Molecular Cloning and Functional Characterization of
Brefeldin A-ADP-ribosylated Substrate
A NOVEL PROTEIN INVOLVED IN THE MAINTENANCE OF THE GOLGI
STRUCTURE*
Stefania
Spanfò
,
Maria Giuseppina
Silletta
,
Antonino
Colanzi,
Saverio
Alberti,
Giusy
Fiucci,
Carmen
Valente,
Aurora
Fusella,
Mario
Salmona§,
Alexander
Mironov,
Alberto
Luini, and
Daniela
Corda¶
From the Istituto di Ricerche Farmacologiche "Mario Negri",
Department of Cell Biology and Oncology, Consorzio Mario Negri Sud,
66030 Santa Maria Imbaro (Chieti) and the § Department of
Biochemistry and Molecular Pharmacology, Via Eritrea 62, 20157 Milano,
Italy
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ABSTRACT |
Brefeldin A (BFA) is a fungal metabolite that
disassembles the Golgi apparatus into tubular networks and causes the
dissociation of coatomer proteins from Golgi membranes. We have
previously shown that an additional effect of BFA is to stimulate the
ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa (brefeldin
A-ADP-riboslyated substrate (BARS)) and that this effect greatly
facilitates the Golgi-disassembling activity of the toxin. In this
study, BARS has been purified from rat brain cytosol and
microsequenced, and the BARS cDNA has been cloned. BARS shares high
homology with two known proteins, C-terminal-binding protein 1 (CtBP1)
and CtBP2. It is therefore a third member of the CtBP family. The role
of BARS in Golgi disassembly by BFA was verified in permeabilized cells. In the presence of dialyzed cytosol that had been previously depleted of BARS or treated with an anti-BARS antibody, BFA potently disassembled the Golgi. However, in cytosol complemented with purified
BARS, or even in control cytosols containing physiological levels of
BARS, the action of BFA on Golgi disassembly was strongly inhibited.
These results suggest that BARS exerts a negative control on Golgi
tubulation, with important consequences for the structure and function
of the Golgi complex.
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INTRODUCTION |
The Golgi complex, which plays a key role in intracellular
trafficking and sorting, is composed of a constellation of stacks of
flat cisternae bound together through tubular-reticular connecting zones into an overall ribbon-like shape. There has always been great
interest among cell biologists in understanding the molecular mechanisms responsible for Golgi architecture and dynamics.
Unfortunately, although significant progress has recently been made by
studying the process of disassembly and reassembly of the Golgi complex that occurs during treatments with toxins such as ilimaquinone (1) and
brefeldin A (BFA)1 (2) or
during mitosis (3), the present knowledge of these processes is still fragmentary.
The focus of this study is on the molecular factors involved in the
Golgi disassembly induced by BFA, a fungal toxin that causes the
massive transformation of Golgi stacks into a tubular-reticular network. The effects of BFA have been attributed to at least two mechanisms. One is the release of coat proteins, including the coatomer
(a major protein complex involved in coat protein I (COPI)-coated vesicle formation) and the small GTP-binding protein ARF
(ADP-ribosylation factor) (4, 5) from Golgi membranes. The second
mechanism is the activation of the endogenous ADP-ribosylation of two
cytosolic proteins of 38 kDa (glyceraldehyde-3-phosphate dehydrogenase, a multifunctional protein involved in several cellular processes), and
50 kDa (BARS, a protein of unknown function) (6-8). The role of
coatomer in preserving the Golgi structure has been attributed to its
function as a major membrane scaffold protein (2), but the significance
of the ADP-ribosylation of BARS and glyceraldehyde-3-phosphate dehydrogenase is less well understood. In a previous report, we provided evidence that the cytosol contains factors that prevent Golgi
disassembly by BFA. Because the activity of such factors appeared to be
abolished by ADP-ribosylation, we suggested that the inhibitory
components of the cytosol might be identical with the ADP-ribosylation
substrates, glyceraldehyde-3-phosphate dehydrogenase and BARS (9).
In this study, we have undertaken the purification and molecular
cloning of BARS. We report the primary sequence of BARS and the
characterization of the function of this protein in the Golgi disassembly induced by BFA. BARS appears to be involved in controlling the equilibrium between tubular and stacked structures in the Golgi complex.
