From the Department of Biochemistry and Molecular Biology, Thoracic Diseases Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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Glucosylceramide synthase (GCS) catalyzes the
transfer of glucose from UDP-glucose to ceramide to form
glucosylceramide, the precursor of most higher order
glycosphingolipids. Recently, we characterized GCS activity in highly
enriched fractions from rat liver Golgi membranes (Paul, P., Kamisaka,
Y., Marks, D. L., and Pagano, R. E. (1996) J. Biol. Chem. 271, 2287-2293), and human GCS was cloned by others
(Ichikawa, S., Sakiyama, H., Suzuki, G., Hidari, K. I.-P. J.,
and Hirabayashi, Y. (1996) Proc. Natl. Acad. Sci.
U. S. A. 93, 4638-4643). However, the polypeptide responsible for GCS activity has never been identified or characterized. In this
study, we made polyclonal antibodies against peptides based on the
predicted amino acid sequence of human GCS and used these antibodies to
characterize the GCS polypeptide in rat liver Golgi membranes. Western
blotting of rat liver Golgi membranes, human cells, and recombinant rat
GCS expressed in bacteria showed that GCS migrates as an ~38-kDa
protein on SDS-polyacrylamide gels. Trypsinization and
immunoprecipitation studies with Golgi membranes showed that both the C
terminus and a hydrophilic loop near the N terminus of GCS are
accessible from the cytosolic face of the Golgi membrane. Treatment of
Golgi membranes with N-hydroxysuccinimide ester-based
cross-linking reagents yielded an ~50-kDa polypeptide recognized by
anti-GCS antibodies; however, treatment of ~10,000-fold purified
Golgi GCS with the same reagents did not yield cross-linked GCS forms.
These results suggest that GCS forms a dimer or oligomer with another
protein in the Golgi membrane. The migration of solubilized Golgi GCS
in glycerol gradients was also consistent with a predominantly oligomeric organization of GCS.
Glucosylceramide is synthesized by UDP-glucose:ceramide
glucosyltransferase (glucosylceramide synthase
(GCS)1) (1), a resident
integral membrane protein of the cis/medial-Golgi membrane (2-4). Glucosylceramide is the common precursor of most higher order glycosphingolipids, which are important cell membrane constituents and have been implicated as important factors in development, differentiation, tumor progression, and pathogen/host interactions (5-11). Thus, GCS may play significant roles in several biological processes by regulating the overall synthesis of
glucosylceramide-derived glycosphingolipids. However, surprisingly
little is known about the polypeptide responsible for GCS activity.
We recently solubilized and partially purified (~10,000-fold)
enzymatically active rat liver GCS (12). We found that
detergent-solubilized GCS peaked in glycerol gradients at an apparent
molecular mass of ~60 kDa, but were unable to conclusively identify
the GCS polypeptide on SDS-polyacrylamide gels. Since then, GCS was
cloned from a human cDNA library by rescue of a mutant mouse cell
line deficient in GCS activity (13). The predicted amino acid sequence
of the cloned enzyme encodes a protein with a calculated molecular mass of ~45 kDa, but the cloned enzyme was not visualized by
SDS-polyacrylamide gel electrophoresis. The difference between the
predicted molecular mass of GCS based on its amino acid sequence and
its apparent size in glycerol gradients suggests that GCS may be
organized in a dimer or oligomer; however, this possibility has not yet been investigated. In addition, no homology was found between the
predicted amino acid sequence of GCS and any previously known protein.
Thus, except for the identification of a predicted N-terminal membrane-spanning domain (13) and observations by us (2) and others (3,
4) that the active site of GCS is accessible to proteases applied to
the cytosolic face of the Golgi membrane, no information is available
on the secondary structure of GCS.
Given the unresolved issues concerning the GCS polypeptide, we prepared
polyclonal antibodies against peptides based on the predicted amino
acid sequence of human GCS and used these antibodies to identify the
GCS polypeptide, to investigate the topology of GCS in rat liver Golgi
membranes, and to provide evidence that GCS is organized as a dimer or
oligomer in the Golgi membrane.
Preparation of Rat Liver Golgi Fractions--
Golgi membrane
fractions were prepared from male Sprague-Dawley rats (5 weeks old) as
described previously (12, 14, 15) with the following modifications.
