From the Department of Molecular and Cell Biology,
Boston University Goldman School of Dental Medicine, Boston,
Massachusetts 02118, the ¶ Department of Biochemistry and
Molecular Biology, University of Massachusetts Medical Center,
Worcester, Massachusetts 01655, and the
Department of
Biochemistry, University of Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
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Glycosylation of glycoproteins, proteoglycans,
and glycosphingolipids occurs mainly in the lumen of the endoplasmic
reticulum and the Golgi apparatus. Nucleotide sugars, donors of all the sugars involved in Golgi glycosylation reactions, are synthesized in
the cytoplasm and require specialized transporters to be translocated into the lumen of the Golgi apparatus. By controlling the supply of
sugar nucleotides in the lumen of the Golgi apparatus, these transporters directly regulate the glycosylation of macromolecules transiting the Golgi. We have identified and purified the rat liver
Golgi membrane UDP-N-acetylgalactosamine transporter. The transporter was purified to apparent homogeneity by a combination of
conventional and dye color chromatography. An ~63,000-fold purification (6% yield) was achieved starting from crude rat liver Golgi membranes and resulting in a protein with an apparent molecular mass of 43 kDa. The transporter was active when reconstituted into
phosphatidylcholine vesicles and could be specifically photolabeled with
P3-(4-azidoanilido)-uridine-5'-[P1-32P]triphosphate,
an analog of UDP-N-acetylgalactosamine. Native functional
size determination on a glycerol gradient suggested that the
transporter exists as a homodimer within the Golgi membrane.
Glycoproteins, proteoglycans, and glycosphingolipids play
fundamental roles in physiologic and pathologic processes, including cell growth, development, modulation of growth factors and
transmembrane signals, oncogenesis, immune response, control of
permeability in kidney basement membrane, and coagulation. These
glycoconjugates also act as receptors for hormones, viruses, and
bacterial toxins (reviewed in Refs. 1-7). The common characteristic of
these complex cellular constituents is the presence of a carbohydrate
chain attached to a protein or lipid core, which often confers a
specific physiological or pathological role.
The glycosylation reactions involved in the assembly of the above
macromolecules occur mainly in the lumen of the endoplasmic reticulum
and the Golgi apparatus (reviewed in Refs. 8 and 9). Nucleotide sugars,
donors of all of the sugars involved in Golgi glycosylation reactions,
are synthesized in the cytoplasm and require specialized transporters
to be translocated into the Golgi apparatus to participate in these
biosynthetic events (reviewed in Refs. 8 and 9). These transporters are
antiporters; they translocate the nucleotide sugar to the inside of the
Golgi apparatus while the corresponding nucleoside monophosphate,
generated in the lumen of the Golgi apparatus through the action of a
glycosyltransferase and a nucleoside diphosphatase, exits (reviewed in
Refs. 8 and 9).
Nucleotide sugar transporters are of physiologic importance because
mutant mammalian, yeast, and Leishmania cells deficient in
these transport activities have a defect in the biosynthesis of the
corresponding macromolecules (9-12). This defect seems to be not only
quantitative but also qualitative. A mutant MDCK cell line (10, 11)
deficient by 98% in UDP-galactose transport into the Golgi lumen
showed a marked reduction in galactosylation of glycoproteins,
glycosphingolipids, and those proteoglycans containing galactose in
their polymer, like keratan sulfate. Other proteoglycans, like
chondroitin sulfate and heparan sulfate, which contain galactose solely
in the linkage region between the sugar polymer and the protein
backbone, were not reduced. Changes in the sulfation pattern of
chondroitin sulfate, probably a secondary effect, were also observed.
Similar results were also observed in Saccharomyces
cerevisiae (12) where a defect in the entry of GDP-mannose
into the lumen of the Golgi apparatus resulted in a significant
decrease in the mannosylation of lipids and O-linked mannoproteins and in the elongation of N-linked
carbohydrates of vacuolar proteins. A partial defect in the
biosynthesis of N-linked mannan chains of secreted proteins
was also observed. Together, the above results support the hypothesis
that by limiting the supply of sugar nucleotides in the lumen of the
Golgi apparatus, these transporters directly regulate the synthesis of
glycoconjugates. Finally, in Leishmania donovani, a defect
in the transport of GDP-mannose into the Golgi apparatus resulted in an
absence of virulence and in transmission of the parasite, offering an
attractive target for chemotherapy (13).