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EXPERIMENTAL PROCEDURES |
ADP-ribosylation Assay--
Cytosol and membranes were prepared
from fresh rat brains (300-400-g male Sprague-Dawley rats) according
to Malhotra et al. (10), except that the 60% ammonium
sulfate precipitation was omitted. Aliquots were frozen in liquid
nitrogen and stored at
80 °C. The ADP-ribosylation assay included
5 mg/ml cytosol (substrate source), 1.2 mg/ml membranes (enzyme
source), and 60 µg/ml BFA incubated for 2 h with 250 µM NAD+/[32P]NAD+,
as described previously (6).
BARS Purification--
All purification steps (see Table I) were
performed at 4 °C by fast protein liquid chromatography (Amersham
Pharmacia Biotech) starting from ADP-ribosylated cytosol (240 mg)
precipitated by 35% ammonium sulfate. The amount of BARS present at
each step was evaluated by 10% SDS-PAGE followed by autoradiography
(Instant Imager, Packard). The precipitate was applied to a hydrophobic column (Phenyl-Sepharose H.P., Amersham Pharmacia Biotech) followed by
a hydroxylapatite column (Bio-Gel HT, Bio-Rad). The recovered fractions
were concentrated (Centriplus Concentrators 10, Amicon), and loaded
onto a gel filtration column (Superose 12, Amersham Pharmacia Biotech).
The BARS-containing fractions (eluted at an apparent molecular mass of
170 kDa) were concentrated and subjected to 2D isoelectric
focusing-SDS-PAGE (11). The radiolabeled spots stained by Coomassie
Blue were subjected to in situ tryptic digestion. Peptides
were separated by reverse-phase high pressure liquid chromatography and
sequenced by the Protein Structure Laboratory, University of
California, Davis. The obtained peptide sequences were compared with
sequence data banks using the BLAST similarity search algorithm at the
National Center for Biotechnology Information site.
Polyclonal Antibodies--
Peptides were synthesized as
described previously (12). After the collection of preimmune sera, male
HY/CR rabbits were immunized with the BARS synthetic peptide conjugated
through the MAP system (Novabiochem) via subcutaneous injections,
following a previously described procedure (13). IgGs were purified by affinity chromatography on protein A-Sepharose (Amersham Pharmacia Biotech). Anti-peptide-specific antibodies were purified by affinity chromatography on peptide-coupled NHS-activated columns (HiTrap, Amersham Pharmacia Biotech).
Construction of BARS Probes and Screening of a Rat Brain cDNA
Library--
mRNA was obtained from male rat brain using a
Quickprep mRNA purification kit (Amersham Pharmacia Biotech)
according to the manufacturer's instructions. Poly(A) mRNA was
reverse-transcribed with Moloney murine leukemia virus reverse
transcriptase using d(T)18 or random hexameric primers
(First Strand cDNA synthesis kit, Amersham Pharmacia Biotech). The
resulting cDNA first strand was subjected to polymerase chain
reaction. Degenerate primers were designed on the basis of the
sequences of peptide 36-2 (probe 1 sense primer,
GCHACHGTGGCHTTYTGYTGYGA), peptide 61 (probe 1 antisense primer,
CRTAGAAVAGCACGTTRAABCC), peptide 55-1 (probe 2 sense primer,
TGYGTGACHCTSCAYTGYGG), and peptide 55-2 (probe 2 antisense primer,
CCYTCCACAGCDGCDGGDAT). A
ZAPII rat brain cDNA library
(Stratagene) was screened essentially as indicated by the manufacturer.
The polymerase chain reaction products and cDNA inserts were
subjected to automated nucleotide sequencing (Nucleic Acid Facility,
Istituto Dermopatico Dell' Immacolata, Rome).
Transient Transfection of BARS (CtBP3/BARS, see below) in COS7
Cells--
The 1290-base pair coding sequence of BARS cDNA was
amplified through polymerase chain reaction using specific primers and cloned in EcoRI- and NotI-cleaved pcDNA3
expression vector (Invitrogen) to obtain pcDNA3-BARS. COS7 cells at
50-70% confluence were transfected with pcDNA3 or pcDNA3-BARS
using LipofectAMINE (Life Technologies, Inc.).
Fluorescence Microscopy--
Experiments were performed as
described previously (9).
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RESULTS |
Purification, Microsequencing, and Molecular Cloning of
BARS--
The purification strategy of BARS is summarized in Table
I. Cytosol was prepared from rat brain,
where BARS is relatively abundant (Fig.
