From the Veterans Administration Medical Center and the Department
of Biological Chemistry, University of Michigan,
Ann Arbor, Michigan 48105
We previously identified a protein from rat liver
that binds CTP:phosphocholine cytidylyltransferase (CT). We have now
purified this protein (cytidylyltransferase-binding protein (CTBP))
from rat liver. The purification involved precipitation at pH 5 and extraction of the precipitate with buffer, followed by sequential chromatography on DEAE-Sepharose and butyl-agarose. Final purification was accomplished by either preparative electrophoresis or
hydroxylapatite chromatography. Amino acid sequences from six peptides
derived from pure CTBP matched sequences in transcytosis-associated
protein (TAP) with 98% identity. Thus, CTBP was positively identified to be TAP. Purified CTBP increased the activity of purified CT measured
with phosphatidylcholine (PC)/oleic acid. In the absence of PC/oleic
acid, CTBP did not stimulate CT activity. Dilution of CT to reduce the
Triton X-100 concentration produced a loss of CT activity. The lost
activity was recovered by the addition of CTBP plus PC/oleic acid to
the assay, but not by the addition of either PC/oleic acid or CTBP
alone. Removal of CTBP from purified preparations by
immunoprecipitation with CTBP antibodies eliminated the activation of
CT. Both CT and CTBP were shown to bind to PC/oleic acid liposomes. The
formation of complexes between CT and CTBP in the absence of PC/oleic
acid liposomes could not be demonstrated. These results suggest that
CTBP functions to modify the interaction of CT with PC/oleic acid
liposomes, resulting in an increase in the catalytic activity perhaps
by the formation of a ternary complex between CT, CTBP, and lipid.
Overall, these results suggest that CTBP (TAP) may function to
coordinate the biosynthesis of phosphatidylcholine with vesicle
transport.
 |
INTRODUCTION |
CTP:phosphocholine cytidylyltransferase
(CT)1 is a major regulatory
enzyme in the biosynthesis of phosphatidylcholine. CT exists intracellularly in inactive and active forms. Regulation of CT involves
interconversion of these forms. There is abundant evidence indicating
that the active form of CT is associated with membranes (reviewed in
Refs. 1-3). A variety of mechanisms have been suggested to explain the
activation of CT in response to a variety of stimulatory treatments.
These include changes in the state of CT phosphorylation (4-8),
changes in the content of phosphatidylcholine in membranes (9-11),
alterations in the physical form of phospholipid at specific membrane
domains (12), and the promotion of CT binding and activation by
specific lipids (6, 13-16). The involvement of other cellular proteins
in the overall regulation of CT is an additional possibility that has
not been fully explored. The identification of a 110-kDa protein that
binds CT (17) led us to consider in more detail the potential
involvement of this protein in the regulation of CT. We subsequently
found that complexes between CT and the cytidylyltransferase-binding protein (CTBP) were formed by incubation of liver cytosol with oleic
acid (18). Further insight into the function of this protein required
the isolation of purified protein.
We have now purified CTBP from rat liver cytosol. Amino acid sequences
from six peptides derived from pure CTBP matched sequences in
transcytosis-associated protein (TAP) from rat liver (19). Studies on
the effects of purified CTBP on the activity of CT suggested that CTBP
enhances the activation of CT by lipids.
 |
EXPERIMENTAL PROCEDURES |
Materials--
We obtained
[methyl-14C]phosphocholine from American
Radiolabeled Corp. Protogel (30% acrylamide and 0.8% bisacrylamide)
purchased from National Diagnostics, Inc. was used to prepare
polyacrylamide gels. The ECL reagent was purchased from Amersham Corp.
Polyvinylidene difluoride membrane and a minipreparative
electrophoresis cell were purchased from Bio-Rad. DEAE-Sepharose Fast
Flow and butyl-agarose were purchased from Pharmacia Biotech Inc.
Hydroxylapatite (Bio-Gel HTP) was from Bio-Rad. Peflabloc was from
Boehringer Mannheim. Phosphatidylcholine, oleic acid, sodium oleate,
CTP, phosphocholine, protease inhibitors, and all other biochemicals
were from Sigma.
Antiserum (N-1) raised against a synthetic peptide corresponding to the
N-terminal sequence of CT was provided by Dr. Claudia Kent (University
of Michigan) (20). The production and preparation of antiserum (AS-II)
that detects CTBP were described previously (17).
