 |
INTRODUCTION |
Much of the intracellular transport of proteins between the
various compartments of the secretory pathway is mediated by small membrane-bounded vesicles (1-4). These vesicles bud from one compartment and transport their cargo to the next compartment of the
secretory pathway. A multitude of factors collaborate in this process:
coat proteins (e.g. COPI (coatomer) or COPII) shape the
membrane into a bud culminating in vesicle release (5); a number of
integral membrane proteins of the p24 family function in cargo
selection during budding (6); and a set of soluble and integral
membrane proteins mediate target selection and membrane fusion
(7).
Many of the proteins involved in vesicular transport have been
identified and/or characterized through use of a well characterized in vitro system that measures intra-Golgi protein traffic
(8). The assay measures the glycosylation of a cargo protein (vesicular stomatitis virus (VSV) G protein) present in one population of Golgi
stacks by a glycosyltransferase present in another population of Golgi.
In this system, COPI-coated transport vesicles form under the direction
of a small GTPase termed ADP-ribosylation factor and a cytosolic coat
protein complex termed coatomer (9). The docking and fusion phases of
the assay require a number of other proteins. For example,
N-ethylmaleimide-sensitive fusion protein
(NSF)1 and soluble NSF
attachment proteins (SNAPs) are required at a time point
just prior to fusion and thus have been thought for some time to be
integral components of the membrane fusion apparatus (10, 11). Another
protein identified with this system is p115 (12), which is also thought
to act at the docking phase (13-15) and may act to tether the vesicle
to the target membrane before assembly of the v/t-SNARE targeting
complex (16, 17). Rab6 (18), which is a Golgi-associated member of a
large family of low molecular weight GTP-binding proteins (19, 20), is
also required in this cell-free system and most likely acts at the membrane docking step (21). Last, a protein termed p16 has recently been identified and was suggested to have a role in the docking and/or
fusion phase of early Golgi transport (22).
The final fusion event in this assay system is likely to be mediated by
members of a family of integral membrane proteins called SNAP
receptors (SNAREs) (23-26). Specific members of the SNARE
family can be found on distinct target membranes (t-SNAREs) or on
vesicles (v-SNAREs). Cognate v-SNAREs and t-SNAREs interact to form
stable complexes (23, 27-30), perhaps via coiled-coil domains (28, 31,
32). This cross-membrane v/t-SNARE complex has recently been termed a
"SNAREpin," and it has been shown that SNAREs in this topological
configuration can induce fusion of the docked membranes in the absence
of other protein factors (33). Recent studies of vacuole-vacuole
docking and fusion in yeast, where the membranes involved in fusion
possess both v- and t-SNAREs, have shown that NSF acts to disassemble
v/t-SNARE complexes in the same membrane prior to, and as a
prerequisite for, vesicle-target membrane docking and fusion (34-36).
Thus, the requirement for NSF and SNAP in the intra-Golgi in
vitro system at a point temporally close to membrane fusion may
reflect the requirement for unassociated v- and t-SNAREs in both the
donor and acceptor membranes for subsequent SNAREpin formation
(33).
This cell-free system used to identify NSF, SNAP, p115, and p16 has
been suggested to reconstitute the entire vesicular transport cycle,
including vesicle budding, targeting, and fusion (7). Extensive
electron microscopic and biochemical analyses have clearly indicated
that vesicles form during the assay, and it has been suggested that
they carry the VSV-G protein from the "donor" Golgi to the
"acceptor" Golgi, where it is glycosylated, resulting in the signal
obtained in the biochemical assay (3). However, several studies have
shown that the assay signal is not affected by removal of
ADP-ribosylation factor (13, 37, 38), which is required for vesicle
formation (9), suggesting that the transfer of VSV-G protein from donor
to acceptor Golgi occurs via a partial transport reaction encompassing
only the membrane docking and fusion steps. In this regard, it is
noteworthy that the four proteins purified based on their activity in
this in vitro system (NSF, SNAPs, p115, and p16) impact on
the docking or fusion phases of transport, as shown by both biochemical
studies (10, 11, 13, 14, 22) and genetic analyses of their homologs in
yeast (15, 39). In addition, removal of Rab proteins, which are
generally thought to be involved in membrane docking (40), inhibits the
in vitro assay (41).