1A). Prior to purification,
the cytosolic BARS was ADP-ribosylated with
[32P]NAD+ in the presence of BFA. It was then
precipitated with 35% ammonium sulfate and passed through a series of
chromatographic columns. After each step, the
[32P]ADP-ribosylated protein was identified by SDS-PAGE
and autoradiography (see "Experimental Procedures"). This procedure
achieved an overall 900-fold purification with a 40% yield. The last
step was a gel filtration column, from which the protein eluted with an
apparent molecular mass of about 170 kDa. BARS was then concentrated
and subjected to 2D isoelectric focusing electrophoresis. Three well resolved spots (at a molecular mass of 46 kDa and isoelectric points of
6.05, 6.10, and 6.15; see Fig. 1, B and C
(inset)) that were clearly
[32P]ADP-ribosylated and Coomassie Blue-stained were
isolated and subjected to microsequencing after in situ
digestion (see "Experimental Procedures"). Nine non-overlapping
peptides, corresponding to internal sequences, were obtained in three
separate preparations (Fig.
2A). These sequences were
compared with protein data bases. The comparison indicated strong
homology with members of the CtBP family of proteins (14-18). The
alignment of the BARS peptides with one such protein, CtBP1, was used
to design two pairs of degenerate primers, which were then used to
amplify a pool of rat brain mRNA by reverse
transcriptase-polymerase chain reaction. The two stretches of DNA thus
obtained were used as probes to screen a rat brain cDNA library.
Five clones, strongly hybridizing with both probes, were isolated and
sequenced. The longest of these cDNA clones was 2430 base pairs
long. It contained a full-length ORF coding for a 430-amino acid
protein (predicted mass 47 kDa). The deduced amino acid sequence (BARS;
GenBankTM/EBI accession number AF067795) included the nine peptides
(Fig. 2A) obtained from the microsequencing, with only one
mismatch at residue 175 (Gly instead of Ser). The cloned protein was
compared with known protein data bases. As expected, it was found to be
highly similar to the two known mammalian members of the CtBP family
(CtBP1 and CtBP2). Both of these proteins have been cloned in both
human and mouse (14-16). The identity was 97% with human and mouse
CtBP1 (GenBankTM/EBI accession numbers U37408 and AJ010483,
respectively) and 79% with human and mouse CtBP2 (GenBankTM/EBI
accession numbers AF016507 and AF059735, respectively). The only
significant region of diversity between CtBP1 and BARS was the
N-terminal stretch (Fig. 2B), where the two proteins differ
markedly in sequence and length. At the nucleotide level, the BARS
cDNA was 94% identical to mouse CtBP1 (86% to human CtBP1) and
72% identical to human and mouse CtBP2. Interestingly, a long sequence
at the 5' end of the BARS cDNA was absent in the CtBP1 and CtBP2
cDNAs. This sequence was present in two mouse sequences
(GenBankTM/EBI accession numbers AA212717 and AI006262) in the EST
data base. In fact, in the AA212717 sequence, a 449-base pair-long
region (which included the 5' untranslated region, the ATG start codon, and 273 base pairs of the BARS sequence) was 98% identical to the BARS
cDNA. This strongly indicates that BARS is a third form of CtBP
that exists both in rat and in mouse. BARS and CtBP1 may be encoded by
an alternatively spliced gene or by two different genes. Studies are in
progress to clarify this point. Provisionally, we will call the new
protein CtBP3/BARS.
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Table I
Chromatographic purification of BARS from rat brain cytosol
BARS was ADP-ribosylated with [32P]NAD+ prior to
purification. The 32P-labeled protein was quantified in each
chromatographic fraction by SDS-PAGE followed by electronic
autoradiography (Packard Instant Imager). ADP-ribosylated cytosol was
subjected to precipitation with 35% ammonium sulfate, then dissolved
in Buffer A (25 mM Hepes, pH 8, 5% glycerol, 0.5 M ammonium sulfate, 1 mM dithiothreitol),
applied to a Phenyl-Sepharose H.P. column, and eluted with a decreasing
linear gradient of ammonium sulfate in Buffer B (25 mM
Hepes, pH 8, 5% glycerol, 1 mM dithiothreitol). Fractions
containing BARS were applied to a hydroxylapatite column
pre-equilibrated in Buffer B and were eluted with an increasing linear
gradient of sodium phosphate in Buffer C (25 mM Hepes, pH
8, 5% glycerol, 0.2 M sodium phosphate, 1 mM
dithiothreitol). Fractions containing BARS were pooled, concentrated,
and applied to a gel filtration column (Superose 12 H.R.)
pre-equilibrated with Buffer D (25 mM Hepes, pH 8, 5%
glycerol, 150 mM NaCl). Superose 12 H.R. was calibrated
with the following molecular mass standard proteins (Bio-Rad):
-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and
vitamin B12 (1.350 kDa). The void volume of the column was
determined with blue dextran.