Cytidylyltransferase Purification and Assay--
Cultures of Sf9
insect cells infected with baculovirus cloning vector carrying the
full-length coding sequence for CT (21) were provided by Dr. Claudia
Kent and Joel Clement (University of Michigan). We purified
cytidylyltransferase from baculovirus-infected Sf9 cells using the
method previously reported for rat liver (21-23). Cytidylyltransferase
activity was determined as described previously (24). Assays contained
1.6 mM [14C]phosphocholine (1000 dpm/nmol), 6 mM CTP (adjusted to pH 7.0), 24 mM magnesium
acetate, 2 mM EDTA, and 50 mM imidazole, pH
7.0. All assays contained 100 µM
phosphatidylcholine/oleic acid (1:1 molar ratio) unless otherwise
indicated.
SDS Slab Gel Electrophoresis--
SDS-polyacrylamide gel
electrophoresis was performed on 10% polyacrylamide slab gels (12 cm × 16 cm × 1.5 mm) using a Bio-Rad Protean II system as
described previously (17). The samples contained 2% SDS and 5%
mercaptoethanol and were boiled for 5 min.
Protein Staining of Slab Gels--
Silver stain analysis of
SDS-polyacrylamide gels was performed using the method of DeMoreno
et al. (25). Molecular mass standards (Sigma M-5505) were
used to calibrate the gels. SDS-polyacrylamide slab gels were stained
overnight using 0.1% Amido Black prepared in 30% methanol and 1.0%
acetic acid. The gel was destained in 30% methanol and 0.3% acetic
acid.
Measurement of CTBP--
We detected CTBP immunologically on
either Western blots or slot blots. Proteins separated by SDS gel
electrophoresis were transblotted onto polyvinylidene difluoride
membranes as described previously (17). For slot blots, 10-50-µl
aliquots were diluted to 200 µl with TBS (25 mM Tris, pH
7.5, and 500 mM NaCl) and applied to nitrocellulose
membranes using a microfiltration manifold (Bio-Rad). The detection of
CTBP in glycerol and sucrose gradients was done by direct application
of 5-10 µl to nitrocellulose membranes. The dried membrane was
submerged briefly in 10% trichloroacetic acid, washed with TBS, and
dried.
Membranes were blocked with 5% nonfat milk in TBS for 3 h at room
temperature. The blot was probed overnight at 4 °C with N-1 or AS-II
antiserum diluted in TBS containing 3% nonfat milk, 0.1% bovine serum
albumin, and 0.1% Tween 20. For all subsequent procedures, we used
glass roller bottles in a Techne hybridizer oven. The blot was washed
four times with 100 ml of TBS containing 0.1% Tween 20. We probed the
blot with goat anti-rabbit IgG conjugated to horseradish peroxidase
(1:10,000 dilution) in TBS containing 3% nonfat milk, 0.1% bovine
serum albumin, and 0.1% Tween 20 for 2 h at room temperature. We
washed the blot three times with 100 ml of TBS containing 0.3% Tween
20 and three times with 100 ml of TBS containing 0.1% Tween 20. Immunoreactive bands were detected by chemiluminescence using the ECL
reagent. We measured the relative optical densities and areas of the
bands on a microcomputer imaging device (Imaging Research Inc.,
Ontario, Canada). We defined a scan density unit as the relative
optical density multiplied by the area of the band.
Purification of CTBP--
Male rats (200-250 g) were
anesthetized with chloral hydrate, and livers were perfused with 0.9%
NaCl. All subsequent procedures were performed at 4 °C. We used
80-100 g of liver for each preparation. In the purification described
below, we used 80 g of liver. Livers were homogenized at 4 ml/g of
liver in Buffer A (150 mM NaCl, 50 mM Tris, 2 mM EDTA, 2 mM DTT, 0.025% sodium azide, pH
7.5, and protease inhibitors (0.1 mM Peflabloc, 2.5 µg/ml
leupeptin, 2.0 µg/ml pepstatin A, 10 µg/ml benzamidine, 10 µg/ml
-aminobenzamidine, and 1.0 µg/ml antipain)). The cytosol was
isolated by sequential centrifugation at 17,000 × g
for 20 min and at 125,000 × g for 60 min.
We diluted the cytosol to 8 mg/ml protein with Buffer A. The pH of the
cytosol was reduced to 5.0 by the addition of 1.0 M acetic
acid. We stirred the mixture on ice for 20 min and centrifuged the
suspension at 12,500 × g for 20 min. We extracted the
precipitate in a volume of Buffer A equal to the volume of the original
cytosol. After stirring at 4 °C for 20 min, we centrifuged the
mixture at 17,000 × g for 20 min to recover the
soluble extract. Under these conditions, nearly all of the CTBP was
solubilized along with ~35% of the CT activity.