Because the output of the intra-Golgi cell-free system is glycosylation
of VSV-G, it is also possible that factors that effect glycosylation
could exhibit activity. Indeed, it has been shown that the inclusion of
uridine monophosphate kinase enhances the signal obtained by indirectly
facilitating the uptake of radiolabeled UDP-sugar into the Golgi lumen,
which is utilized by the glycosylation machinery (42). Thus, this
cell-free system is a powerful tool for identification and analysis of
transport factors and may be primarily dependent on those involved in
membrane docking and/or fusion and perhaps glycosylation of VSV-G.
In this report, we describe the purification and initial
characterization of a novel Golgi-associated protein complex containing at least five polypeptide chains, ranging from 71 to 110 kDa, that
stimulates transport in this in vitro intra-Golgi transport system.
| |
MATERIALS AND METHODS |
General Procedures
pH measurements were done at room temperature; other
manipulations were performed at 0-4 °C, unless otherwise noted.
Dialysis membranes had a molecular mass cutoff of 12-14 kDa (BioDesign Inc.). Salt concentrations were determined by conversion from conductivity using a standard curve generated from 25 mM
Tris-Cl, pH 8.0, 1 mM DTT, 10% glycerol (w/v) with 0-1
M KCl or from 25 mM Tris-Cl, pH 7.4, 100 mM KCl, 1 mM DTT, 10% glycerol (w/v) with 0-500 mM KPi (potassium phosphate). Protein
concentrations were determined with the Bradford protein assay (43)
(Bio-Rad) using a Beckman DU-64 spectrophotometer (Beckman
Instruments). Unless otherwise noted, all fractions that were analyzed
in the transport assay were first dialyzed into assay buffer (20 mM Hepes-KOH, pH 7.4, 100 mM KOAc, 1 mM DTT).
Purification of Bovine Serum Albumin (BSA)
Six ml of 40 mg/ml BSA (fraction V; Sigma) in 25 mM
Tris-Cl, pH 7.4, 100 mM KOAc, 1 mM DTT were
loaded onto six 10.5-ml 10-25% glycerol (w/v) gradients in the same
buffer. The gradients were centrifuged in a Beckman SW41 rotor (Beckman
Instruments Inc.) at 40,000 rpm (198,000 × gavg) for 24 h. The gradients were then fractionated into ~0.8-ml fractions, and the
A280 peak was pooled, avoiding the leading edge
of the peak that sedimented deeper into the gradient. The pool was
loaded at 1.5 ml/min onto an 8-ml Mono-Q column (HR 16/10; Amersham
Pharmacia Biotech) equilibrated in 25 mM Tris-Cl, pH 7.4, 100 mM KCl, 1 mM DTT. The column was washed with 16 ml of equilibration buffer and eluted with a 60-ml gradient from 100 mM to 1 M KCl in the same buffer.
Fractions (1.5 ml) were collected, and those with a minimum of
contaminating proteins, as determined by SDS-PAGE and Coomassie
blue-staining, were pooled and was referred to as purified BSA.