Considerable progress has been made in the past few years on the
molecular cloning of genes encoding some of these Golgi membrane transporters. Genes encoding CMP-sialic acid (14, 15), UDP-galactose (16, 17), and UDP-N-acetylglucosamine (18) transporters were
cloned from mammalian species, and a gene encoding a GDP-mannose transporter (13, 19) was identified in L. donovani. Also, genes encoding transporters for UDP-N-acetylglucosamine (20) and UDP-galactose (21) were cloned from yeast. All of these genes were
identified by complementation cloning in glycosylation mutant cells.
However, no cells have been identified that display a defect in the
transport of UDP-N-acetylgalactosamine into the Golgi
apparatus, suggesting that approaches other than complementation cloning must be used to identify the Golgi membrane UDP-GalNAc transporter. We chose to identify the UDP-GalNAc transporter by classical protein purification and biochemical reconstitution techniques. We previously showed that detergent extraction of Golgi
membranes followed by reconstitution of the extract into phosphatidylcholine liposomes resulted in proteoliposomes that were
active in specific nucleotide sugar transport (22). Here we used this
reconstituted proteoliposome system to monitor the purification of the
UDP-GalNAc transport activity from a rat liver Golgi membrane
preparation. Column chromatography and photoaffinity labeling were used
to identify a 43-kDa protein as the UDP-GalNAc transporter.
Proteoliposomes containing this protein were active in UDP-GalNAc but
not in other uridine nucleotide sugar transport, and native functional
size determination on a glycerol gradient suggested that the
transporter exists as a homodimer in the membrane.
Materials
Frozen rat livers were purchased from Pel-Freez Biologicals.
[3H]UDP-GalNAc (5-15 Ci/mmol) was purchased from
American Radiolabeled Chemicals, Inc. and Na125I (350-600
µCi/ml) from Amersham Pharmacia Biotech. Extracti-Gel G was purchased
from Pierce. All other chemicals were obtained from Sigma.
Methods
Purification of the Rat Liver Golgi Membrane UDP-GalNAc
Transporter
All of the operations 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. (23). 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), and 0.3% Triton X-100 (v/v) with protease inhibitors
(0.5 mM 4-(2-aminoethyl)benzene-sulfonyl 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 described 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, which was saved. The pellet was resuspended, and the extraction was repeated as described above. The
two Triton X-100 extracts were combined and diluted with buffer without
Triton X-100 to a final Triton X-100 concentration of 0.5%. NaCl up to
a final concentration of 200 mM was added.
Step 2: First DEAE-Sephacel Column--
The above described
Triton X-100 extract was applied to three DEAE-Sephacel columns (Sigma;
25 × 5 cm each) equilibrated with Buffer A (10 mM
Tris·HCl, pH 7.0, 1 mM MgCl2, 1 mM dithiothreitol, 1% glycerol, and 0.5% Triton X-100)
containing 200 mM NaCl. Elution was with 6 column volumes
of equilibration buffer followed by 4 column volumes of Buffer A
containing 700 mM NaCl. The salt-eluted material,
containing the UDP-GalNAc transport activity, was then concentrated and
diluted to a 300 mM NaCl final concentration using a
Minitan Ultrafiltration System (Millipore).
Step 3: Blue-Sepharose Column--
The above described fraction
was applied to three Blue-Sepharose columns (Amicon; 20 × 5 cm
each) equilibrated in Buffer A containing 300 mM NaCl. The
UDP-GalNAc transport activity eluted in the flow-through.
Step 4: Second DEAE-Sephacel Column--
The flow-through from
the Blue-Sepharose column was diluted to 100 mM NaCl with
Buffer A and applied to eight DEAE-Sephacel columns (Sigma; 23 × 2.7 cm each) equilibrated in Buffer A containing 100 mM
NaCl. The UDP-GalNAc transport activity eluted in the flow-through.
Step 5: Carboxymethylcellulose Column--
The second
DEAE-Sephacel flow-through was applied to 12 carboxylmethylcellulose
columns (Pharmacia; 23 × 3 cm each) equilibrated in Buffer A
containing 100 mM NaCl. The UDP-GalNAc transport activity eluted in the flow-through. This fraction was concentrated using a
Minitan Ultrafiltration System (Millipore) and adjusted to a final
concentration of 20% glycerol and 3% Triton X-100.
