From the Department of Molecular and Cell Biology,
Boston University Goldman School of Dental Medicine, Boston,
Massachusetts 02118 and ¶ Department of Biochemistry and Molecular
Biology, University of Massachusetts Medical Center,
Worcester, Massachusetts 01655
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ABSTRACT |
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Phosphorylation of secretory and integral membrane proteins and of proteoglycans also occurs in the lumen of the Golgi apparatus. ATP, the phosphate donor in these reactions, must first cross the Golgi membrane before it can serve as substrate. The existence of a specific ATP transporter in the Golgi membrane has been previously demonstrated in vitro using intact Golgi membrane vesicles from rat liver and mammary gland.
We have now identified and purified the rat liver Golgi membrane ATP
transporter. The transporter was purified to apparent homogeneity by a
combination of conventional ion exchange, dye color, and affinity
chromatography. An ~70,000-fold purification (2% yield) was achieved
starting from crude rat liver Golgi membranes. A protein with an
apparent molecular mass of 60 kDa was identified as the putative
transporter by a combination of column chromatography, photoaffinity
labeling with an analog of ATP, and native functional size
determination on a glycerol gradient. The purified transporter appears
to exist as a homodimer within the Golgi membrane, and when
reconstituted into phosphatidylcholine liposomes, was active in ATP but
not nucleotide sugar or adenosine 3'-phosphate 5'-phosphosulfate transport. The transport activity was saturable with an apparent Km very similar to that of intact Golgi vesicles.
Post-translational modifications of proteins, occurring in the
lumen of the Golgi apparatus, include glycosylation, sulfation, and
phosphorylation. Although the significance and general mechanistic features of the first two reactions are well understood, very little is
known about the latter one. Secreted proteins such as caseins (1, 2)
and vitellogenin (3, 4) and integral Golgi membrane proteins (5) have
been shown to undergo phosphorylation in the Golgi lumen. The
occurrence of phosphorylated proteoglycans, including heparan sulfate,
chondroitin sulfate, and proteodermatan sulfate (6-8), has also been
reported. In these macromolecules, the phosphate is attached to the
protein core and, in some cases, to xylose (7). Although it has been
suggested that phosphorylation can contribute to maintaining the
stability of the protein by protecting it against proteolytic
degradation in situ (1, 2) or, in the case of
proteoglycans, as a specific targeting signal (7), the
significance of these post-translational modifications remains to
be determined.
ATP, the phosphate donor in the above phosphorylation reactions, is
synthesized mainly in the mitochondrial matrix. To be accessible to the
lumen of the Golgi apparatus, where these reactions occur, it must
first cross the mitochondrial membranes through a specific and well
characterized transporter and then the Golgi membrane. The existence of
a specific transporter in the latter membrane has been demonstrated
in vitro using intact rat liver and mammary gland Golgi
membrane vesicles (5). Transport was found to be
temperature-dependent, saturable at micromolar
concentrations of ATP and appears to be via an antiporter mechanism (9,
10), with AMP being the most likely antiporter (5). Casein
phosphorylation was found to occur subsequent to transport of ATP into
the lumen of rat mammary gland Golgi vesicles (5). ATP transport has also been shown to occur across the membrane of the rough endoplasmic reticulum (ER)1 (11), where
it is involved in energy-requiring (reviewed in Ref. 12) and
phosphorylation reactions such as of BiP (11, 13). The former include
also dissociation of complexes between chaperones and correctly folded
and assembled proteins in the lumen of the ER, disulfide bond
formation, and protein polymerization (reviewed in Ref. 14). Recently,
it has also been shown that ATP is required for protein translocation
into the ER of yeast and mammalian cells (reviewed in Refs. 15 and
16).
To understand the importance of phosphorylation as a post-translational
Golgi lumenal event and to analyze the possibility that regulation of
ATP transport into the Golgi lumen can affect the biosynthesis and
function of macromolecules being synthesized in the Golgi apparatus, we
need initial knowledge of the amino acid and nucleic acid sequences of
such transporter. Moreover, the existence of specific ATP transporters
located in the membranes of three different intracellular organelles,
mitochondria, Golgi apparatus, and ER, raises the question of how these
proteins with the same function are localized in different organelles
and whether or not they share common structural features.