A DEAE-Sepharose column (2.2 × 12 cm) was packed and equilibrated
with Buffer A at a flow rate of 90 ml/h. The extract from the pH 5.0 precipitate (350 mg of protein in 320 ml) was applied to the column at
a flow rate of 45 ml/h. The column was washed with 250 ml of Buffer A. The column was eluted with a 500-ml gradient of NaCl (150-400
mM) prepared in Buffer A. CTBP coeluted with a small peak
of protein at ~300 mM NaCl. A large protein peak eluted
just before CTBP.
Fractions containing CTBP were pooled from the DEAE-Sepharose column.
The pool (19 mg of protein in 100 ml) was adjusted to 800 mM phosphate by adding 2.0 M ammonium
phosphate, pH 7.4, containing 2 mM EDTA, 2 mM
DTT, and protease inhibitors. All subsequent phosphate buffers
contained 2 mM EDTA, 2 mM DTT, and protease
inhibitors. The sample was applied to a butyl-agarose column (1.6 × 12 cm) equilibrated with 800 mM ammonium phosphate, pH
7.4. The column was then washed with 100 ml of 800 mM
ammonium phosphate, pH 7.4. CTBP was eluted from the column with a
250-ml gradient (800 to 20 mM ammonium phosphate, pH 7.4).
The gradient was followed by 100 ml of 20 mM ammonium
phosphate, pH 7.4. CTBP was eluted at ~100 mM phosphate.
More than 90% of the applied protein and the remaining CT were eluted
ahead of CTBP.
CTBP-containing fractions from the butyl-agarose column were pooled and
concentrated by centrifugal ultrafiltration in a 30K Macrosep (Filtron
Technology Corp.). We changed the buffer content of the CTBP
preparation by repeatedly concentrating the preparation after dilution
with 62 mM Tris, pH 6.8. The final concentrated pool
contained 2.7 mg of protein in 1.3 ml.
Preparative native electrophoresis was performed using a Bio-Rad
minipreparative cell. The lower gel (7.5 cm) contained 4% polyacrylamide and 0.376 M Tris, pH 8.8. The upper gel
(1.25 cm) contained 4% polyacrylamide and 0.124 M Tris, pH
6.8. The electrode and elution buffers contained 25 mM Tris
and 192 mM glycine, pH 8.3. The sample buffer contained 62 mM Tris, pH 6.8. A 450-µl sample (900 µg of protein) of
concentrated CTBP was mixed with 75 µl (v/v) of 25% glycerol and
bromphenol blue tracking dye in 0.30 M Tris, pH 6.8. This
mixture was applied to the gel column. Electrophoresis was conducted at
voltage and current limits of 300 V and 2.5 mA, respectively. The
elution flow rate was 4.8 ml/h. The majority of the contaminating
protein eluted with the dye front after ~10 h. The elution of CTBP
was complete after ~22 h. The combined fractions contained 110 µg
of protein.
Hydroxylapatite chromatography, which was used by Waters et
al. (26) to purify p115, proved to be a useful alternative to preparative native electrophoresis for the final purification step of
CTBP. This method was used after CTBP was found to be identical to
p115. CTBP from the butyl-agarose column was concentrated 20-fold by
centrifugal ultrafiltration in a 30K Macrosep. A Bio-Gel HTP column
(1.2 × 7 cm) was packed and equilibrated with Buffer B (200 mM KCl, 10% (v/v) glycerol, 10 mM sodium
phosphate, 1 mM DTT, pH 7.4, and protease inhibitors).
Concentrated CTBP (1.5 mg of protein) was diluted 10-fold with Buffer B
and applied to the Bio-Gel HTP column at a flow rate of 5 ml/h. The
column was washed with 12 ml of Buffer B. CTBP was eluted from the
column with an 80-ml gradient of sodium phosphate (10-500
mM) in Buffer B. CTBP was eluted from the Bio-Gel HTP
column at ~90 mM phosphate. The combined fractions
contained 630 µg of protein.
We used preparative electrophoresis in the presence of SDS to isolate
the 110-kDa form of CTBP. CTBP-containing fractions from the
butyl-agarose column were pooled and concentrated by centrifugal
ultrafiltration in a 30K Macrosep. We changed the buffer content of the
CTBP preparation by repeatedly concentrating the preparation after
dilution with 62 mM Tris, pH 6.8. CTBP was recovered from
the concentrator using 60 mM Tris, pH 6.8, containing 2%
SDS. Preparative SDS electrophoresis was performed using the Bio-Rad
minipreparative cell. The lower gel (7.5 cm) contained 6%
polyacrylamide, 0.1% SDS, and 0.376 M Tris, pH 8.8. The
upper gel (1.25 cm) contained 4% polyacrylamide, 0.1% SDS, and 0.124 M Tris, pH 6.8. The electrode buffer contained 0.1% SDS,
25 mM Tris, and 192 mM glycine, pH 8.3. The
sample buffer contained 2% SDS and 62 mM Tris, pH 6.8. We
applied a 450-µl sample of CTBP (900 µg of protein) to the gel.