Step 6: Third DEAE-Sephacel Column--
The above described
fraction was applied to 12 DEAE-Sephacel columns (Sigma; 23 × 3 cm each) equilibrated in Buffer A containing 20% glycerol and 3%
Triton X-100 (Buffer B). Elution was with 4 column volumes of the
equilibration buffer followed by 4 column volumes of Buffer B
containing 300 mM NaCl and 3 column volumes of Buffer A
containing 1.5 M NaCl. This last elution volume was recovered in fractions of 18.5 ml each, and the UDP-GalNAc transport activity was eluted in fractions 1-30, which were pooled, desalted, and concentrated up to a final concentration of 20% glycerol and 1.5%
Triton X-100.
Step 7: Fractogel-EMD Column--
The above fraction was applied
to three Fractogel-EMD columns (EM Science; 15 × 1.5 cm each)
equilibrated with Buffer A containing 20% glycerol and 1.5% Triton
X-100 (Buffer C). Elution was with 4 column volumes of the
equilibration buffer followed by 4 column volumes of Buffer C
containing 1 M NaCl and, finally, 3 column volumes of
Buffer A containing 1.5 M NaCl. The UDP-GalNAc transport activity was eluted with 1.5 M NaCl.
Glycerol Gradient
The apparent functional weight of the UDP-GalNAc transporter was
estimated by analytical ultracentrifugation using an 8-30% glycerol
gradient in Buffer A. An active fraction obtained either from
purification Step 4 or Step 7 was concentrated and exchanged to 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 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. Isolation and Topography of Rat Liver Golgi Vesicles
For the photoaffinity radiolabeling studies, rat liver Golgi
vesicles were isolated as described (23) and resuspended in cryoprotective buffer (24). Sialyltransferase activity was enriched ~50-fold over crude homogenate. Approximately 90% of the vesicles were sealed and were of the same membrane topographical orientation as
those found in vivo (25).
Photoaffinity Radiolabeling with
[32P]AAUTP1
All of the following experiments were performed in a dark room
in the presence of a filtered safe-light. The photoaffinity reagent,
[32P]AAUTP, was synthesized as described previously (26).
Fractions to be photolabeled were incubated with
[32P]AAUTP (0.2 µM final concentration) at
0 °C for 1 min in 25 µl of Buffer A in the presence or absence of
nonradioactive UDP-GalNAc. 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 Transport Assay
Transport of solutes into intact rat liver Golgi vesicles was
assayed as described before (27) in the presence or absence of
nonradioactive AAUTP. To follow the transporter purification, the
UDP-GalNAc transport activity was reconstituted in phosphatidylcholine liposomes (22, 28) and incubated in the presence of
[3H]UDP-GalNAc (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 (29). Fractions
of 300 µl were collected, and the radioactivity was determined by
liquid scintillation spectrometry.
Protein Visualization
The purity of the various active and inactive fractions was
determined by SDS-PAGE. Visualization was done by Coomassie-silver nitrate staining (OWL separation system) or by labeling proteins with
300 uCi of Na125I using chloramine T (30). Protein was
quantified using the BCA protein assay kit (Pierce).
Purification of the UDP-GalNAc Transporter--
The rat liver
Golgi membrane UDP-GalNAc transporter was purified ~63,000 over the
crude Golgi preparation with a yield of 6.3% (Table
I). We began the purification with a
crude Golgi membrane preparation; the UDP-GalNAc 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
completely solubilize the membrane proteins. Approximately 90% of the
total UDP-GalNAc 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 and dye color columns. Details of the
different chromatographic steps are given under "Experimental Procedures," and the main results of each step are given in Table I.
To monitor the purification through the different chromatographic steps, membrane proteins were reconstituted into phosphatidylcholine liposomes by freeze-thawing and then assayed for their ability to
translocate radiolabeled UDP-GalNAc in vitro. The purity of the UDP-GalNAc transporter during the purification was determined by
SDS-PAGE (Fig. 1).