Here we used a reconstituted phosphatidylcholine proteoliposome system
(17) to monitor the purification of the ATP transport activity from a
rat liver Golgi membrane preparation. Column chromatography and
photoaffinity radiolabeling followed by SDS-PAGE electrophoresis were
used to identify a 60-kDa protein as the ATP transporter. Proteoliposomes containing this protein were active in ATP but not in
nucleotide sugars or PAPS transport; a similar apparent Km of ATP transport than previously reported for
intact Golgi vesicles was determined. Finally, native functional size determination on a glycerol gradient suggested that the ATP transporter exists as a homodimer in the membrane of the Golgi apparatus.
Materials
Frozen rat livers were purchased from Pel-Freez Biologicals.
[2,8-3H]ATP (15-30 Ci/mmol) was purchased from American
Radiolabeled Chemicals, Inc., [125I]NaI (350-600 mCi/ml)
was from Amersham Pharmacia Biotech, and 8-azido[ Methods
Purification of the Rat Liver Golgi Membrane ATP Transporter
All the operations described below were performed at
4 °C.
Step 1: Detergent Extraction--
A crude Golgi fraction was
prepared from 6 kg of frozen rat livers according to the procedure of
Leelavathi et al. (18). This fraction was resuspended in 10 mM Tris·HCl, pH 7.2, 1 mM MgCl2,
1 mM dithiothreitol, 0.3 mM sucrose, 20%
glycerol (v/v), 0.3% Triton X-100 (v/v) with protease inhibitors (0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.1 µg/ml pepstatin A, 0.1 µg/ml chymostatin, 0.1 µg/ml antipain, 0.1 µg/ml leupeptin, and 1 µg/ml aprotinin). The suspension was stirred
for 45 min at 4 °C and centrifuged at 100,000 × g for 45 min. The supernatant solution was discarded, and the pellet was
resuspended in the above buffer containing a final concentration of
1.1% Triton X-100. The mixture was stirred and centrifuged again as
described above. The supernatant was decanted, yielding a Triton X-100
extract that was saved. The pellet was resuspended, and the extraction
was repeated as described above. Both detergent extracts were combined
together and adjusted to a final concentration of 0.5% Triton X-100
and 0.2 M NaCl.
Step 2: First DEAE-Sephacel Column--
The above Triton X-100
extract was applied to three DEAE-Sephacel columns (Sigma; 25 cm ×5 cm
each) equilibrated in buffer A (10 mM Tris·HCl, pH 7.0, 1 mM MgCl2, 1 mM dithiothreitol, 1% glycerol, 0.5% Triton X-100) containing 0.2 M NaCl.
Elution was with six column-volumes of equilibration buffer followed by
four column-volumes of buffer A containing 0.7 M NaCl. The
salt-eluted material containing the ATP transport activity was then
concentrated and diluted to 0.3 M NaCl final concentration
by use of a Minitan ultrafiltration system (Millipore).
Step 3: Blue-Sepharose Column--
The above fraction was
applied to three Blue-Sepharose columns (Amicon; 20 cm × 5 cm,
each) equilibrated in buffer A containing 0.3 M NaCl. The
ATP transport activity eluted in the flow-through.
Step 4: Second DEAE-Sephacel Column--
The flow-through from
the Blue-Sepharose column was diluted to 0.1 M NaCl with
buffer A and applied to 8 DEAE-Sephacel columns (Sigma; 23 cm ×2.7 cm
each) equilibrated in buffer A containing 0.1 M NaCl. The
ATP transport activity eluted in the flow-through.
Step 5: Carboxymethylcellulose Column--
The second
DEAE-Sephacel flow-through was applied to 12 carboxymethylcellulose
columns (Amersham Pharmacia Biotech; 23 cm ×3 cm each) equilibrated in
buffer A containing 0.1 M NaCl. After washing with four
column-volumes of the equilibration buffer, elution was achieved with a
linear gradient of 0.1-1.5 M NaCl and 20-1% glycerol.