Livers were homogenized (25%, w/v) in 0.25 M sucrose in
buffer A (50 mM Tris-HCl, pH 7.4, and 25 mM
KCl) with protease inhibitors (10 µg/ml each leupeptin, tosylarginylmethyl ester, and aprotinin; 1 µg/ml each pepstatin and
antipain; and 25 mM 4-amidophenylmethanesulfonyl fluoride (all from Sigma)) using a Polytron (Brinkmann Instruments) at setting 1 for ~30 s. The homogenate was filtered through cheesecloth and then
adjusted (150 parts homogenate and 95 parts 2 M sucrose in
buffer A (v/v)) to a final concentration of 1.07 M sucrose. The adjusted homogenate (19 ml/tube) was loaded into Beckman SW 28 tubes; 9 ml each of 0.9 and 0.2 M sucrose in buffer A were then sequentially overlaid above the homogenate. The tubes were then
centrifuged in an SW 28 rotor for 2 h at 83,000 × g. Golgi fractions were collected at the 0.2/0.9
M sucrose interface.
To prepare right-side-out "intact" Golgi membranes, Golgi fractions
were mixed 1:1 (v/v) with buffer B (50 mM HEPES, pH 7.4, 100 mM KCl, and the protease inhibitors as indicated above)
with 20% glycerol and centrifuged for 90 min at 200,000 × g. Golgi membrane pellets were resuspended at one-twentieth
of their original volume in 0.25 M sucrose in buffer B. N-Lauroylsarcosine (NLS)-washed Golgi membranes were
prepared as described previously (12). In some experiments, NLS-washed
Golgi membranes were solubilized with either 1% Igepal CA-630 (a
Nonidet P-40 equivalent from Sigma) or 1% CHAPS and 0.2% Triton X-100
in buffer B. The extract was then centrifuged at 200,000 × g, and the pellet was discarded. Fractions ~10,000-fold
enriched in GCS were prepared from NLS-washed Golgi membranes using
dye-agarose columns as described previously (12) and then concentrated
and equilibrated with buffer B containing 1% Igepal CA-630 using
Centricon 50 filters (Amicon, Inc., Beverly, MA).
Preparation of Polyclonal Antibodies--
Four synthetic
peptides were synthesized based on hydrophilic regions of the predicted
amino acid sequence of human GCS (13). The peptides used were
CTISWRTGRYRLRCGGTAEEILDV (referred to as GCS-1),
AMQNSGSYSISQFQSRMIRWTKLRC-NH2 (GCS-2),
KPLKGVDPNLINNLETFFELDYC-NH2) (GCS-5), and
TRLHLNKKATDKQPYSKLPGC-NH2 (GCS-6), which correspond to amino acids 372-394, 257-280, 57-78, and 33-52, respectively, of
the human GCS sequence (13), except that cysteine was added to the N
terminus of GCS-1, and cysteine-NH2 was added to the C
terminus of GCS-2, -5, and -6 to facilitate peptide conjugation. Fractions of each peptide were conjugated to keyhole limpet hemocyanin. To prepare polyclonal antibodies, New Zealand White rabbits (two rabbits/peptide) were injected with peptides (500 µg of conjugate plus 100 µg of the corresponding free peptide for initial injection) in Freund's adjuvant and boosted (100 µg each of conjugate and free
peptide) every 20 days. Bleeds were tested for immunoreactivity to GCS
by immunoprecipitation assays and Western blotting. For some studies,
antibodies were affinity-purified using the corresponding peptide
conjugated to Aminolink Plus columns (Pierce).
Immunoprecipitation and Immunopurification Methods--
For
immunoprecipitation and immunopurification of active GCS, Golgi samples
were solubilized in 1% CHAPS and 0.2% Triton X-100 in buffer B. For
immunoprecipitation to be followed by Western blotting, samples were
solubilized in 150 mM NaCl, 1.0% Igepal CA-630, 0.5%
sodium deoxycholate, 0.1% SDS, and 50 mM Tris-HCl, pH 8.0 (radioimmune precipitation assay buffer) (16), or in 1.0% Triton
X-100, 0.2% SDS, 150 mM NaCl, 0.5 mM EDTA, and
10 mM Tris-HCl, pH 8.0 (17). Samples were incubated for
2 h at room temperature with affinity-purified antibodies
conjugated to protein A-Sepharose CL-4B beads (Sigma) prepared using
dimethyl pimelimidate (16). The samples were then centrifuged; the
supernatant was saved; and the pellet was washed three times in the
solubilizing buffer. Pellets and supernatants were analyzed by GCS
activity assays or by Western blotting (see below). Human GCS was
immunoprecipitated from lysates of differentiated cultured human
keratinocytes as described (10). For immunopurification, solubilized
rat Golgi GCS was eluted from antibody-bead complexes with 3 M MgCl2 and 25% ethylene glycol, pH 7.2 (18).