The Triton X-100 extract (Fig. 1, lane 1) was loaded onto a
first DEAE-Sephacel column followed by elution with 700 mM
NaCl in Buffer A. 78% of the transport activity was recovered with a
150-fold purification over the crude Golgi preparation (Fig. 1,
lane 2). In the next chromatographic step, we used a tandem sequence of three consecutive negative columns, respectively, Blue-Sepharose, DEAE-Sephacel, and carboxymethylcellulose. An important factor in this step was the Triton X-100 concentration. When
these columns were run in the presence of 0.5% Triton X-100, the
UDP-GalNAc transport activity was always found in the flow-through (negative chromatography), whereas when used in the presence of 3%
Triton X-100, the activity always bound to the matrix (positive chromatography). These three negative columns combined together provided important and substantial purification (Fig. 1, lanes 3-6), resulting in the binding of most of the applied
proteins but not the UDP-GalNAc transport activity. 62% of the
activity found in the active fraction from the first DEAE-Sephacel
column was recovered after these steps with a 17,560-fold overall
purification. In the next step we again used a DEAE-Sephacel column but
this time at higher Triton X-100 concentration (positive
chromatography). The active fraction obtained from the
carboxymethylcellulose column was concentrated to obtain a 3% Triton
X-100 concentration and applied to the third DEAE-Sephacel column. The
transporter activity was eluted with 1.5 M NaCl in Buffer A
and recovered in the first 30 fractions of the elution volume (Fig.
2) with a 35,000-fold overall
purification. This represented an important concentration step because
from the initial ~11 liters of the applied sample, the transporter
activity was recovered in ~500 ml. Silver staining of the active
fraction from the third DEAE-Sephacel column shows eight predominant
bands (Fig. 1, lane 7). In the next chromatographic step we
used a Fractogel-EMD column in the presence of 1.5% Triton X-100; this
resulted in the binding of all of the transporter activity to the
column. Although most of the proteins were eluted with 1 M
NaCl, the transporter activity was found in the 1.5 M NaCl
eluate. The Fractogel-EMD chromatography gave a high specific activity
transporter fraction. In order not to use high volumes of this active
fraction, small aliquots of the sample were subjected to
radioiodination with chloramine T prior to electrophoresis and
visualization with autoradiography. For comparison, the active sample
from the third DEAE-Sephacel column (sample applied to the
Fractogel-EMD) is shown after silver staining (Fig. 1, lane 7) and iodination (Fig. 1, lane 8). The SDS-gel profile
(Fig. 1, lane 10) of the active fraction from the
Fractogel-EMD chromatography showed two bands of 98 and 43 kDa. Only
the 43-kDa band was not visualized in fractions inactive (Fig. 1,
lane 11) for the transport activity, suggesting that the
UDP-GalNAc transport activity had a mobility of 43 kDa.
Photoaffinity Radiolabeling with
[32P]AAUTP--
Photoaffinity radiolabeling in the
presence or absence of nonradioactive UDP-GalNAc was used as a
different criterion to demonstrate that the transport activity is a
43-kDa protein. AAUTP is a membrane-impermeant photoaffinity reagent
that has been shown to bind and label various glycosyltransferases and
to inhibit uridine nucleotide sugar transport into endoplasmic
reticulum vesicles (26). When used at different concentrations in the
absence of UV light, AAUTP inhibited transport of UDP-GalNAc into Golgi
membrane vesicles (Table II) without affecting the integrity of the vesicles (results not shown). Under the
same conditions AAUTP also inhibited other uridine nucleotide sugars
but not CMP-sialic acid transport (results not shown). We then
subjected Golgi membrane vesicles to photolabeling with [32P]AAUTP in the absence or presence of nonradioactive
UDP-GalNAc. Although several bands could be visualized (Fig.
3, lane 1), the 43-kDa band
was the only one to be protected after preincubation with
nonradioactive UDP-GalNAc used at different concentrations (Fig. 3,
lanes 2 and 3); 5 µM UDP-GalNAc was
able to almost completely protect the 43-kDa band from photoaffinity
labeling. Neither UV irradiation, used without the photoprobe, nor the
photoprobe by itself, without UV irradiation, resulted in photolabeling
of bands (results not shown). We finally reconstituted active and
inactive fractions from the Fractogel-EMD column into proteoliposomes
and subjected them to photolabeling. Only fractions that were active in
the UDP-GalNAc transport showed a radiolabeled band of 43 kDa (Fig.
4, lane 4) whereas inactive
fractions did not (Fig. 3, lane 5). When the highly purified
active fraction from the Fractogel-EMD column was assayed for
nucleotide sugar transport activities, no other uridine nucleotide
sugars were active (Table III),
indicating that the UDP-GalNAc transporter is highly
substrate-specific.