Electrophoresis was conducted at voltage and current limits of 300 V
and 2.5 mA, respectively. The elution flow rate was 4.8 ml/h.
Protein Assays--
Protein was determined with a modification
(27) of the procedure of Lowry et al. (42) or with the
Coomassie protein assay reagent (Pierce). Bovine serum albumin was used
as standard.
 |
RESULTS |
Purity of CTBP--
The protein composition of the final
preparation was examined by SDS-polyacrylamide electrophoresis (Fig.
1). The major protein component migrated
to a position just beyond the 116-kDa protein standard, corresponding
to a molecular mass of 110 kDa. This protein band was detected by CTBP
antiserum (AS-II) on a Western blot. There was a small amount of a
95-kDa protein contaminant. The 95-kDa protein was not detected by CTBP
antiserum, suggesting that it was not a product of proteolytic
digestion of CTBP. The overall purity of CTBP from the hydroxylapatite
column was similar to that obtained by native gel electrophoresis (Fig.
2). The hydroxylapatite preparation
contained a small amount of a 50-kDa protein, but did not contain the
95-kDa protein that was present in the native electrophoresis
preparation. The CTBP preparations did not contain CT activity or CT
detected by CT antiserum on Western blots.

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Fig. 1.
SDS gel analysis of CTBP purified by
preparative native gel electrophoresis. The protein composition
and immunostain pattern of the native CTBP preparation were assessed by
SDS-polyacrylamide gel electrophoresis on an 8% polyacrylamide slab
gel. An aliquot of the CTBP preparation containing 3.2 µg of protein
was applied to both lanes A and B. Lane
A was detected by silver staining. Lane B was detected
by Western blot immunostaining with CTBP antiserum.
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Fig. 2.
SDS gel analysis of CTBP purified by
hydroxylapatite chromatography. The protein composition of CTBP
was assessed by SDS electrophoresis on a 10% polyacrylamide slab gel.
Protein was detected by silver staining. Lane A, 9 µg of
butyl-agarose preparation; Lane B, 2 µg of hydroxylapatite
preparation.
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Determination of Amino Acid Sequence--
For sequence analysis,
we prepared CTBP by gel column electrophoresis in the presence of SDS.
The purity of this CTBP preparation is shown in Fig.
3. Silver staining of SDS gels indicated
a small amount of contaminating protein that migrated slightly above
and below the band of CTBP. Immunostain analysis with CTBP antiserum revealed only one immunoreactive band. CTBP migrated to a position just
ahead of the 116-kDa protein standard, with a calculated molecular mass
of 110 kDa.

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Fig. 3.
SDS gel analysis of CTBP purified by
preparative SDS gel electrophoresis. The protein composition and
immunostain pattern of CTBP were assessed by SDS electrophoresis on a
10% polyacrylamide slab gel. An aliquot of the CTBP preparation
containing 4.5 µg of protein was applied to both lanes A
and B. Lane A was detected by silver staining.
Lane B was detected by Western blot immunostaining with CTBP
antiserum.
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A 50-µl sample of the CTBP preparation, containing 25 µg of
protein, was applied to one lane on an SDS-8% polyacrylamide slab gel.
After electrophoresis, we detected the band of CTBP by staining the gel
with Amido Black. We then excised the band with a scalpel. We submitted
the gel segment to the Protein Sequencing Facility at the University of
Michigan. Peptide fragments were prepared by cyanogen bromide cleavage.
Amino acid sequence analysis was performed on six different
peptides.
As shown in Fig. 4, the sequences from
each peptide (18-31 amino acids) generated from CTBP matched regions
in the sequence of TAP from rat liver (19). The six peptide sequences
of CTBP contained a total of 164 amino acids, and 161 amino acids were identical to the corresponding region in TAP. The calculated molecular mass of TAP was 107,161 Da (19). This value closely matches the
molecular mass of CTBP (110 kDa) obtained by SDS gel electrophoresis. Barroso et al. (19) found that TAP was 92% identical to a
vesicular transport protein (p115) from bovine brain (28).

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Fig. 4.