Cross-linking Studies--
Bis(sulfosuccinimidyl) suberate (BS),
disuccinimidyl suberate (DSS), dithiobis(succinimidyl propionate)
(DSP), 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), and
sulfosuccinimidyl 6-(biotinamido)hexanoate were from Pierce.
Cross-linking reagents dissolved in Me2SO were added to
Golgi fractions or detergent extracts (in 50 mM HEPES, pH
7.4) to a final concentration of 5 mM cross-linker with
10% Me2SO. Samples were then incubated for 1 h at
room temperature, and the cross-linkers were quenched with 100 mM Tris at 4 °C for 30 min. Finally, the samples were
stored at Expression of Recombinant Rat GCS--
The full coding region of
rat GCS was amplified by polymerase chain reaction from a rat brain GCS
cDNA clone.2 The
polymerase chain reaction product was cloned into the TA vector
(Invitrogen, Carlsbad, CA). To delete a portion of the N terminus of
GCS, the position of the initiating ATG codon was altered using the
QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).
GCS cDNAs were subcloned into the pET-3d expression vector and
expressed in BL21(DE3) bacteria (Novagen) according to the
manufacturer's instructions. After 30 min at 37 °C, bacterial cell
pellets were harvested by centrifugation and then stored at Controlled Proteolysis and Immunoisolation
Studies--
Right-side-out Golgi membranes were prepared as described
above, except that the membranes were resuspended to a final
concentration of ~3 mg/ml protein in trypsinization buffer (0.25 M sucrose, 50 mM HEPES, pH 7.4, 100 mM KCl, 1 µg/ml pepstatin, and 2 mM EDTA). Trypsin (Worthington) was added at a ratio of 1:8 (trypsin/Golgi protein (w/w)), and samples were incubated at 37 °C. At various time
points, samples were placed on ice, and soybean trypsin inhibitor (Sigma) was added at a ratio of 20:1 (soybean trypsin inhibitor/trypsin (w/w)). Aliquots of the samples were tested for GCS and sphingomyelin synthase (SMS) activity or subjected to Western blotting as described below. For immunoisolation of Golgi vesicles, right-side-out Golgi membranes (see above) were incubated with anti-GCS antibody-bead complexes for 2 h at room temperature. Samples were washed in 0.25 M sucrose in buffer A, loaded onto a 0.9 M
sucrose cushion in 1.5-ml microcentrifuge tubes, and centrifuged at
11,000 × g for 5 min. Under these conditions,
membranes bound to beads were pelleted, whereas unbound membranes were
retained at the 0.25/0.9 M sucrose interface. The
distribution of Golgi membranes was assessed by measuring GCS activity
in pellets and supernatants
Glycerol Gradient Fractionation--
Aliquots of
N-lauroylsarcosine-washed Golgi membranes (0.5 ml) were
solubilized with 1% Igepal CA-630 (see above); adjusted to 5%
glycerol; and loaded onto 10.5-ml 8-25% glycerol gradients prepared
in 0.3% Igepal CA-630, 50 mM HEPES, pH 7.4, 50 mM KCl, 2 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml tosylarginylmethyl ester, 1 µg/ml pepstatin, and 25 mM 4-amidophenylmethanesulfonyl fluoride. The gradients
were centrifuged for 18 h at 200,000 × g in a
Beckman SW 41 rotor at 4 °C. After centrifugation, gradient
fractions (0.8 ml) were collected and analyzed for GCS activity or by
Western blotting. In some cases, Igepal CA-630-solubilized samples were treated with cross-linking reagents either before or after gradient fractionation.
Miscellaneous Methods--
Total microsomes were prepared as
described previously (19). GCS and SMS activities were measured using
N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl-D-erythro-sphingosine as a substrate as described (12). Total protein was measured with
Coomassie Blue dye reagent (Bio-Rad) as described (20) using bovine
serum albumin as a standard. For SDS-polyacrylamide gel
electrophoresis, samples were solubilized in sample buffer (200 mM Tris base, pH 6.8, 2% SDS, 20% glycerol, 8 M urea, 0.4 mg/ml bromphenol blue, ±7.5 mg/ml
dithiothreitol) and incubated for 15 min at 37 °C.
SDS-polyacrylamide gel electrophoresis was performed as described (21).
SDS-polyacrylamide gels were transferred to polyvinylidene difluoride
membranes (Millipore Corp.) using transfer buffer with 20% methanol as
described (22). Western blotting of the polyvinylidene difluoride
membranes with anti-GCS antibodies and horseradish
peroxidase-conjugated goat anti-rabbit secondary antibody was performed
as described (10). Signals on Western blots were visualized with
chemiluminescent reagents (NEN Life Science Products) and exposed to
XAR film (Eastman Kodak Co.).