Glycerol Gradient--
A glycerol gradient was used to estimate
the functional size of the UDP-GalNAc transporter. The rationale for
using the glycerol gradient was based on the fact that Golgi nucleotide
derivative transporters are homodimers in the membrane (9, 28), and when solubilized in the presence of 0.5% Triton X-100, they also appear to behave as dimers.2
Separate aliquots from active fractions of the second DEAE-Sephacel column or the Fractogel-EMD column were loaded on top of a 8-30% glycerol gradient and centrifuged for 40 h, as described under "Experimental Procedures." Fig. 4 shows the profile of the
transporter activity throughout the gradient. A peak in the 80-90-kDa
area was observed consistent with the proposal that the 43-kDa band is
recovered as a dimer in Triton extracts and may function as a dimer
within the Golgi membrane.
We have identified and purified the UDP-GalNAc transporter
activity from rat liver Golgi membranes. The transporter showed an
apparent molecular mass of 43 kDa, and its identity was confirmed by
functional reconstitution of purified material as well as photoaffinity labeling.
To identify the UDP-GalNAc transporter by column chromatography, we had
to obtain a ~63,000-fold purification. This was expected because a
similar apparent fold of purification was required for other low
abundance Golgi membrane proteins, such as the PAPS transporter (28)
and the heparan sulfate
N-deacetylase/N-sulfotransferase (31). A key
element during the purification was the use of three different negative
chromatographic columns. They were specifically designed to obtain a
rapid and substantial purification of the transporter activity. The
nature of the influence of the final Triton X-100 concentration on the
chromatographic behavior of the transporter during these negative
chromatographic steps is unclear. It is possible that the precise
Triton X-100 concentration affects the physical environment of the
protein in such a way so as to modulate the exposure of charged
residues on the protein and the efficiency with which they interact
with the various chromatographic resins (32). The UDP-GalNAc
transporter, like other Golgi membrane transporters, is an integral
membrane protein and would be expected to have a highly hydrophobic
character. The hydrophobicity of the environment as caused by the
Triton X-100 concentration can probably determine the number of charges
exposed and ultimately the possible interaction between the protein and
the active group of the resin (32).
The Fractogel-EMD column eluate (Fig. 1, lane 10) showed two
bands of 98 and 43 kDa, respectively. However, the 43-kDa band was not
present in the inactive fractions (Fig. 1, lane 11).
Although not reported under "Results," another column,
Macro-Prep-Q-Anion (Bio-Rad), also gave similar results showing that
the 43-kDa band was the only band always found in the active fractions
and never in the inactive fractions. In conclusion, the results
obtained by column chromatography suggest that the UDP-GalNAc
transporter is a 43-kDa protein.
Additional independent evidence suggesting that the 43-kDa band is
indeed the UDP-GalNAc transporter was obtained with photoaffinity radiolabeling using AAUTP, an azido-anilido derivative of UTP. When we
photolabeled Golgi membrane vesicles with [32P]AAUTP,
several bands were visualized, but only one band of 43 kDa was
protected from labeling when nonradioactive UDP-GalNAc was included in
the labeling reaction. This result is in strong agreement with previous
studies showing that nucleotide sugar transporters are highly specific
(reviewed in Refs. 8 and 9). A 43-kDa band could also be visualized
from a fraction that was active for the UDP-GalNAc transport activity.
The absence of radiolabeling of fractions that were inactive in
UDP-GalNAc transport activity is consistent with these results.
Nucleotide derivative transporters appear to be arranged in the Golgi
membrane as homodimers. Using glycerol gradient ultracentrifugation
(33), we showed that the UDP-GalNAc transporter has a size of 80-90
kDa, roughly twice its apparent molecular mass as determined by
reducing gel electrophoresis. It is well known that the assembly state
of a membrane protein in the presence of a detergent also depends on
the concentration and physical characteristics of the detergent and the
ionic strength of the medium (33). Although the results we obtained
cannot by themselves demonstrate the assembly state of the transporter and therefore the subunit size, they are consistent with the results obtained after column chromatography, i.e. the UDP-GalNAc
transporter, like other nucleotide derivative transporters, may be
functional as a homodimer in Golgi membranes. Our interpretation is
supported by the evidence that the PAPS transporter, a 75-kDa protein
that has been shown to oligomerize as a homodimer (28), migrated in the
150-kDa area of a glycerol gradient performed under exactly the same conditions.