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Fig. 1.
Purification and 2D SDS-PAGE analysis of
BARS. A, cytosols (250 µg) from rat lung (lane
1), liver (lane 2), spleen (lane 3), and
brain (lane 4), and control membranes (lane 5)
were [32P]ADP-ribosylated in the presence of BFA and
separated by SDS-PAGE (see "Experimental Procedures").
B, the brain tissue was the richest in BARS and was employed
as the source for purification (see text and Table I). A 900-fold
purified preparation of BARS was analyzed by 2D isoelectric
focusing-SDS-PAGE and stained by Coomassie Blue. The overlapping
radiolabeled- and Coomassie Blue-stained spots (C
inset show a magnification of the area of interest) were subjected
to microsequencing as described under "Experimental
Procedures."
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Fig. 2.
Microsequenced peptides and deduced amino
acid sequence of CtBP3/BARS. Panel A shows the sequence of 9 peptides obtained by microsequencing of the spots in Fig.
1C. Panel B shows the alignment of the CtBP3/BARS
N terminus with that of the known mammalian members of the CtBP family.
Identical residues are boxed. The GenBankTM/EBI accession
numbers for the sequences reported are the following: AF067795 (BARS);
AA212717 (mouse EST data base sequence); AJ010483 (mouse CtBP1); U37408
(human CtBP1); AF059735 (mouse CtBP2); and AF016507 (human
CtBP2).
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To verify that the cloned rat CtBP3/BARS is indeed BARS (operationally
defined as the 50-kDa cytosolic substrate ADP-ribosylated by BFA), we
used several experimental approaches. First, CtBP3/BARS was
overexpressed in COS7 cells. The amount of BARS was then measured in
the transfected population. If CtBP3/BARS is BARS, the
ADP-ribosylatable 50-kDa protein should be increased after
transfection. Fig. 3A shows
that indeed the cytosol of CtBP3/BARS-transfected COS7 cells contains a
much larger amount of the 50-kDa protein ADP-ribosylated by BFA than of
mock-transfected controls. This finding indicates that CtBP3/BARS is
BARS. To verify whether the other CtBPs might have BARS properties,
human CtBP1 was also overexpressed in COS7 cells, and the cytosol from
these cells was ADP-ribosylated. Again, high levels of the 50-kDa
ADP-ribosylatable protein were found (data not shown). Similar data
were obtained in cells overexpressing mCtBP2 (data not shown), which
indicates that indeed the known CtBPs are BARS. Second, antibodies
against CtBP proteins were obtained. Two were generated against
peptides VSQAVALR (present also in CtBP1) and SVEQIREVASGAARIR (present
also in CtBP1 and CtBP2), corresponding to regions 174-181 and
147-162 of CtBP3/BARS (denominated anti-BARS/9 and anti-BARS/SN1
antibodies, respectively). A third antibody, raised against the whole
human CtBP1 protein (anti-CtBP1) (14), was obtained as a gift from Dr.
Chinnadurai (St. Louis University Medical Center, St. Louis, MO). All
of these antibodies gave detectable signals in brain cytosol in
immunoblotting experiments, and the antibody against the whole CtBP1
protein also efficiently immunoprecipitated all of the mammalian CtBPs (not shown). Brain cytosol was ADP-ribosylated and used to test whether
the 32P-labeled cytosolic BARS was recognized by antibodies
raised against the CtBP proteins. Fig. 3B, lane 4, shows
that the anti-BARS/SN1 antibody recognized a 50-kDa band in Western
blots. The band is more evident after enrichment of BARS by ammonium
sulfate precipitation of the cytosol (Fig. 3B, lane
5). That this band corresponds to BARS is indicated by the fact
that it precisely co-runs in SDS-PAGE with brain BARS (shown in Fig.
3A, lane 4) in both the presence and absence of
urea (we have previously reported that BARS exhibits the urea shift in
SDS-PAGE; see Ref. 7). Similar results were obtained with the
anti-BARS/9 antibody. Moreover, the anti-whole CtBP1 antibody
quantitatively immunoprecipitated [32P]BARS from the
[32P]ADP-ribosylated cytosol (Fig. 3C).