Development of Polyclonal Antibodies against GCS--
To develop
antibodies against the GCS protein, we synthesized four peptides
(referred to as GCS-1, -2, -5, and -6; see Fig. 1A) based on
hydrophilic regions of the predicted amino acid sequence of human GCS
and injected these into rabbits. Antibodies against peptides GCS-1, -5, and -6 each recognized an ~38-kDa polypeptide in rat liver Golgi
membrane fractions and in fractions of enzymatically active GCS
purified from solubilized Golgi membranes on an anti-GCS-1 antibody
affinity column (Fig. 1B),
slightly lower than the predicted molecular mass (44.9 kDa) of human
GCS (13). The migration of this immunoreactive band was unchanged by
the inclusion of reducing agents (data not shown). Antisera made
against the GCS-2 peptide did not recognize the ~38-kDa band or other
specific bands in Western blotting or immunoprecipitations (data not
shown). Western blots showed that anti-GCS-1 and anti-GCS-6 antibodies
immunoprecipitated the ~38-kDa band from Golgi fractions under mildly
denaturing conditions (radioimmune precipitation assay buffer);
however, the anti-GCS-5 antibody immunoprecipitated the ~38-kDa band
only under strongly denaturing conditions (data not shown). These data demonstrate that rat Golgi GCS runs as an ~38-kDa polypeptide on
SDS-polyacrylamide gels. GCS was not detected by Western blotting in
crude liver fractions (homogenates or total microsomes) loaded at 20 µg/lane (Fig. 1B), probably due to the low overall
expression of GCS in liver.
To further characterize the GCS polypeptide, we compared the migration
on SDS-polyacrylamide gels of rat Golgi GCS with that of recombinant
rat GCS expressed in Escherichia coli and GCS
immunoprecipitated from differentiated human keratinocytes, a cell type
that expresses a high level of GCS (10). Rat GCS is 97% identical to
human GCS at the amino acid level, with a predicted molecular mass of 44.8 kDa.2 Western blotting showed that rat Golgi GCS
migrated identically (~38 kDa) on SDS-polyacrylamide gels compared
with rat GCS expressed in E. coli and GCS immunoprecipitated
from human keratinocytes. Immunoprecipitation and Western blotting with
the anti-GCS-1 antibody, which recognizes the C-terminal amino acids of
GCS, indicated that the C terminus of GCS is present in the mature rat
and human proteins (Fig. 1). To investigate the possibility that the
N-terminal region of GCS is missing (due to either post-translational
cleavage or GCS translation initiating from a second methionine (amino acid 11 of the rat GCS sequence), we expressed a deletion mutant that
lacks the first 10 amino acids of GCS. This mutant form of GCS ran
lower on Western blots than wild-type recombinant rat GCS (Fig.
1C) and, in addition, had only 4% of the activity of the
wild-type form.2 Together, these data indicate that both
rat and human GCS proteins migrate anomalously as ~38-kDa
polypeptides and that the full amino acid sequence, including the N
terminus, is present in the mature forms.
GCS Topology--
We then used the anti-GCS antibodies to further
investigate the topology of GCS in the Golgi membrane. Golgi membrane
fractions were incubated with a low concentration of trypsin over time; samples were then tested for GCS and SMS activities, and the integrity of the GCS polypeptide was assessed by Western blotting. In agreement with our previous study (2), GCS activity decreased rapidly over time,
whereas SMS activity was relatively unaffected (Fig. 2A). When incubations were
performed in the presence of 0.02% Triton X-100, SMS was also
rapidly degraded (data not shown). These results demonstrate that,
under the experimental conditions used here, the Golgi membranes are
predominantly nonpermeable to trypsin and are right-side out (GCS, but
not SMS, is accessible to trypsin) (2, 23).
Western blotting using three different anti-GCS antibodies showed that
GCS was rapidly degraded by trypsin, with little of the intact
~38-kDa form present after 5 min of incubation (Fig. 2B).
The anti-GCS-1 antibody recognized only intact GCS and detected no
lower molecular mass forms after trypsin treatment, suggesting that the
GCS-1 epitope is readily accessible to the protease. At 5 min and later
time points, an ~35-kDa GCS fragment was recognized by both the
anti-GCS-5 and anti-GCS-6 antibodies (Fig. 2B). Finally, a
third fragment (~33 kDa), which peaked in appearance at 10 min, was
recognized by only the anti-GCS-5 antibody (Fig. 2B). At
later time points, the ~33-kDa fragment was apparently degraded as
well. These results show that both the GCS-1 and GCS-6 epitopes may be
cleaved by trypsin, leaving the ~33-kDa fragment, and suggest that
this fragment may be partially protected from proteolysis by its
secondary structure.