Cis to Medial Intra-Golgi Transport Assay
The transport assay used in this purification is a modification
of previously published assays (8, 12, 44), with the primary changes
being inclusion of transport factors shown to be active in this assay,
specifically NSF (45), 
-SNAP (44), and p115 (12), and the use of the
N-acetylglucosaminyl transferase-deficient mutant Chinese
hamster ovary cell line Lec1 (46), rather than 15B (8), for the
preparation of VSV-G-bearing donor membranes (47). The 25-µl
reactions, which were incubated at 37 °C for 1 h, contained 15 µl of chromatographic fractions to be assayed, 3 µl of Chinese
hamster ovary (Lec1) VSV-infected donor Golgi and 2 µl of Chinese
hamster ovary (wild type) acceptor Golgi membranes (~3 µg of
protein), 25 ng of His6-NSF (48), 200 ng of
His6--SNAP (48), 125 ng of p115 (12), 0.3 µCi of
UDP-[3H]N-acetylglucosamine (American
Radiochemicals), 20 mM Hepes-KOH, pH 7.4, 16 mM
Tris-Cl, pH 7.4, 60 mM KCl, 2.5 mM
Mg(OAc)2, 250 µM UTP, 100 µM
ATP, 5 mM creatine phosphate, 12 IU/ml creatine kinase, 10 µM palmitoyl-coenzyme A (49), 40 µg/ml nucleotide monophosphate kinase (42), 200 mM sucrose, 4.8% (w/v)
glycerol, and 0.6 mM DTT.
[3H]N-acetylglucosamine incorporation was
quantitated by immunoprecipitation of VSV-G protein and scintillation
counting (8).
For the inhibition of in vitro transport with
affinity-purified antibodies (Fig. 8), affinity-purified anti-GTC-90
(see below) was dialyzed extensively into assay buffer without DTT. The
antibody was then serially diluted in assay buffer without DTT and
added to a standard transport assay containing serial dilutions of
either BBC-30, or the CHT2-I pool (see below). A preimmune IgG fraction was prepared on a protein A-Sepharose column according to the manufacturer's instructions, dialyzed into assay buffer without DTT,
and used as a control for the inhibition experiments.
Purification of GTC
Preparation of Bovine Brain Cytosol and Ammonium Sulfate
Precipitation--
Five cow brains were obtained immediately after
slaughter, and bovine brain cytosol (BBC) was prepared and ammonium
sulfate-precipitated as described previously (12), except that ammonium
sulfate was added to a final concentration of 30% saturation at
0 °C, and the pellet was resuspended with and dialyzed against 25 mM MES-KOH, pH 6.8, 50 mM KCl, 1 mM
DTT, 10% glycerol (w/v). The dialysate (228 ml) had a protein
concentration of 36.7 mg/ml.
SP-Sepharose Chromatography--
The protein concentration of
the dialysate was adjusted to 10 mg/ml with 25 mM MES-KOH,
pH 6.8, 50 mM KCl, 1 mM DTT, 10% glycerol (w/v) and was loaded at 7.0 ml/min onto a 450-ml SP-Sepharose Fast Flow
(Amersham Pharmacia Biotech) column equilibrated in the same buffer.
The column was washed with the same buffer, and the protein that flowed
through the column was collected and dialyzed extensively against 25 mM Tris-Cl, pH 8.0, 1 mM DTT (T/D, pH 8.0) with
50 mM KCl. The material was then clarified in a Sorvall GS3 rotor at 8500 rpm for 15 min, resulting in a 1.2-liter pool with a
protein concentration of 4.1 mg/ml.
DEAE-Sepharose Chromatography--
The dialyzed SP-Sepharose
flow-through was loaded at 4.0 ml/min onto a 250-ml DEAE-Sepharose Fast
Flow (Amersham Pharmacia Biotech) column equilibrated in 25 mM Tris-Cl, pH 8.0, 1 mM DTT, 10% glycerol
(w/v) (T/D/G, pH 8.0) with 50 mM KCl. The column was washed
with 300 ml of equilibration buffer, followed by elution with a 300-ml
gradient from 50 to 350 mM KCl in T/D/G, pH 8.0, followed
by a 150-ml gradient from 350 mM to 1 M KCl in
T/D/G, pH 8.0. Fractions (15 ml) were collected, and an aliquot of each was assayed for transport activity after dialysis into assay buffer. The active fractions, usually from 55 to 170 mM KCl, were
pooled (133 ml) and had a protein concentration of 2.4 mg/ml.