Fractions of 16 ml each were collected. Transport activity was eluted
at approximately 0.6 M (fractions 12-15). The active
fractions were pooled, desalted, and adjusted up to a final
concentration of 10% glycerol and 0.5% Triton.
Step 6: 3',5'-ADP-Agarose Column--
The above fraction was
applied to 19 3',5'-ADP-agarose columns (Sigma; 5 cm ×1.5 cm each)
equilibrated in buffer A containing 10% glycerol. After washing with
four column-volumes of the equilibration buffer, elution was achieved
with a linear gradient of 0-1.5 M NaCl and 10-1%
glycerol. Fractions of 2.5 ml each were collected, and the transport
activity was eluted at approximately 0.25 M (fractions
5-15).
Glycerol Gradient
The apparent functional mass of the ATP transporter was
estimated by analytical ultracentrifugation using an 8-30% glycerol gradient in buffer A. The active fraction obtained from purification Step 6 was concentrated and dialyzed against buffer A in Centricon filters (Amicon) to obtain a final glycerol concentration of 8%. The
10-ml glycerol gradient was equilibrated at 4 °C for 17 h before loading of the sample (0.5 ml) and then centrifuged in a SW 50 rotor at 46,000 rpm for 40 h at 4 °C. Fractions of 0.35 ml were
collected. Photoaffinity Radiolabeling with
8-Azido[ Isolation and Topography of Rat Liver Golgi Vesicles
For the characterization of the ATP transporter, rat liver Golgi
vesicles were isolated as described (18) and resuspended in
cryoprotective buffer (19). Sialyltransferase activity was enriched
~50-fold over crude homogenate. Approximately 90% of the vesicles
were sealed and of the same membrane topographical orientation as
in vivo (20).
Transport Assay
Transport of solutes into intact rat liver Golgi vesicles was
assayed as described before (21). To follow the transporter purification, the ATP transport activity was reconstituted in phosphatidylcholine liposomes (17, 22, 23) and incubated in the
presence of [3H]ATP (4 µM; 400 cpm/pmol)
for 5 min at 30 °C. The reaction mixture was then applied to a 3-ml
Dowex 1 × 2-100 column (Sigma) as described previously (22, 23).
Fractions of 300 µl were collected, and the radioactivity was
determined by liquid scintillation spectrometry.
Protein Visualization
The purity of the various fractions active and inactive in ATP
transport activity was determined by SDS/PAGE. Visualization was done
by Coomassie Blue/silver nitrate staining (OWL Separation System) or by
labeling proteins with 300 µCi of Na125I and chloramine T
(22, 23). Protein was quantified using the BCA protein assay kit (Pierce).
Purification of the ATP Transporter--
The rat liver Golgi
membrane ATP transporter was purified ~70,000 over the crude Golgi
membrane preparation with a yield of 2% (Table
I). To monitor the purification through
the different purification steps, membrane proteins were reconstituted
into phosphatidylcholine liposomes by freeze-thawing and then assayed for their ability to translocate radiolabeled ATP in vitro.
The purity of the ATP transporter during the purification was
determined by SDS/PAGE (Fig. 1). We began
the purification with a crude Golgi membrane preparation; the ATP
transport activity was extracted after a two-step solubilization with
Triton X-100. In the first step, Golgi membranes were treated with a
low concentration of Triton X-100 (0.3%, v/v). These conditions
removed the peripheral membrane proteins and did not result in a
significant loss of transport activity or in its extraction from the
membrane. In the second step, a higher concentration of Triton X-100
was used (1.1%, v/v) to almost completely solubilize the membrane
proteins. Approximately 85% of the total ATP transporter activity from
the Golgi membrane preparation could be solubilized under these
conditions, with a 15-fold purification over the crude Golgi membrane
preparation.
The Triton X-100 extract was then applied to a combination of
conventional ion exchange, dye color, and affinity chomatography columns. Details of the different chromatographic steps are given under
"Experimental Procedures," and the results of each step are given
in Table I.