Finally, we used the known property of CtBP proteins of binding to the
C terminus of the adenoviral protein E1A-243R (14, 19). A bacterially
expressed GST-(C-ter)-E1A fusion protein (20) was linked to
glutathione-agarose beads and used to selectively extract E1A
C-terminal-binding proteins from brain cytosol
[32P]ADP-ribosylated in the presence of BFA. If the
ADP-ribosylated BARS in this cytosol is a protein of the CtBP family,
it should bind to E1A. Indeed, 70% of the
[32P]ADP-ribosylated cytosolic BARS was found to
specifically and tightly bind to the GST-(C-ter)-E1A (data not shown).
Conversely, it was shown that cytosolic proteins that bind to the
GST-(C-ter)-E1A are ADP-ribosylated in the presence of BFA (data not
shown). Thus, the above collective evidence shows that the cloned rat
CtBP3 is a BARS and suggests that all of the CtBPs have the features of
BARS proteins. These proteins will thus henceforth be referred to as
CtBP/BARS.

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Fig. 3.
BFA-dependent ADP-ribosylation of
CtBP3/BARS in COS7 cells and immunoprecipitation of CtBP/BARS in rat
brain cytosol. Cytosols prepared from CtBP3/BARS-transfected COS7
cells were [32P]ADP-ribosylated, analyzed by SDS-PAGE,
transferred to nitrocellulose, and revealed either by autoradiography
(panel A) or by an anti-CtBP/BARS antibody (anti-BARS/SN1)
with peroxidase-based detection (panel B) (see
"Experimental Procedures"). The lanes contain 10 µg of
cytosolic proteins from: untransfected COS7 cells (Untrans.)
(lane 1); mock-transfected cells (Mock-trans.)
(lane 2); CtBP3/BARS-transfected cells
(BARS-trans.) (lane 3); 100 µg of rat brain
cytosol, for comparison (Control) (lane 4); and
100 µg of rat brain cytosol after precipitation with 35% ammonium
sulfate (A.S. Prec.) (lane 5). The
[32P]ADP-ribosylated CtBP3/BARS in lane 3 is
clearly recognized by the anti-BARS/SN1 antibody. Panel C
shows CtBP/BARS that has been [32P]ADP-ribosylated and
then immunoprecipitated from rat brain cytosol with the anti-CtBP1
antibody and revealed by SDS/PAGE followed by autoradiography. The
ADP-ribosylated CtBP/BARS is quantitatively recovered in the
immunoprecipitate. Lane 1 shows the crude ADP-ribosylated
cytosol prior to precipitation (Control); lane 2,
the immunoprecipitate (Pellet); and lane 3, the
supernatant left after immunoprecipitation (Supernat.). The
higher molecular mass (46 kDa) band is CtBP/BARS and the 38-kDa band is
glyceraldehyde-3-phosphate dehydrogenase (see Ref. 6). Data are
representative of at least three independent experiments.
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The Role of CtBP/BARS in the Golgi Disassembly Induced by
BFA--
We have previously shown that brain cytosol contains factors
that prevent the Golgi disassembly induced by BFA. We have also proposed that BARS is one of these factors, based on the fact that the
ADP-ribosylation of BARS by BFA correlates with the loss of inhibitory
activity of the cytosol on Golgi disassembly (9). This hypothesis can
now be tested directly by exploiting the molecular tools
(anti-CtBP/BARS antibodies and purified CtBP/BARS) developed in the
course of this study. To assay Golgi disassembly by BFA we used
streptolysin O-permeabilized rat basophilic leukemia cells, which
maintain a near normal Golgi morphology and respond to BFA under
appropriate conditions in the presence of cytosol (9). The position and
overall morphology of the Golgi was monitored by immunofluorescence
using an antibody to mannosidase II, a well characterized Golgi
resident protein. Cytosol was prepared from rat brain as described (9)
and dialyzed extensively to remove NAD+. Because
NAD+ is the ADP-ribose donor in all ADP-ribosylation
reactions, its removal is absolutely necessary in these experiments to
prevent the ADP-ribosylation of BARS and, therefore, the possible
consequent inactivation of this protein (see above and Ref. 9). Fig.
4, B and C,
illustrates the importance of removing NAD+ (see also Ref.