We noted that after 10 min of proteolysis, almost no intact GCS was
recognized by the anti-GCS-1 antibody, although large fragments
recognized by the other antibodies were present, and GCS activity had
decreased to ~35% of initial levels (Fig. 2). These results suggest
that the C-terminal region recognized by the anti-GCS-1 antibody may be
important for optimal GCS activity. Expression of a recombinant GCS
mutant lacking the 8 last amino acids of the C terminus showed that it
had only ~4% activity compared with wild-type GCS,2
supporting the hypothesis that the C terminus of GCS is important for activity.
We then tested the ability of different anti-GCS antibodies to interact
in situ with GCS in right-side-out Golgi membranes. GCS-1
immunoprecipitated most (~80%) of the GCS activity in Golgi vesicles; GCS-6 partially immunoprecipitated activity (~50%); but
the anti-GCS-5 antibody did not immunoprecipitate activity (Fig.
2C). Taken together with the controlled proteolysis studies, these data support a model for GCS in which the C terminus (GCS-1 epitope) extends into the cytosol from the outer face of the Golgi membrane and the GCS-6 epitope forms a loop or hinge that is also partially accessible to the cytosol, whereas the GCS-5 epitope is
somewhat protected by the protein secondary structure or interactions with the Golgi membrane.
GCS Oligomerization--
We next began studies to determine if GCS
is organized in oligomers. Experiments in which a series of
bifunctional cross-linking agents were reacted with Golgi membranes
demonstrated that cross-linkers based on
N-hydroxysuccinimide esters (BS, DSS, DSP, and DTSSP) consistently induced the appearance of an ~50-kDa polypeptide recognizable by anti-GCS antibodies on Western blots (Fig.
3). Additional higher cross-linked forms
were also detectable, especially with the anti-GCS-1 antibody (Fig.
3A). In general, cross-linking caused an increase in the
overall signal of immunoreactive material on blots, possibly due to
changes in immunoreactivity after protein derivatization by
cross-linkers or increased efficiency of electrophoretic transfer of
higher molecular mass derivatized forms relative to the unaltered GCS
monomer.
When Golgi fractions were incubated with the cleavable cross-linker
DTSSP, the ~50-kDa band formed was recognized by the anti-GCS-5 antibody, but not by GCS-1 (Fig. 3B). However, cleavage of
DTSSP with dithiothreitol in these samples eliminated the ~50-kDa
band and restored the anti-GCS-1 antibody-immunoreactive monomeric GCS
band. These results are consistent with the hypothesis that the
~50-kDa form is a dimer or oligomer that contains GCS, rather than an
artifact caused by reaction of the cross-linker with some other
protein. Additional control experiments demonstrated the following. 1)
The cross-linked forms were not recognized by nonimmune sera; and 2)
higher molecular mass forms were not generated by reaction of Golgi
proteins with sulfosuccinimidyl 6-(biotinamido)hexanoate, a reagent
with reactivity (i.e. primary amines) similar to that of the
bifunctional cross-linkers used, but that does not form cross-links
(data not shown).
We next compared the cross-linking of GCS in four different
Golgi-derived fractions. We found that reaction of BS or DSS with intact Golgi membranes, Golgi membranes washed with
N-lauroylsarcosine (~75% of total proteins (but little
GCS) are removed by this step (12)), and Igepal CA-630 extracts of
NLS-washed Golgi membranes all yielded similar patterns of
GCS-immunoreactive cross-linked forms (Fig. 3C). Treatment
of dye-agarose-purified GCS with cross-linking reagents, however, did
not result in the formation of the ~50-kDa form or other
immunoreactive cross-linked species (Fig. 3C). Similarly, reaction of Igepal CA-630-solubilized recombinant rat GCS expressed in
bacteria with cross-linkers also did not generate any cross-linked GCS
forms (data not shown). These results suggest that GCS in Golgi
membranes specifically cross-links to a closely associated protein,
which is absent in dye-agarose-purified GCS and bacterially expressed
GCS (i.e. GCS in the Golgi membrane is a heterodimer or heterooligomer).
To provide further characterization of GCS oligomers, we fractionated
detergent-solubilized Golgi membranes on a glycerol gradient and then
cross-linked each sample with 5 mM BS after gradient
fractionation to "trap" any oligomeric GCS in higher molecular
mass forms that would be visible by Western blotting. Without
cross-linking, only the GCS monomer (~38-kDa form) was visible by
Western blotting; this form peaked early in the gradient (approximately
fractions 3-5) and coincided with the peak of GCS activity (Fig.