The Triton X-100 extract (Fig. 1, lane 1) was loaded onto a
first DEAE-Sephacel column followed by elution with 0.7 M
NaCl in buffer A. 72% of the transport activity was recovered with a
130-fold purification over the crude Golgi preparation (Fig. 1,
lane 2). In the next two chromatographic steps, the
Blue-Sepharose and the second DEAE-Sephacel column, the transport
activity was found in the flow-through (negative columns). These two
negative columns combined together, resulted in the binding of most of the applied proteins but not the ATP transport activity (Fig. 1,
lanes 3 and 4). 41% of the initial activity was
recovered after these two steps with a 2,700-fold overall purification.
The active fraction obtained from the second DEAE-Sephacel was then
loaded onto a carboxymethylcellulose column and eluted with a linear gradient of 0.1-1.5 M NaCl and 20-1% glycerol (Fig.
2) with a 30,000-fold overall
purification (Fig. 1, lanes 5 and 6). In the next
step we used a 3'-5'-ADP-agarose column, which provided an important
and substantial purification (Fig. 1, lanes 7 and
8). The transport activity was eluted with a linear gradient
of 0-1.5 M NaCl and 10-1% glycerol (Fig.
3). This strategy resulted in a
60,000-fold overall purification with a recovery of 8% of the initial
transport activity. In order not to use high volumes of this active
fraction, small aliquots of the sample were subjected to
radioiodination with chloramine T before electrophoresis and visualization by autoradiography. The SDS-gel profile of the active fraction (Fig. 1, lane 7) showed two protein bands of 60- and 58-kDa. These were not visualized in fractions inactive for ATP transport activity (Fig. 1, lane 8).
Glycerol Gradient--
A glycerol gradient was used as a last step
of purification and to estimate the functional size of the ATP
transporter. The rationale for this was based on the fact that other
Golgi nucleotide sugar and nucleotide sulfate transporters appear to be
homodimers in the membrane (9, 10) and, when solubilized in the
presence of 0.5% Triton X-100, also behave as dimers (22, 23). The pooled active fraction from the 3'-5'-ADP-agarose column was loaded on
top of a 8-30% glycerol gradient and centrifuged for 40 h, as
described under "Experimental Procedures." Fig.
4A shows the profile of the
transporter activity throughout the gradient; a peak in the 120-kDa
area, corresponding to the native protein, was observed with a single
protein band of 60 kDa in the denaturating gel (Fig. 1, lane
9, and Fig. 4B), which correlated with the ATP transport activity (Fig. 4). This latter strategy gave a 70,000-fold overall purification and a recovery of 2% of the initial transport activity.
Photoaffinity Radiolabeling with
8-Azido[ Characterization of the ATP Transporter--
To characterize the
ATP transporter, we reconstituted the highly purified 3'-5'-ADP-agarose
fraction (Fig. 1, lane 7) into phosphatidylcholine
liposomes. Transport of ATP into proteoliposomes was saturable with an
apparent Km of 3.3 µM (Fig.
6), very similar to that of intact Golgi
vesicles (1.3 µM; Fig. 6, inset). The same
fraction was inactive in transport of PAPS, CMP-sialic acid, and
UDP-N-acetylgalactosamine (Table
II).
We have identified, purified, and characterized the ATP transport
activity from rat liver Golgi membranes. The transporter showed an
apparent molecular mass of 60 kDa, and its identity was confirmed by
functional reconstitution of the purified protein into liposomes as
well as photoaffinity labeling.
To purify the ATP transporter protein by column chromatography to
apparent homogeneity, a ~70,000-fold purification was required. This
fold of purification was expected because a similar apparent fold was
required for other low-abundance Golgi membrane proteins such as the
UDP-GalNAc transporter (23), the PAPS transporter (22), and the heparan
sulfate N-deacetylase/N-sulfotransferase (24).
After glycerol gradient ultracentrifugation, the transporter migrated
in the 120-kDa area, twice its apparent molecular mass as determined by
reducing gel electrophoresis, suggesting that the ATP transporter is
functional as a homodimer in Golgi membranes. These results are
consistent with previous reports showing that some nucleotide
derivative transporters are arranged in the Golgi membrane as
homodimers (9, 10) and with analogous results obtained by us with the
PAPS (22) and UDP-GalNAc transporters (23). Under the exact conditions
used in this work, the PAPS transporter, a 75-kDa protein that has been
shown to oligomerize as a homodimer (22), migrated in the 150-kDa area
of a glycerol gradient, whereas the UDP-GalNAc transporter, a 43-kDa
protein (23), migrated in the 80-90-kDa area (23).