9). In dialyzed, NAD+-deprived cytosol, BFA lost its
ability to disassemble the Golgi (compare panel A, showing a
"normal" bright central Golgi spot, with panel F,
showing the massive Golgi-disassembling action of the toxin in intact
cells). When 400 µM NAD+ was re-added to this
cytosol, BFA regained its activity. The same effect (regain of BFA
activity) was observed when cytosol was pre-ADP-ribosylated
(panels B and C, showing a completely diffuse
Golgi fluorescence), confirming that NAD+ functions here as
the ADP-ribosyl donor (see Ref. 9). These results are consistent with
the idea that ADP-ribosylation inactivates the inhibitory effect of
BARS on BFA (9). The role of CtBP/BARS in Golgi disassembly was then
tested directly by quantitatively immunodepleting dialyzed cytosol of
CtBP/BARS. The rationale of this experiment is that if CtBP/BARS is a
cytosolic factor that prevents the tubular transformation of the Golgi
by BFA, then depleting the cytosol of CtBP/BARS should greatly
facilitate the Golgi disassembling action of the toxin. Fig. 4,
D and E, shows that, indeed, although in control
(mock-depleted) cytosol BFA was inactive, in CtBP/BARS-depleted cytosol
it induced its full effect of Golgi disassembly. Thus, BARS is indeed
an inhibitor of the tubular transformation of the Golgi induced by BFA.
Notably, even though the extent of the disassembly by BFA under these
conditions was comparable with that induced by the toxin in intact
cells (shown in Fig. 4F for comparison), the toxin
concentrations required for activity were somewhat higher than those
effective in the intact system. Similar observations of loss of potency
of BFA in permeabilized cells have been reported several times
previously (see Ref. 9 and references therein). The origins of this
effect are still unclear. Perhaps inhibitory factors other than BARS are present in the cytosol or permeabilization induces the partial loss
of a component needed for the Golgi response to BFA. Next, CtBP/BARS-depleted cytosol was complemented with CtBP/BARS, using the
chromatographically purified protein (a mixture of the cytosolic CtBPs/BARS) at a final concentration 5-fold higher than calculated to
be present in control cytosol. The effect of BFA on the Golgi was
nearly completely suppressed (Fig. 4G), again consistent
with an inhibitory effect of CtBP/BARS on Golgi disassembly.
Interestingly, in parallel experiments BARS did not inhibit another
effect of BFA, namely, the dissociation of coatomer proteins from the
Golgi complex (data not shown, see Ref. 9). This finding indicates that
BARS does not generically block the action of BFA, but rather it exerts
an opposing action on the Golgi tubular disassembly. Finally,
mock-depleted, NAD+-depleted cytosol was treated with
anti-BARS/9 antibody. If the antibody has neutralizing properties, it
should inhibit CtBP/BARS and thereby abolish the ability of the cytosol
to prevent the effects of BFA. Indeed, in cytosol treated with the
anti-BARS/9 antibody (Fig. 4H), BFA potently disassembled
the Golgi complex. Preimmune IgGs had no effect (data not shown).
Similar experiments were carried out to check the ultrastructure of the
Golgi complex under the above experimental conditions. The Golgi
maintained a nearly normal stacked structure in the presence of
dialyzed cytosol, whereas in CtBP/BARS-depleted cytosol or in cytosol
treated with anti-BARS/9 antibody it was transformed into a tubular
network or redistributed into the endoplasmic reticulum (not shown, see Ref. 9). Taken together, these findings demonstrate that the previously
reported ability of brain cytosol to inhibit the effects of BFA on
Golgi morphology is mediated largely, if not completely, by CtBP/BARS.
Whether this activity is shared by all three of the CtBPs/BARS or
belongs exclusively to one of the isoforms remains to be clarified.
Moreover, these results, together with our previous observations that
NAD+ rapidly induces the ADP-ribosylation of CtBP/BARS in
the presence of BFA and concomitantly abolishes the ability of dialyzed
cytosol to prevent the effect of BFA on Golgi disassembly (9), indicate that CtBP/BARS is inactivated by ADP-ribosylation.

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Fig. 4.
CtBP/BARS prevents the BFA effects on the
Golgi complex in permeabilized cells. RBL cells were permeabilized
with 1 unit/ml streptolysin O and incubated for 15 min at 32 °C in
the presence of 3.3 µg/ml BFA and one of the following: dialyzed
cytosol (A, Control Cyt.), dialyzed cytosol and 400 µM NAD+ (B, Cyt. + NAD+), pre-ADP-ribosylated cytosol (C,
Pre-ADP-Rib. Cyt.), mock-depleted cytosol
(D, Mock Depleted Cyt.), CtBP/BARS-depleted cytosol
(E, CtBP/BARS Depleted Cyt.), cytosol and purified CtBP/BARS
(G, Cyt.+ CtBP/BARS), or cytosol treated with the
affinity-purified anti-BARS/9 antibody (H, Cyt.+
anti-BARS Ab). Panel F shows the effects of
BFA in intact cells (Control Intact Cells) for comparison.