4). Note that without cross-linking, any
GCS oligomers present in these fractions are dissociated by the
conditions of SDS-polyacrylamide gel electrophoresis so that only the
GCS monomer is visualized on Western blots.
In cross-linked fractions (Fig. 4A, +BS), the GCS
monomer peaked earlier (fraction 3) in the glycerol gradient than the
peak of GCS activity, and most GCS-immunoreactive material appeared as
cross-linked ~50-, ~70-, and ~120-kDa forms that peaked later in
the gradient (fractions 5 and 6). The higher molecular mass forms,
including aggregated immunoreactive material present at the top of the
gel, were not recognized by nonimmune antisera, but were recognized by
both anti-GCS-1 (Fig. 4A) and anti-GCS-6 (data not shown)
antibodies, confirming that they represent cross-linked forms of GCS.
These data show that a significant portion of solubilized GCS migrated
farther in the gradient than the monomer and was cross-linkable with
BS, consistent with an oligomeric structure.
This study reports the development of peptide-specific anti-GCS
antibodies and their use to characterize the GCS protein in rat liver.
We have used the anti-GCS antibodies to visualize GCS by Western
blotting for the first time and show that it runs as an ~38-kDa
protein on SDS-polyacrylamide gels. We used Western blotting and
activity assays for GCS in conjunction with controlled proteolysis and
immunoprecipitation of intact Golgi vesicles to generate new
information on the topology of GCS in the Golgi membrane. Finally, we
used cross-linking reagents and glycerol gradients to provide evidence
that GCS is organized as a heterodimer or heterooligomer.
Recognition of GCS by Antibodies--
The predicted molecular mass
of both the human (13) and rat GCS polypeptides is ~45 kDa. We
demonstrate here that rat Golgi GCS, recombinant rat GCS expressed in
bacteria, and human GCS run as ~38-kDa polypeptides on
SDS-polyacrylamide gels as detected by Western blotting with
peptide-specific antibodies against GCS. We have shown that the C
terminus of GCS is present in these polypeptides by the use of an
antibody (GCS-1) against the C-terminal 23 amino acids of GCS. We also
expressed a mutant rat GCS lacking the first 10 amino acids at its N
terminus. This mutant ran lower than wild-type GCS on
SDS-polyacrylamide gels and had little activity, in contrast to the
wild-type form, suggesting that the N terminus of GCS must also be
present in the native forms. Thus, we have ruled out the possibility
that the inconsistency between the predicted and empirical molecular
masses of GCS is due to the loss of a portion of the N- or C-terminal
region of GCS during the processing of the protein. A likely
explanation for the discrepancy in molecular mass is that, because GCS
is an extremely hydrophobic protein, it runs anomalously on
SDS-polyacrylamide gels, as do some other hydrophobic proteins
(e.g. see Ref. 24).
GCS Topology--
Previous results from our laboratory (2) and
others (3, 4) have established that the active site of GCS is on the cytosolic face of the Golgi membrane by showing that the GCS activity of right-side-out Golgi fractions is readily accessible to proteases. Here we extend these data by demonstrating that, in right-side-out Golgi vesicles, the GCS-1 epitope at the carboxyl terminus of the
protein is rapidly degraded by trypsin and is accessible to antibodies
(for immunoprecipitation of Golgi vesicles). The GCS-6 epitope was
slightly less accessible to trypsin and could also be used to
immunoisolate Golgi vesicles, although with lower efficiency. Finally,
the GCS-5 epitope appeared to be somewhat protected against trypsin and
was not accessible to antibodies for immunoprecipitation except under
denaturing conditions. These results suggest that the carboxyl terminus
of GCS (GCS-1 epitope) and a loop (containing the GCS-6 epitope) just
after the putative N-terminal transmembrane domain of GCS protrude into
the aqueous environment on the cytosolic face of the Golgi membrane,
whereas the region containing the GCS-5 epitope is shielded from the
aqueous environment by other portions of the protein or membrane interactions.
GCS Oligomerization--
We have shown that bifunctional
cross-linking reagents shift the size of GCS to ~50 kDa and higher
forms. The ~50-kDa form could be generated from both solubilized and
intact Golgi membranes, suggesting that GCS is organized in
dimers/oligomers; however, treatment of dye-agarose-purified GCS or
bacterially expressed rat GCS with cross-linkers did not yield any
cross-linked GCS products. These results suggest that there is a
specific association of GCS with a small (~15 kDa) polypeptide
normally present in Golgi membranes, but absent in the
dye-agarose-purified GCS fractions. In preliminary experiments, we
attempted to identify such a "GCS-associated polypeptide" by trying
to immunoprecipitate GCS (and associated proteins) from both
metabolically 35S-labeled proteins from cultured cells and
125I-labeled rat liver Golgi proteins; however, we could
not visualize GCS or associated proteins above background radioactivity
in either case (data not shown), presumably because of the low levels
of GCS expression. Thus, the isolation of GCS-associated
polypeptide(s) will probably require an affinity purification
approach using overexpressed recombinant GCS.