In addition to the results obtained after column chromatography and
glycerol gradient ultracentrifugation (see Figs. 1 and 4), independent
evidence suggesting that the 60 kDa is indeed the ATP transporter was
obtained by functional reconstitution of the transporter into
proteoliposomes and photoaffinity radiolabeling using
8-azido[ The possibility that the 60-kDa protein we purified as the Golgi
membrane ATP transporter is instead the ATP transporter from a
different membrane, i.e. mitochondria or the ER, is very
unlikely because the mitochondrial ATP transporter has a different
molecular mass, ~35 kDa (25), and the one from the ER is not
functional when reconstituted into proteoliposomes by the freeze-thaw
procedure as used in this work (Ref. 26; see "Experimental
Procedures").
Although it has been clearly shown that phosphorylation is one of the
post-translational modifications that both secreted and integral Golgi
membrane proteins and proteoglycans undergo during their transit
through the Golgi apparatus (1-8), its functional importance is
largely unknown. If indeed, as it has been suggested, it contributes to
maintaining the stability of the protein backbone from proteolytic
degradation in situ (1, 2) or serves as a specific targeting
signal, as in the case of proteoglycans (7), remains to be determined.
Understanding the importance of phosphorylation as a post-translational
event and evaluation of how the possible regulation of ATP transport
into the Golgi lumen can affect the biosynthesis/modification and
function of macromolecules represents a major biological question. The
purification of the ATP transporter constitutes an important step
toward this direction. It will enable us to obtain the peptide sequence
of the transporter, and from this, it will allow us to proceed toward
its cloning. This in turn will enable us to study how the transporter
is arranged in the membrane, if it is structurally related to the ATP
transporter from mitochondria and ER, and whether its expression can
regulate the post-translational modifications of the above macromolecules.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (2-15 Ci/mmol) was from ICN
Pharmaceuticals, Inc. Extracti-Gel G was purchased from Pierce. All
other chemicals were obtained from Sigma.
-Amylase (200 kDa), alcohol dehydrogenase (150 kDa),
-galactosidase (120 kDa), phosphorylase B (100 kDa), tumor necrosis
factor
-convertase (80 kDa), and bovine serum albumin (66 kDa) were
used as internal molecular markers.
-32P]ATP
All the following experiments were performed in a dark room in
the presence of a filtered safe-light. Fractions to be photolabeled were incubated with 8-azido[
-32P]ATP (0.2 µM final concentration) at 0 °C for 1 min in 25 µl of buffer A. The mixture was irradiated on ice for 1 min in a Stratalinker UV 2400 oven (Stratagene; 5 cm of distance, maximum energy), and the reaction was stopped by the addition of loading buffer. Samples were then immediately subjected to 10% SDS/PAGE, and
the autoradiography of the dried gel was done at
80 °C on Kodak film.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Purification of the Golgi membrane ATP transporter
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Fig. 1.
SDS-PAGE of the different chromatographic
steps of the ATP transporter purification. Lane 1, Triton
X-100 extract; lane 2, first DEAE-Sephacel active fraction;
lane 3, Blue-Sepharose active fraction; lane 4,
second DEAE-Sephacel active fraction; lane 5,
carboxymethylcellulose active fraction (fraction 13 of Fig. 2);
lane 6, carboxymethylcellulose inactive fraction (fraction 7 of Fig. 2); lane 7, 3'-5'-ADP-agarose active fraction
(fraction 9 of Fig. 3); lane 8, 3'-5'-ADP-agarose inactive
fraction (flow-through); lane 9, glycerol gradient active
fraction (fraction 12 of Fig. 4); lane 10, glycerol gradient
inactive fraction (fraction 2 of Fig. 4). Lanes 1-6 and
8 were visualized with Coomassie-silver nitrate
staining, whereas lanes 7, 9, and 10 were
visualized by autoradiography after radioiodination.
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Fig. 2.