The cells were fixed and labeled with anti-mannosidase II antibody as
described previously (9). The slight lack of sharpness of the images is
because of the permeabilization and the fact that RBL cells are small
and roundish. Similar results were obtained in three independent
experiments.
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The Effect of ADP-ribosylation on the Physicochemical Properties of
CtBP/BARS--
To study how the ADP-ribosylation of CtBP/BARS might
inactivate its function in regulating Golgi architecture, we
investigated whether the ADP-ribosylated CtBP/BARS exhibits different
physicochemical properties than the native protein. Such differences
may be revealed by the chromatographic behavior of the protein. First,
the tendency of CtBP/BARS to associate with itself or other proteins
was examined by gel chromatography. The native protein eluted mainly in
fractions corresponding to a calculated molecular mass of 50 kDa, thus, most likely, it was in the monomer state. In contrast, as mentioned in
the description of the biochemical purification, the main peak of the
ADP-ribosylated protein shifted to a calculated molecular mass of 170 kDa (Fig. 5), which indicates that
ADP-ribosylation modulates the ability of CtBP/BARS to oligomerize.
Second, on a hydrophobic Phenyl H.P. column the native CtBP/BARS eluted
at lower salt concentrations than the ADP-ribosylated protein,
suggesting a different degree of hydrophobicity (not shown). Together,
these results indicate that ADP-ribosylation causes a conformational change in CtBP/BARS that results in homo- or hetero-oligomerization of
the protein and in an altered exposition of hydrophobic surfaces. Such
changes might explain the loss of activity of the protein in
antagonizing the effect of BFA on Golgi disassembly.

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Fig. 5.
Chromatographic properties of native and
ADP-ribosylated CtBP/BARS. Fractions (0.2 ml) were collected from
a Superose 12 H.R. 10/30 column and analyzed by autoradiography after
SDS-PAGE separation. Native CtBP/BARS (closed squares,
Native) mainly eluted as a monomer of approximately 50 kDa,
whereas the ADP-ribosylated form (open squares,
ADP-Ribosylated) elutes as a complex at 170 kDa. The
ADP-ribosylation assay was carried out either before (ADP-ribosylated
form) or after (native form) chromatographic separation of CtBP/BARS.
Data obtained by Western blot analysis using the anti-BARS/SN1 antibody
confirmed the same pattern of protein elution. Data are representative
of three independent experiments.
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DISCUSSION |
In this study, we have purified and cloned CtBP3/BARS, a protein
substrate of BFA-dependent ADP-ribosylation. The primary sequence of CtBP3/BARS is highly similar to that of CtBP1, a protein originally identified based on its property to bind the C terminus of
the transforming adenoviral protein E1A (14, 19). In fact, CtBP3/BARS
and CtBP1 are nearly identical throughout most of their sequence,
except that CtBP3/BARS lacks the 11 terminal amino acids of CtBP1.
Whether this finding represents a functionally significant difference
remains to be ascertained. While this work was in progress, another
mammalian homologue of CtBP, CtBP2, was cloned in human and mouse (15,
16), which also differs from CtBP1 mostly at the N terminus. Moreover,
at least two CtBP homologues have been reported in
Drosophila (17, 18). Thus, the newly cloned protein can be
considered as the third member of the mammalian CtBP family (see
"Results"). To indicate this fact, we have called the protein CtBP3/BARS. Interestingly, CtBP1 and -2 can also be considered BARS
because they are substrates of BFA-dependent
ADP-ribosylation.
We have previously reported that BARS behaves as a cluster of proteins
in 2D gels and that some of these proteins bind GTP (7). This is
consistent with the presence of several BARS isoforms and/or
post-translational modifications. CtBP3/BARS however is not a classical
GTP-binding protein, because its sequence does not have homology to
known G proteins, nor contains a canonical GTP-binding motif. This in
itself is not surprising because proteins that bind GTP but do not
possess this motif have already been reported (21). However,
preliminary binding studies using the recombinant CtBP3/BARS have so
far failed to reveal specific GTP binding (data not shown). The
explanation for this failure could be that either not all of the BARS
isoforms bind GTP, or an as yet undefined post-translational
modification of BARS is required for GTP binding.