Cibacron Blue 3GA Chromatography--
The salt concentration of
the DEAE pool was adjusted to 100 mM KCl by the addition of
T/D/G, pH 8.0, 3.0 M KCl and loaded onto a 30-ml Cibacron
Blue 3GA (Sigma) column equilibrated in T/D/G, pH 8.0, 100 mM KCl. The column was washed with 50 ml of equilibration
buffer, followed by elution with 90 ml of T/D/G, pH 8.0, 500 mM KCl. The eluted fractions were pooled (43 ml) and had a
protein concentration of 2.4 mg/ml.
Butyl-Sepharose Chromatography--
The salt concentration of
the Blue 3GA pool was adjusted to 1.5 M KCl by the addition
of T/D/G, pH 8.0, 3 M KCl and loaded at 0.5 ml/min onto a
5-ml butyl-Sepharose Fast Flow (Amersham Pharmacia Biotech) column
equilibrated in T/D/G, pH 8.0, 1.5 M KCl. The column was
washed with 10 ml of equilibration buffer followed by elution with 25 ml of T/D/G, pH 8.0, 3 mM KCl. The eluted material (9.0 ml)
had a protein concentration of 4.3 mg/ml.
Ceramic Hydroxylapatite (CHT2-I) Chromatography--
The salt
concentration of the butyl pool was adjusted to 100 mM KCl
by the addition of T/D/G, pH 7.4. Potassium phosphate (KPi), pH 7.4, was then added to a final concentration of 2 mM, and the pool was loaded at 1.0 ml/min onto a 2-ml
CHT2-I column (Bio-Rad) equilibrated in T/D/G, pH 7.4, 100 mM KCl, 2 mM KPi. The column was
washed with 4 ml of the same buffer followed by the elution of the
bound material with a 14-ml gradient from 2 to 125 mM
KPi in the same buffer. Fractions (0.5 ml) were collected in "low adhesion" polypropylene microcentrifuge tubes
(USA/Scientific Plastics) and immediately dialyzed in a microdialyzer
(Life Technologies, Inc.) against assay buffer to remove the phosphate
(extended exposure to phosphate seemed to reduce the activity of the
fractions). An aliquot (7.5 µl) of each fraction was then assayed for
transport activity. The active fractions, usually eluting from 50-75
mM KPi, were pooled (3 ml) and had a protein
concentration of 0.96 mg/ml.
Superose 6 Size Exclusion Chromatography--
The CHT2-I pool
was concentrated to a volume of 0.5 ml using an Ultrafree-4 centrifugal
filter device (Millipore, Biomax-10K NMWL). The concentrated material
was centrifuged for 10 min in a microcentrifuge to remove any insoluble
material and then sieved at 0.25 ml/min through a 24-ml Superose 6 column (Amersham Pharmacia Biotech) equilibrated in T/D, pH 7.4, 100 mM KOAc, 0.01% (v/v) Tween 20, 250 µg/ml purified BSA.
Fractions (0.5 ml) were collected in low adhesion microcentrifuge
tubes, and 15 µl of each fraction were assayed without prior
dialysis. The active fractions, usually eluting between 9 and 11.5 ml,
were pooled (2.5 ml). Because the column was run in the presence of
BSA, protein concentrations from this point forward were not determined.
Mono-Q Chromatography--
The Superose 6 pool was loaded at 0.3 ml/min onto a 1-ml Mono-Q column (HR5/5, Amersham Pharmacia Biotech)
equilibrated in T/D, pH 7.4, 100 mM KCl. The column was
washed with 2.5 ml of the same buffer followed by elution with a 7.5-ml
gradient from 0.1 to 1.0 M KCl in the same buffer.
Fractions (0.25 ml) were collected in low adhesion tubes, and 3 µl of
each fraction were assayed without prior dialysis. The active
fractions, usually eluting from 250-300 mM KCl, were
pooled (0.7 ml). This column acted primarily as a concentration step.