Better understanding of how and to what extent the regulation of
nucleotide sugar transport into the Golgi lumen can affect the
glycosylation of macromolecules in the Golgi apparatus requires knowledge of the amino acid and nucleic acid sequences of such transporters. This can only be obtained by the identification of more
mutants defective in transport and cloning of the respective genes or
by the purification of such transporters via the approach outlined
here. The purification of the UDP-GalNAc transporter constitutes an
important step toward understanding the role of N-acetylgalactosamine in the above glycoconjugates and will
enable us to obtain the peptide sequence of the transporter and then proceed toward its cloning. This cloning in turn will allow us to study
in more detail how the transporter is arranged in the membrane, whether
it is structurally related to other nucleotide sugar transporters, and
most importantly, whether its expression can regulate the biosynthesis
and modifications of Golgi lumenal glycoproteins, proteoglycans, and
glycosphingolipids containing N-acetylgalactosamine in their
carbohydrate chains. The purification will also permit us to make
specific antibodies and to determine the localization of the
transporter within the Golgi apparatus and whether it forms a complex
with the corresponding transferases and nucleotide diphosphatase in the
Golgi membrane.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-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.
80 °C on Kodak film.
RESULTS
Purification of the rat liver Golgi membrane UDP-GalNAc transporter
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Fig. 1.
SDS-PAGE of the different chromatographic
steps of the UDP-GalNAc 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; lane 6,
carboxymethylcellulose inactive fraction; lane 7, third
DEAE-Sephacel active fraction; lane 8, third DEAE-Sephacel
active fraction; lane 9, third DEAE-Sephacel inactive
fraction; lane 10, Fractogel-EMD active fraction; lane
11, Fractogel-EMD inactive fraction. Lanes 1-7, 9, and
11 were visualized with Coomassie-silver nitrate staining,
whereas lanes 8 and 10 were visualized by
autoradiography after radioiodination.
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Fig. 2.
Elution profile of the UDP-GalNAc transport
activity from the third DEAE-Sephacel column. Elution was with 3 column volumes of Buffer A containing 1.5 M NaCl. 18.5-ml
fractions were collected, and 100-µl aliquots were used to assay
UDP-GalNAc transport activity, as described under "Experimental
Procedures."
Effect of AAUTP on UDP-GalNAc transport into Golgi membrane vesicles
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Fig. 3.
SDS-PAGE and autoradiography of intact Golgi
vesicles and proteoliposomes subjected to UV photolabeling with
[32P]AAUTP. Rat liver Golgi membrane vesicles and
reconstituted proteoliposomes were prepared as described under
"Experimental Procedures." [32P]AAUTP was always used
at 0.2 µM final concentration, and photolabeling was
performed at 0 °C for 1 min (5 cm, maximum energy). Lane
1, rat liver Golgi vesicles (8 µg of protein) photolabeled in
the absence of nonradioactive UDP-GalNAc; lane 2, rat liver
Golgi vesicles (8 µg of protein) preincubated for 1 min at 30 °C
with 1 µM nonradioactive UDP-GalNAc prior to
photolabeling; lane 3, rat liver Golgi vesicles (8 µg of
protein) preincubated for 1 min at 30 °C with 5 µM
nonradioactive UDP-GalNAc prior to photolabeling; lane
4, proteoliposomes from Fractogel-EMD active fraction (see Fig. 1,
lane 10); lane 5, proteoliposomes from
Fractogel-EMD inactive fraction (see Fig. 1, lane 11).
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Fig. 4.
Glycerol gradient sedimentation profile of
the UDP-GalNAc transport activity. An active fraction from either
purification Step 4 or Step 7 was loaded onto an 8-30% glycerol
gradient and centrifuged as described under "Experimental
Procedures." 0.35-ml fractions were collected and assayed for the
UDP-GalNAc transport activity. Representative sedimentation profiles of
four different gradients are shown. The bars indicate the
sedimentation positions 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).
Uridine nucleotide sugar transport activities from the active fraction
of the Fractogel-EMD column
DISCUSSION
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM34396 (to C. B. H.) and GM55427 (to A. K. M.) 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 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.: 617-414-1050; Fax: 617-414-1041; E-mail: chirschb{at}bu.edu.
The abbreviations used are: [32P]AAUTP, P3-(4-azidoanilido)-uridine-5'-[P1-32P]triphosphate; AAUTP, P3-(4-azidoanilido)-uridine-5'-triphosphate; PAPS, adenosine 3'-phosphate 5'-phosphosulfate; PAGE, polyacrylamide gel electrophoresis.
2 L. Puglielli, E. C. Mandon, and C. B. Hirschberg, unpublished results.
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REFERENCES |
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