The function of the mammalian CtBP2 protein and the
Drosophila CtBP have recently become partially understood.
These proteins appear to function in the regulation of transcription as
co-repressors interacting with a broad range of transcription factors.
This finding of course raises the intriguing question as to whether the
CtBP/BARS family might play two different roles, one in transcription and one in Golgi maintenance. In principle, it is possible that one of
the CtBPs/BARS might have evolved a radically different function than
that of other CtBPs/BARS and that such function might be limited to the
Golgi. This hypothesis seems unlikely, however, in view of the high
degree of homology among the CtBPs/BARS. The alternative is that the
CtBPs/BARS might have a dual role, one in the nucleus and one in the
cytoplasm. A multiple role for proteins ("moonlighting", see Ref.
22) is not a new concept. Indeed, the examples are numerous, and it has
even been proposed that "moonlighting" might be a common property
of proteins (22). What might be the significance of a dual role in the
case of CtBP/BARS? An attractive hypothesis is that this protein might
provide a link, during mitosis, between transcriptional events and
Golgi function. This conjecture would fit previous observations that the Golgi complex disassembles during mitosis (3) and that CtBPs/BARS
are phosphorylated during the mitotic phase of the cycle (19). Further
work is required to clarify this important point.
The central problem posed by the present findings in the context
of the issue of Golgi dynamics and architecture is what might be the
precise role of CtBP/BARS in Golgi maintenance. Our observation that
this protein antagonizes the tubular-reticular disassembly of the Golgi
complex by BFA suggests that the physiological role of CtBP/BARS is to
stabilize this organelle. The inhibition of BFA is not the result of a
trivial mechanism such as, for instance, direct binding and
neutralization of the toxin. This possibility is ruled out by the facts
that: (a) the concentrations of BFA used in our experiments
largely exceed (at least by 150-fold) those of BARS; and (b)
another effect of BFA, namely, the dissociation of coatomer proteins
from the Golgi complex, is not antagonized by BARS (see also Ref. 9).
Thus, BARS exerts a specific negative regulation on the tubular
transformation of the Golgi complex. Our working hypothesis is that the
physiological function of BARS is to exert a negative control on the
formation of Golgi tubules. This premise could explain the ability of
BARS to prevent the effect of BFA, and it might also have significant
consequences in the physiology of the secretory pathway. Tubules are
very abundant and dynamic structures, with a potentially important role
in traffic; however, their precise function is still obscure.
Elucidating the molecular mechanisms of action of CtBP/BARS might
provide new insights into the mechanisms controlling the dynamics of
Golgi tubules and the equilibrium between tubular and stacked
structures in the Golgi complex.
 |
ACKNOWLEDGEMENTS |
The authors thank Drs. G. Chinnadurai (St.
Louis University, St. Louis, MO) and C. Svensson (Uppsala University,
Uppsala, Sweden) for the generous gift of anti-CtBP antiserum and
GST-(C-ter)-E1A cDNA, respectively; C. Rossi (Consorzio Mario Negri
Sud) for help in raising rabbit antibodies; Dr. P. James (ETH-Zentrum,
Zurich) for initial help in protein sequencing; and Dr. C. P. Berrie for critical reading of the text. We also thank Dr. M. Pietrarelli and R. Zampone for collaborating in the purification of
BARS.
 |
FOOTNOTES |
*
This study was supported in part by the Italian Association
for Cancer Research (Milano, Italy), the Italian Foundation for Cancer
Research (Milano, Italy), Telethon Grant E.841 (Italy), and the Italian
National Research Council (Rome, Italy) Progetto Finalizzato
"Biotecnologie" Grant 97.01297.49.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF067795.
The first two authors contributed equally to this paper.
¶
To whom correspondence should be addressed. Tel.:
39-0872-570-353; Fax: 39-0872-570-412; E-mail:
corda{at}cmns.mnegri.it.
 |
ABBREVIATIONS |
The abbreviations used are:
BFA, brefeldin A;
BARS, BFA-ADP-ribosylated substrate;
CtBP, C-terminal-binding protein;
GST, glutathione S-transferase;
GST-(C-ter)-E1A, GST-E1A
fusion protein containing E1A C-terminal 44 amino acids;
2D, two-dimensional;
PAGE, polyacrylamide gel electrophoresis.
 |
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