Glycerol Gradient Sedimentation--
The Mono-Q pool was layered
onto a 4.7-ml linear 10-25% glycerol (w/v) gradient in T/D, pH 7.4, 100 mM KOAc. S value standards (2-macroglobulin (Sigma), 20 S; catalase (Amersham
Pharmacia Biotech), 11.4 S; BSA (Sigma), 4.6 S) were loaded onto a
second gradient. As a control for minor contaminants that may have
remained in the purified BSA, 625 µg of purified BSA (approximately
equivalent to the amount of BSA in the Mono Q pool) was applied to a
third gradient. The gradients were centrifuged in a Beckman SW55 rotor at 55,000 rpm for 6.5 h with slow acceleration and deceleration. Fractions (0.6 ml) were collected into low adhesion microcentrifuge tubes and analyzed by electrophoresis and silver staining (50). In
addition, 7.5 µl of each fraction were assayed directly for transport
activity. The active fractions, sedimenting at approximately 13 S, were
pooled. The activity co-purified with a hetero-oligomeric complex
composed of at least five subunits (see below), which we have termed
the GTC.
Sequence Determination of Tryptic Peptides of the 90-kDa GTC
Subunit
Pools from three GTC purifications (150 µg of total protein,
estimated) were prepared for peptide sequencing by 6% trichloroacetic acid precipitation in the presence of 0.02% deoxycholate to
concentrate them for SDS-8% polyacrylamide gel electrophoresis. After
electrophoresis, the gel was washed for 1 min in HPLC grade water and
copper-stained (Bio-Rad) according to the manufacturer's instructions.
Each of the bands of the GTC complex was excised along with control gel slices of the same volume and relative molecular weight range from an
adjacent lane in which no protein had been loaded. The gel slices were
washed twice in 50% acetonitrile (Pierce) for 10 min, gently vortexing
every 2 min. The supernatant was discarded, and the wet gel slices were
frozen and stored at 
20 °C.
Sequences of three tryptic peptides derived from the 90-kDa subunit
(GTC-90) were obtained at the Harvard Microchemistry Facility (Harvard
University, Cambridge, MA), where trypsin digestion, HPLC purification,
and sequencing of the peptides by either Edman degradation (one
peptide) or mass spectroscopy (two peptides) were performed.
Cloning and Sequencing of a GTC-90 cDNA
Sequence of Human Expressed Sequence Tags (ESTs) Corresponding to
GTC-90--
The three peptide sequences obtained from GTC-90 were used
to search the dBEST data base with the BLAST algorithm (51). Three
overlapping I.M.A.G.E. Consortium (LLNL) cDNA clones (52) were
found that coded for the three peptides. Two of these, I.M.A.G.E. Consortium clone numbers 53376 and 626427 were obtained from Genome Systems (St. Louis, MO), and the complete nucleotide sequences of the
two cDNAs were determined.
PCR Amplification of the 5'-End of the GTC-90 cDNA--
To
obtain the 5'-end of the GTC-90 cDNA, a HeLa cDNA library
(Stratagene) in Bluescript SK() was PCR-amplified in two rounds using
a pair of primers nested in the 5'-end of clone 626427 and one primer
in the Bluescript polylinker (M13-2) (53). In the first round of PCR,
the outside GTC-90-specific primer and M13-2 were used to amplify an
aliquot of the above library, which had been lysed by heating for 5 min
at 98 °C in water. The second round of PCR was performed with the
product from the first round using M13-2 and the inside
GTC-90-specific primer. The product of round 2 was digested with
XbaI and BglII and subcloned into a vector
containing the 5'-end of EST-626427 linearized with the same enzymes.