Comparison of CTBP peptide sequences with the
amino acid sequence of TAP. The amino acid sequences of six
separate peptides from CTBP (boldface) are aligned with the
corresponding regions in the full-length 959-amino acid sequence of TAP
(19). Dashes indicate unidentified amino acids in the CTBP
peptides. Underlined amino acids do not match the
corresponding amino acids in TAP.
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Effects of CTBP on CT Activity--
In these experiments, we used
purified CT. We store purified CT at
40 °C in buffer containing 50 mM Tris, 200 mM ammonium phosphate, 150 mM NaCl, 2 mM EDTA, 2 mM DTT,
0.025% sodium azide, and 0.03-0.06% Triton X-100, pH 7.5. Under
these conditions, CT is stable, and the activity remains constant for
many months. Since purified CT contained high activity, it was
necessary to dilute the preparation for routine experimental use. We
diluted purified CT 1:40 either in Buffer C (50 mM Tris,
200 mM ammonium phosphate, 150 mM NaCl, 2 mM EDTA, and 0.025% sodium azide, pH 7.5) or in Buffer C
containing 0.06% Triton X-100. Dilution of CT in Buffer C without
Triton X-100 resulted in a 70-80% decrease in CT activity, measured
with optimal concentrations of PC/oleic acid. The addition of purified
CTBP to the assay produced an increase in CT activity (Fig.
5A). Thus, the addition of
CTBP resulted in the recovery of the CT activity that was lost by the
dilution. Dilution of CT with Buffer C containing 0.06% Triton X-100
did not produce a decrease in CT activity (Fig. 5B). Under
these conditions, CTBP increased CT activity to values that were nearly
70% higher than in the original undiluted CT preparation. Half-maximal
activation was obtained with 10 ng of CTBP. Maximal stimulation of CT
activity, in the presence of 100 µM PC/oleic acid, was
obtained at an ~1:1 molar ratio of CTBP to CT, assuming that both CT
and CTBP were present as homodimers. CT activity after dilution in the
presence or absence of Triton X-100 was not increased by ovalbumin,
thyroglobulin, or bovine serum albumin (Fig. 5, A and
B). Thus, the effect was specific for CTBP. CTBP produced a
similar increase in CT activity in the original undiluted preparation
(Fig. 5C). These assays contained 16 times more CT than the
dilution experiments. Maximal activation was obtained with ~16 times
more CTBP. Thus, the molar ratios of CT to CTBP required for optimal
activation were similar to those for diluted CT.

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Fig. 5.
Effect of CTBP on CT activity. Purified
CT (12 µl) was diluted to 480 µl in Buffer C (A) or in
Buffer C containing Triton X-100 (B). The diluted mixtures
were kept on ice for 30 min. CT activity was measured in 5-µl
aliquots of the diluted mixtures. CT activity was also measured in
2-µl aliquots of undiluted CT (C). All CT assay mixtures
contained 100 µM PC/oleic acid. Varied amounts of CTBP
( ), ovalbumin, ( ), thyroglobulin ( ), or bovine serum albumin
( ) were added to the assay mixtures immediately before the addition
of CT. The results are calculated as the percent recovery of the
activity in undiluted CT. The activity in undiluted CT was determined
in each experiment in the presence of 100 µM PC/oleic
acid. The average activity of purified CT, determined in 13 separate
experiments, was 1.06 ± 0.09 nmol/min/µl. This activity was
used to calculate the percent of the total.
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The results shown in Fig. 5 were obtained with CTBP purified by
hydroxylapatite chromatography. Similar results were obtained using
CTBP purified by native polyacrylamide electrophoresis. Each
preparation contained >80% CTBP. However, the population of
contaminating proteins was different in the two preparations. The
similarity in the results obtained with the two preparations suggested
that the contaminating protein(s) were not responsible for the
stimulatory activity. To obtain direct evidence that CTBP was
responsible for the activation of CT, we specifically removed CTBP from
the preparation with CTBP antibodies. Two separate approaches were
used. In the first, we used protein A-Sepharose to isolate IgG
fractions from CTBP antiserum and preimmune serum. IgG recovered from
each was coupled to Affi-Gel 10 (Bio-Rad). Treatment of CTBP preparations with Affi-Gel containing CTBP antibodies removed CTBP
(determined by dot-blot assay). The immunodepleted preparation produced
an 8% increase in CT activity. Treatment of the CTBP preparation with
Affi-Gel containing preimmune IgG did not remove CTBP. The treated CTBP
preparation retained the ability to activate CT (45% increase in CT
activity). In the second set of experiments, CTBP (150 ng) was
incubated with protein A-Sepharose containing IgG from either CTBP
antiserum or preimmune antiserum. A third incubation contained buffer
in place of the protein A-Sepharose. After removal of the Sepharose by
centrifugation, an aliquot of the supernatant was used to activate
diluted CT. CT activity was increased by 30.5 ± 2.1% with the
untreated CTBP preparation. The preparation treated with protein
A-Sepharose containing preimmune IgG produced a 29.9 ± 2.2%
increase in CT activity compared with an 8.8 ± 3.4% increase
with the preparation treated with protein A-Sepharose containing CTBP
IgG. Western blot analysis of the supernatants indicated that protein
A-Sepharose containing CTBP IgG removed CTBP. Protein A-Sepharose
containing preimmune IgG did not remove CTBP. Thus, CTBP IgG
specifically adsorbs the activating protein in preparations of purified
CTBP. Taken together, these results indicated that CTBP/TAP
specifically activates CT.
