophoresis, among them p200 and p190, the amounts of which were
judged to correlate best with activity. After elution from the gel and
renaturation, p200 exhibited BFA-inhibited GEP activity (21), as did
p190 (data not shown).
Although the other components of the isolated complex remain to be
identified, they seem likely to be of importance in the process and/or
regulation of vesicular transport. It has, of course, not been
established that the two GEPs, in fact, reside together in the same
supramolecular structure because it appears probable that the purified
preparation was a heterogeneous population of complexes, derived,
obviously, from a heterogeneous population of cells. In the absence of
other information, it seems plausible to consider that BIG1 and BIG2
may be components of different but similar complexes, with analogous
compositions and parallel functions, probably in different
intracellular pathways and/or in different cells. Separation from the
preparation of a single, homogeneous macromolecular complex so that its
component proteins can be characterized is clearly critical to
understanding the relationship between the two very similar, albeit
different, BIG gene products, their functions, and their
interactions with other molecules, both within and outside of the complex.