Elution profile of the ATP transport activity
from the carboxymethylcellulose column. Elution was with a linear
gradient of 0.1-1.5 M NaCl and 20-1% glycerol. Fractions
of 16 ml were collected, and aliquots of 100 µl were used to assay
ATP transport activity as described under "Experimental
Procedures."
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Fig. 3.
Elution profile of the ATP transport activity
from the 3'-5'-ADP-agarose column. Elution was with a linear
gradient of 0-1.5 M NaCl and 10-1% glycerol. Fractions
of 2.5 ml were collected, and aliquots of 100 µl were used to assay
ATP transport activity, as described under "Experimental
Procedures."
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Fig. 4.
Glycerol gradient sedimentation of the ATP
transport activity and SDS-PAGE profile. An active fraction from
purification Step 6 was loaded onto an 8-30% glycerol gradient and
centrifuged as described under "Experimental Procedures." Fractions
of 0.35 ml were collected and assayed for the ATP transport activity.
A, profile of the transport activity throughout the
gradient. The numbers on the top indicate the sedimentation position of
-amylase (200 kDa), alcohol dehydrogenase (150 kDa),
-galactosidase (120 kDa), phosphorylase B (100 kDa), tumor necrosis
factor
-convertase (80 kDa), and bovine serum albumin (66 kDa).
B, aliquots of active and inactive fractions were
radioiodinated before SDS-PAGE and then subjected to
autoradiography.
-32P]ATP--
Photoaffinity radiolabeling was
used as an independent criterion to demonstrate that the transport
activity is a protein of 60 kDa. We reconstituted into proteoliposomes
active fractions from the glycerol gradient (see Fig. 1, lane
9) and inactive fractions from the carboxymethylcellulose (see
Fig. 1, lane 6); these were then subjected to photolabeling
with 8-azido[
-32P]ATP, an azido derivative of ATP.
Only fractions active in ATP transport showed a radiolabeled protein
band of 60 kDa (Fig. 5, lane
1), whereas inactive fractions did not (Fig. 5, lane
2). Neither UV irradiation, used without the photoprobe, nor the
photoprobe by itself, without UV irradiation, resulted in photolabeling
of protein bands (results not shown).
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Fig. 5.
SDS-PAGE of proteoliposomes subjected to
photolabeling with
8-azido-[ -32P]ATP.
Proteoliposomes were prepared as described under "Experimental
Procedures." 8-Azido[
-32P]ATP was always used at 0.2 µM final concentration. Photolabeling was performed at
0 °C for 1 min (5 cm, maximum energy). Lane 1,
proteoliposomes from the active fraction number 12 of the glycerol
gradient (see Fig. 1, lane 9); lane 2,
proteoliposomes from the inactive fraction number 7 of the
carboxymethylcellulose (see Fig. 1, lane 6).
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Fig. 6.
Rate of ATP transport into proteoliposomes
and intact Golgi vesicles (inset). An active
fraction from the 3'-5'-ADP-agarose column (fraction number 9 from Fig.
3) was reconstituted into proteoliposomes and assayed for ATP
transport. Transport of ATP into intact Golgi vesicles
(inset) was assayed as described before (5).
Substrate specificity of the 3'-5'-ADP-agarose column eluate
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP, an azido anilide derivative of ATP.
When reconstituted into proteoliposomes, the highly purified
transporter was active in ATP transport, and the transport activity was
saturable with an apparent Km very similar to that
of intact Golgi vesicles, suggesting that the two activities are identical.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM34396 (to C. B .H.) and the Italian Consiglio Nazionale delle Ricerche (Contributo di Soggiorno to L. P.).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.
§ Supported by a Human Frontier Science Program Organization (HFSPO) long term fellowship.
To whom correspondence should be addressed: Dept. of Molecular
and Cell Biology (W-200), Boston University Goldman School of Dental
Medicine, 715 Albany St., Boston, MA 02118-2392. Tel.: 617-414-1040;
Fax: 617-414-1041; E-mail: chirschb{at}bu.edu.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; PAPS, adenosine 3'-phosphate 5'-phosphosulfate; PAGE, polyacrylamide gel electrophoresis.
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