The organization of GCS in heterodimers or heterooligomers may play a
significant role in GCS localization and/or function. The
oligomerization of Golgi membrane proteins has been suggested to be
involved in their retention in the Golgi complex (25-29). In addition,
several other glycosyltransferases have been shown to have a dimeric or
oligomeric structure, suggesting a functional role of oligomerization
in such enzymes (30-33). We plan future studies using site-directed
mutagenesis and truncation mutations to identify the oligomerization
domain(s) of GCS and to explore the role of GCS oligomerization in
Golgi targeting.
INTRODUCTION
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ABSTRACT
INTRODUCTION
REFERENCES
EXPERIMENTAL PROCEDURES
80 °C until they were analyzed by Western blotting.
70 °C
until use.
RESULTS
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Fig. 1.
Anti-GCS antibodies recognize rat and human
GCS proteins. Polyclonal antibodies were prepared by injection of
peptides (GCS-1, -2, -5, and -6) into rabbits as described under
"Experimental Procedures." For Western blots, samples were run on
SDS-polyacrylamide gels (10% acrylamide) and transferred to
polyvinylidene difluoride membrane, and blotting was performed using
anti-GCS antibodies. A, diagram of locations within the
human GCS sequence (13) of peptides used as immunogens in the
preparation of anti-GCS antibodies. Peptides (1,
2, 5, and 6) were synthesized and
injected into rabbits as described under "Experimental Procedures."
B, Western blots of rat liver fractions using the anti-GCS
antibodies indicated. Protein fractions were as follows: H,
homogenate; TM, total microsomes; WG,
N-lauroylsarcosine-washed Golgi membranes (all 20 µg of
protein/lane); IP, enzymatically active immunopurified GCS
(protein levels were undetectable). Anti-GCS-5 and anti-GCS-6
antibodies were immunopurified on corresponding peptide columns prior
to Western blotting. Anti-GCS-1, -5, and -6 antibodies all recognize
GCS as a polypeptide migrating at ~38 kDa. The anti-GCS-2 antisera
did not recognize any specific polypeptides. C,
comparison of the migration of rat and human GCS proteins on
SDS-polyacrylamide gels. GCS was detected by Western blotting with the
anti-GCS-5 antibody. Similar results were seen with the anti-GCS-1
antibody (data not shown). 1,
N-lauroylsarcosine-washed rat liver Golgi membranes;
2, the entire rat GCS coding region expressed in E. coli; 3, a rat GCS truncation mutant that lacks the
first 10 amino acids at the N terminus expressed in E. coli;
4, GCS from cultured differentiated human keratinocytes.
Fractions 2-4 are GCS immunoprecipitates from crude lysates.
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Fig. 2.
Topology of GCS in right-side-out Golgi
membranes. Rat liver Golgi membranes were prepared as described
under "Experimental Procedures" and either treated with trypsin
(1:8 (w/w) trypsin/Golgi protein) at 37 °C (A and
B) or immunoisolated with anti-GCS antibodies (C)
as described under "Experimental Procedures." A, GCS and
SMS activities of trypsin-treated Golgi membranes. Enzyme activities
were measured using
N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)-D-erythro-sphingosine
as a substrate as described (12). GCS activity was rapidly lost, but
SMS activity was relatively unaffected over time, indicating that the
Golgi membranes were relatively intact and mainly right-side out.
B, Western blotting of GCS in trypsin-treated Golgi
membranes. GCS (~38 kDa) was recognized by all three anti-GCS
antibodies. Trypsin treatment generated an ~35-kDa fragment
(fragment 1), which was recognized by the anti-GCS-5 and
anti-GCS-6 antibodies, and an ~33-kDa fragment (fragment
2), which was recognized only by the anti-GCS-5 antibody.
C, immunoisolation of Golgi vesicles with anti-GCS
antibodies coupled to protein A beads. Supernatants (Sup.)
and pellets were separated by centrifugation on a 0.9 M
sucrose cushion and then tested for the distribution of Golgi membranes
using GCS activity as a marker. Note that the anti-GCS-1 and anti-GCS-6
antibodies immunoprecipitated Golgi vesicles, but the anti-GCS-5
antibody did not. GlcCer, glucosylceramide.
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Fig. 3.