The activation of CT by CTBP was dependent upon the presence of
PC/oleic acid in the assay (Fig.
6A). CTBP did not increase CT
activity in the absence of PC/oleic acid. In the presence of PC/oleic
acid, CT activity was activated by CTBP to nearly 80% of the activity
in the undiluted preparation (Fig. 6A). This was nearly a
4-fold increase in activity over the activity obtained with PC/oleic
acid alone. When we diluted CT in the presence of 0.06% Triton X-100,
~80% of the activity was recovered with assays containing PC/oleic
acid (Fig. 6B). The addition of CTBP to the assays increased
CT activity to values nearly 50% higher than in the original undiluted
preparation. This is consistent with the results shown in Fig.
5B. CTBP alone did not increase CT activity in the presence
of Triton X-100. The PC/oleic acid dependence for CT activity and for
CTBP stimulation was similar for undiluted CT and for CT diluted with
Triton X-100 in the dilution buffer (Fig. 6, compare C with
B).

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Fig. 6.
CTBP stimulation of CT activity requires
PC/oleic acid. Purified CT (12 µl) was diluted to 480 µl in
Buffer C (A) or in Buffer C containing 0.06% Triton X-100
(B). The diluted mixtures were kept on ice for 30 min. CT
activity was measured in 5-µl aliquots of the diluted mixtures. CT
activity was also measured in 2-µl aliquots of undiluted CT
(C). CT activity was measured in the presence of varied
amounts of PC/oleic acid with ( ) or without ( ) CTBP. In
A and B, 94 ng of CTBP were added to the assay.
In C, 188 ng of CTBP were added to the assay. CTBP was added
to assay mixtures immediately before the addition of CT. The results
are calculated as the percent recovery of the activity in undiluted CT
measured with 100 µM PC/oleic acid.
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CT and CTBP Bind to Liposomes--
Since CTBP activated CT only in
the presence of PC/oleic acid, it seemed likely that CTBP may enhance
or stabilize the binding of CT to PC/oleic acid liposomes. To assess
the effect of CTBP on the binding of CT to PC/oleic acid liposomes, we
incubated CT, CTBP, or CT plus CTBP with PC/oleic acid. We then
separated proteins bound to PC/oleic acid from the unbound proteins by
flotation of the PC/oleic acid liposomes to the top of a sucrose
gradient (29).
The results from these experiments indicated that both CT and CTBP bind
to PC/oleic acid liposomes (Fig.
7A). All of the CTBP was bound
to PC/oleic acid, but a portion of the CT (~25%) did not bind to
PC/oleic acid and remained at the bottom of the gradient. CTBP appeared
to bind to the CT at the bottom of the gradient (Fig. 7A,
compare
and
). The amount of CT bound to PC/oleic acid was not
increased by CTBP (Fig. 7A, compare
and
). The activity of CT bound to PC/oleic acid in the absence of CTBP was measured with and without the addition of CTBP to the assay. The addition of CTBP stimulated CT activity (Fig. 7B, compare
and
). When CT was incubated with CTBP and PC/oleic acid, the
activity of CT bound to PC/oleic acid was increased (Fig. 7, compare
B (
) and C (
)). The increase in CT activity
appeared to be due to activation by CTBP because the mass of CT bound
to PC/oleic acid was not increased (Fig. 7A). Furthermore,
CT activity was not increased by the addition of more CTBP to the assay
(Fig. 7C, compare
and
). These results suggested that
CT, CTBP, and PC/oleic acid formed a relatively stable complex in which
CT was optimally active.

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Fig. 7.