Plasmids containing the largest inserts were sequenced. One of the
inserts contained an additional 492 base pairs of the open reading
frame, which we termed 626427-A. According to splice site prediction
analysis of the genomic DNA (54), the remaining GTC-90 sequence was
contained within a single exon. Thus, the remaining 5' sequence was
obtained by PCR using a human bacterial artificial chromosome encoding
the 5'-end of GTC-90 as a template (bacterial artificial chromosome
RG020D02 from chromosome seven (7q22)). The bacterial artificial
chromosome-derived PCR product was subcloned into 626427-A after
digestion of both with BamHI and SacI. Because
this construct was missing the putative alternatively spliced region,
it was termed GTC-90 (ASR).
Construction of a Putative Full-length GTC-90 cDNA--
The
GTC-90 (ASR) construct does not contain the region coding for amino
acids 557-593, because this region was absent from the original EST
(number 626427) derived from HeLa cells. Because this region contains
one of the peptides found in our purified brain protein, we chose to
insert the exon encoding this region to make the putative full-length
brain construct. To accomplish this, EST 53376, which contains the
region, was subcloned into Bluescript II SK() after digesting both
with PstI and XbaI. This construct, which
represents the 3'-end of the GTC-90 cDNA, was termed 53376-BSSK.
The 5'-end of the GTC-90 cDNA was then subcloned onto 53376-BSSK by
digestion of GTC-90 (ASR) with BamHI and XbaI and ligating it to 53376-BSSK, which was digested with the same enzymes. This putative cDNA construct was termed full-length
GTC-90.
Production of Affinity-purified Anti-GTC-90 Antibodies
A construct encoding an N-terminal hexahistidine partial-GTC-90
fusion protein (His6-53376) was constructed using an
I.M.A.G.E. cDNA clone. Clone 53376 was digested with
HindIII and ligated into linearized pQE-30 (Qiagen) to yield
the plasmid QE-53376. M15 cells (Qiagen) were transformed with the
plasmid QE-53376, and expression was induced for 18 h with 0.2 mM isopropyl-
-D-thiogalactoside. The fusion
protein was purified by Ni2+-nitrilotriacetic acid
chromatography (Qiagen) under denaturing conditions as described in the
manufacturer's instructions, dialyzed into 1× phosphate-buffered
saline, and injected into rabbits for production of polyclonal antisera
according to standard methods (55).
The anti-GTC-90 antisera was affinity-purified on a GTC-90 affinity
column. To generate a GTC-90 affinity column, GST was fused to a
portion of GTC-90 by first digesting EST-626427 with XbaI
and ligating it into 53376-BSSK (see construction of full-length cDNA) linearized with the same enzyme. This new construct was then
digested with BamHI and EcoRV and ligated into
pGEX-3X (Amersham Pharmacia Biotech) linearized with BamHI
and SmaI. The plasmid was transformed into BL21 cells
(Novagen), and expression was induced for 4 h with 0.1 mM isopropyl--D-thiogalactoside. Cell lysates were made in TBS (20 mM Tris-Cl, pH 7.5, 500 mM NaCl) in the presence of 5 mM DTT and 1.5%
sarcosyl (56). After sonication and clarification by centrifugation,
Triton X-100 was added to 4%, and the detergent lysate was incubated
for 45 min with 2 ml of glutathione-Sepharose (Amersham Pharmacia
Biotech) equilibrated in TBS. The mixture was poured into a column and
then washed with 10 ml of TBS followed by 10 ml of 100 mM
HEPES-NaOH, pH 7.4, 150 mM NaCl. The column was eluted with
100 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 0.1%
SDS. The eluate was gel-filtered on a PD-10 column (Amersham Pharmacia
Biotech) and cross-linked to 0.5 ml of Affi-Gel-15 (Bio-Rad) according
to the manufacturer's instructions using 100 mM
HEPES-NaOH, pH 7.4, 150 mM NaCl, 0.1% SDS as a
cross-linking buffer. The antibody was affinity-purified according to
previously published protocols (55) except that the low pH elution used 100 mM glycine, pH 2.5, 10% ethylene glycol (v/v).