Cross-linking of GCS in rat liver Golgi
membranes with N-succinimidyl ester-based cross-linking
reagents. Samples of N-lauroylsarcosine-washed Golgi
membranes were treated with cross-linking reagents (BS, DSS, DSP, and
DTSSP) added in Me2SO (5 mM final concentration
with 10% Me2SO) and then Western-blotted with anti-GCS
antibodies as described under "Experimental Procedures."
A, comparison of cross-linked forms recognized by two
anti-GCS antisera (GCS-1 and GCS-5; see Fig. 1). Each cross-linker used
(BS, DSP, and DSS) generated an ~50-kDa form (XL1)
recognized by anti-GCS antibodies. Some higher molecular mass forms
were also generated. Similar cross-linked polypeptides were detected
with the anti-GCS-6 antibody, but not with nonimmune controls (data not
shown). B, reversible cross-linking with DTSSP. GCS in Golgi
membranes was treated with DTSSP (a thiol reagent cleavable
cross-linker), BS, or 5% Me2SO alone (Control)
and then Western-blotted for GCS. DTSSP (minus dithiothreitol
( DTT)) induced the formation of an ~50-kDa band
(XL1) recognized by the anti-GCS-5 antiserum and decreased
the signal for the GCS monomer. With dithiothreitol (+DTT),
the ~50-kDa band recognized by GCS-5 was cleaved, and the signal of
the GCS monomer was restored. C, effects of cross-linking
reagents on GCS in different Golgi-derived fractions. Intact Golgi
membranes, NLS-washed Golgi fractions, Igepal CA-630
(IGP-630)-solubilized NLS-washed Golgi fractions, and
dye-agarose-purified rat liver Golgi GCS fractions were treated with 5 mM cross-linker (BS or DSS) or 10% Me2SO alone
(Control) and Western-blotted with a mixture of anti-GCS-1
and anti-GCS-5 antisera (diluted 1:2000 each). For Igepal
CA-630-solubilized NLS-washed Golgi fractions and dye-agarose-purified
rat liver Golgi GCS fractions, DSS results (data not shown) were
similar to those shown for BS. Note the occurrence of an ~50-kDa band
(XL1) after treatment with cross-linkers for each fraction,
except for dye-agarose-purified GCS.
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Fig. 4.
Fractionation of GCS oligomers in glycerol
gradients detected by cross-linking and Western blotting. An
Igepal CA-630 extract of N-lauroylsarcosine-washed Golgi
membranes was fractionated on an 8-25% glycerol gradient as described
under "Experimental Procedures." A, Western blots
showing the distribution of GCS immunoreactivity in the glycerol
gradient fractions. Fractions are numbered starting from the least
dense fraction (top). Fractions 1 and 11-15 had almost no
GCS immunoreactivity and are not shown. Upper panel, the
distribution of the GCS monomer without BS treatment; lower
panel, the distribution of GCS immunoreactivity in the same
gradient fractions cross-linked with BS after fractionation. The
amounts of sample loaded ±BS were the same (20 µl/lane).
Cross-linking caused an increase in the total signal detected on
Western blots, possibly due to increased efficiency of electrophoretic
transfer. XL1, XL2, and XL3 are
~50-, 70-, and 120-kDa cross-linked forms, respectively, recognized
by anti-GCS antibodies. B, GCS activity in the same gradient
fractions. Note that the peak of GCS activity (fractions 3 and 4)
coincides with the maximal signal for the GCS monomer detected in
A (upper panel).
DISCUSSION
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM22942 (to R. E. P.). Part of this work was previously presented in preliminary form (Marks, D. L., Paul, P., and Pagano, R. E. (1998) Mol. Biol. Cell 8, 338a (abstr.)).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.
Present address: Synthelabo Research, Genomic Targets and
Biochemistry, 10, rue de Carrières, 92500 Rueil-Malmaison, France.
§ Present address: Applied Microbiology Dept., National Institute of Bioscience and Human-Technology, Tsukuba, Ibaraki 305, Japan.
¶ To whom correspondence should be addressed: Mayo Clinic and Foundation, Guggenheim 621-C, 200 First St., S. W., Rochester, MN 55905-0001. Tel.: 507-284-8754; Fax: 507-266-4413; E-mail: pagano.richard{at}mayo.edu.
2 K. Wu, D. L. Marks and R. E. Pagano, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: GCS, glucosylceramide synthase; NLS, N-lauroylsarcosine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BS, bis(sulfosuccinimidyl) suberate; DSS, disuccinimidyl suberate; DSP, dithiobis(succinimidyl propionate); DTSSP, 3,3'-dithiobis(sulfosuccinimidyl propionate); SMS, sphingomyelin synthase..
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REFERENCES |
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