CT and CTBP binding to PC/oleic acid
liposomes. Purified CT (19 nmol/min), purified CTBP (3.9 µg), or
CT and CTBP were incubated with PC/oleic acid (2 mg of PC and 0.75 mg
of oleic acid, 1:1 molar ratio) containing trace amounts of
sn-1,2-dipalmitoylglycerophosphoryl[N-methyl-3H]choline
in a final volume of 60 µl of buffer containing 50 mM imidazole, 2 mM EDTA, and 2 mM DTT, pH 7.0. After 5 min at 37 °C, 60 µl of 70% sucrose were added. The
mixture (100 µl) was layered under a 15-30% sucrose gradient
(4.0-ml total volume). The gradient was centrifuged in an SW 50.1 rotor
for 16 h at 40,000 rpm. Fractions (200 µl) were collected from
the top. CT and CTBP masses were measured by immunoassays. The amount
of radioactive PC determined in each fraction was used to measure the
distribution of PC/oleic acid (expressed as percent distribution of
radioactive PC; ). CT activity was determined with and without the
addition of 65 ng of CTBP to the assay. A, mass distribution
of CT and CTBP. , CT mass after incubation with PC/oleic acid; ,
CTBP mass after incubation with PC/oleic acid; , CT mass after
incubation with CTBP and PC/oleic acid; , CTBP mass after incubation
with CT and PC/oleic acid. B, distribution of CT activity
after incubation with PC/oleic acid. The activity was measured with and
without CTBP added to the assay. , without added CTBP; , with 65 ng of CTBP. C, distribution of CT activity after incubation
with CTBP and PC/oleic acid. CT activity was measured with and without CTBP added to the assay. , without added CTBP; , with 65 ng of
CTBP.
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CT Does Not Bind to CTBP in the Absence of Lipid--
To assess
the ability of CT to form complexes with CTBP in the absence of lipid,
we incubated purified CT with purified CTBP. A mixture of CT and CTBP
and samples of CT and CTBP alone were then subjected to glycerol
density centrifugation. CT sedimented in the glycerol gradient to a
position corresponding to a molecular mass of 90-100 kDa (Fig.
8, A (activity) and
C (immunoassay)). There was no evidence for the formation of
a stable complex between CT and CTBP because incubation of CT with CTBP
did not change the position of CT in the gradient (Fig. 8B).
Interestingly, CT that was sedimented alone was nearly inactive (Fig.
8A). The addition of CTBP to the assay produced a 4-5-fold
increase in activity. When CT was sedimented with CTBP, the recovery of
CT activity was increased (Fig. 8B). Furthermore, CT
activity was not increased appreciably when CTBP was added to the
assay. This was likely due to the fact that CTBP sedimented to
approximately the same position as CT in the gradient (Fig.
8D). Therefore, CTBP would have been present in the assay
along with CT.

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Fig. 8.
CT does not bind to CTBP in the absence of
lipid. Purified CT (47 nmol/min) or CT and purified CTBP (5.3 µg) were incubated in a final volume of 500 µl of buffer containing
10 mM ammonium phosphate, 20 mM KCl, 1%
glycerol, 50 mM imidazole, 2 mM EDTA, and 2 mM DTT, pH 7.0. After 30 min at 4 °C, the mixture (450 µl) was layered onto a 10-35% glycerol gradient (12-ml total
volume). The gradient was centrifuged in an SW 41 rotor for 16 h
at 40,000 rpm. Fractions (400 µl) were collected from the top. CT and
CTBP masses were measured by immunoassays. CT activity was determined in the presence of 100 µM PC/oleic acid with and without
the addition of 65 ng of CTBP to the assay. A, CT incubated
without CTBP. , CT activity measured with added CTBP; , CT
activity measured without added CTBP. B, CT incubated with
CTBP. , CT activity measured with added CTBP; , CT activity
measured without added CTBP. C, CT incubated without CTBP.
, CT immunomass. D, CT incubated with CTBP. , CTBP
immunomass.
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DISCUSSION |
In previous studies, we identified a protein (CTBP) with
a subunit molecular mass of 110-112 kDa that binds CT, forming
heterogeneous complexes (17, 18). In the present study, we purified
CTBP from rat liver. Amino acid sequences from six peptides derived from purified CTBP exactly matched sequences in TAP that had previously been isolated from rat liver and sequenced (19). The reported sequence
of TAP is 90% identical to p115, a vesicular transport factor isolated
from bovine liver (28). TAP/p115 is homologous to Uso1p, a protein
involved in vesicular transport in yeast (31). Structural analysis of
TAP/p115 indicated an elongated structure consisting of two subunits
that interact, forming an N-terminal head and a C-terminal coiled-coil
tail that terminates in an acidic domain (19, 28). TAP/p115 appears to
be located on Golgi membranes as a peripheral protein that is rather
easily extracted by salt-containing buffers (26). The precise function
of TAP/p115 in vesicular transport has not been conclusively
identified. Current evidence suggests that it likely functions in the
targeting or fusion stage of the vesicular transport cycle (19, 28).
Barroso et al. (19) proposed that TAP may interact with
target membranes and hold the vesicular and target membranes in
proximity. Recent studies on the disassembly and reassembly of the
Golgi apparatus during cell division provided additional evidence for a
function of TAP/p115 in the docking of vesicles to target membranes
(32).
Purified CTBP stimulated CT activity under a variety of conditions. The
increase in CT activity occurred after dilution in the presence of
Triton X-100 or without Triton X-100. Dilution of CT without
maintaining the Triton X-100 concentration resulted in a loss of CT
activity, measured under optimal concentrations of PC/oleic acid.
However, the addition of CTBP to the assay increased CT activity to the
original activity measured before dilution. With both undiluted CT and
CT diluted with Triton X-100, the activity of CT was increased by CTBP
to levels 50-60% higher than those obtained with PC/oleic acid alone.
Under all conditions, stimulation of CT activity by CTBP occurred only
when PC/oleic acid was present. CTBP did not stimulate CT activity in
the absence of PC/oleic acid. Both CT and CTBP bound to PC/oleic acid
liposomes. The isolated complexes contained active CT, but the activity
was increased when CTBP was present. Complexes between CT and CTBP were
not formed in the absence of lipid, in agreement with the observation that CTBP alone did not stimulate CT activity. These results are in
general agreement with our previous observations that oleic acid or
PC/oleic acid promoted the formation of complexes between CT and CTBP
in liver cytosol (17, 18). Overall, the results indicted that the
coincident binding of CT and CTBP to PC/oleic acid formed a ternary
complex in which CT activity was increased above the activity
achievable with PC/oleic acid. At present, the mechanism for this
cooperative effect is unknown.
The finding that CTBP is identical to TAP/p115, together with the
results suggesting a role of CTBP in the regulation of CT activity
leads to the interesting possibility that this protein enables
communication between phosphatidylcholine synthesis and vesicular
transport. Conceptually, this dual function would allow the rate of
vesicular transport to be coordinated with the biosynthesis of
phosphatidylcholine needed for the production of membrane vesicles. There is some relevant information in the literature to support a
connection between vesicular transport and phosphatidylcholine synthesis. Cytidylyltransferase has been reported to be present in
Golgi membranes (33, 34). Studies by Slomiany et al. (35) on
the biogenesis of endoplasmic reticulum transport vesicles in gastric
mucosal tissue suggested that CT was associated with the transport
vesicle. Indirect evidence led the authors to speculate that the
formation of vesicles from the endoplasmic reticulum may be regulated
by the activity of cytidylyltransferase. The results suggesting that CT
is located in the nucleus (6, 20, 36) are difficult to reconcile with a
function of CT in vesicular transport. However, the subcellular
localization of CT is not completely established. For example, recent
studies by Houweling et al. (37), using immunofluorescence,
immunogold electron microscopy, and biochemical techniques, indicate
that CT is not exclusively located in the nucleus in all cells. For
example, CT was found to be distributed throughout the cytoplasm in
isolated rat hepatocytes.
A series of genetic studies with Saccharomyces cerevisiae
provided independent evidence for a regulatory connection between cytidylyltransferase and Golgi function. Sec14p, a
phosphatidylinositol/phosphatidylcholine transfer protein, is bound
peripherally to Golgi membranes and is required for transport from
Golgi compartments (38-40). Skinner et al. (41) provided
both in vitro and in vivo evidence that Sec14p
decreases the CDP-choline pathway for phosphatidylcholine synthesis by
inhibiting cytidylyltransferase. Furthermore, the data suggest that
Sec14p is inhibitory when in the phosphatidylcholine-bound form. This
provides a potential feedback process to regulate the amount of
phosphatidylcholine in Golgi membranes. Thus, it is possible that a
Sec14p-type action and the effects of CTBP/TAP are both examples of a
complex mechanism to coordinate the biosynthesis of phosphatidylcholine
with membrane formation required for vesicular transport. Full
recognition of the importance of CTBP/TAP in regulating cytidylyltransferase activity requires both a detailed understanding of
the mechanisms for the cooperative effect of CTBP on CT activity and
in vivo demonstration of the effects of CTBP on CT.