Briefly, after passing serum over the affinity column and washing the
column, the antibodies were eluted sequentially with pH 2.5 buffer
followed by elution with 100 mM triethylamine, pH 11.5. The
affinity-purified antibodies used in the experiments described here are
anti-His6-53376 antibodies eluted at pH 11.5, and are
referred to as anti-GTC-90.
Immunoblotting, Immunofluorescence Microscopy, and
Immunoprecipitation
For detection of GTC-90 by immunoblots, proteins were separated
by SDS-PAGE and transferred to polyvinylidene difluoride membrane (NEN
Life Science Products). The blots were then processed according to
previously published protocols (55) using 5% nonfat dry milk in
phosphate-buffered saline, 0.1% Tween 20 as a blocking agent. Blots
were incubated overnight at 4 °C with primary antibody at the
indicated dilutions and with secondary antibody diluted 1:6000 in
blocking solution for 1 h at room temperature. Polyclonal
antibodies were detected with goat anti-rabbit HRP (Bio-Rad), and
monoclonal antibodies were detected with goat anti-mouse horseradish
peroxidase (Bio-Rad). The blots were developed with ECL Plus (Amersham
Pharmacia Biotech) and exposed to X-AR5 film (Eastman Kodak Co.).
Indirect immunofluorescence was performed with NRK cells grown on
flame-sterilized 12-mm glass coverslips. The cells were fixed in
methanol for 15 min at 20 °C; washed four times in TBS, pH 8.0;
and treated with 6 M urea in TBS for 5 min at room
temperature. After urea treatment, the cells were washed in TBS and
blocked in 5% nonfat dry milk in TBS for 30 min at room temperature.
The cells were incubated in primary antibody in blocking solution overnight at room temperature, and all secondary antibodies were applied in blocking solution for 1 h at room temperature. Mouse monoclonal antibodies were detected with Texas red-conjugated goat
anti-mouse antibodies, and rabbit polyclonal antibodies were detected
with fluorescein isothiocyanate-conjugated goat-anti-rabbit antibodies.
For GTC-90 detection, after overnight incubation with affinity-purified
antibody, the coverslips were first washed with blocking solution,
incubated with biotinylated goat anti-rabbit antibodies for 1 h at
room temperature, washed with blocking solution, and finally incubated
with fluorescein isothiocyanate-labeled avidin for 1 h at room
temperature. Confocal images were obtained using a Zeiss LSM 510 laser
confocal microscope.
For immunoprecipitation of GTC, Madin-Darby bovine kidney (MDBK) cells
in 100-mm plates were grown to 80% confluency in Dulbecco's modified
Eagle's medium plus 10% fetal calf serum, starved for 30 min in
Dulbecco's modified Eagle's medium without Cys and Met, and then
incubated for 16 h in 90% Dulbecco's modified Eagle's medium
without Cys and Met, 10% Dulbecco's modified Eagle's medium plus
10% fetal calf serum, and 50 µCi Tran35S-label (ICN)/ml.
The cells were washed five times with 1× phosphate-buffered saline and
then scraped off the plate in 750 µl of IP buffer (T/D/G, pH 8.0, with 50 mM KCl) containing the protease inhibitor mixture used for preparing BBC (12). The cell suspension was homogenized in a
2-ml Dounce homogenizer with 10 strokes of the B pestle and centrifuged
for 20 min at 4 °C in a microcentrifuge. The membrane pellet was
solubilized in 1.2 ml of IP buffer plus 1% Nonidet P-40 and clarified
in a microcentrifuge as above. One volume of a 50% protein A-Sepharose
(Amersham Pharmacia Biotech) slurry in IP buffer was added and
incubated for 3 h at 4 °C. The beads were removed, and 600 µl
of lysate was incubated overnight with 200 µl of 50 µg/ml
affinity-purified anti-GTC-90 antibody or preimmune IgG or with 200 µl of IP buffer alone. Protein A-Sepharose beads (30 µl of a 50%
slurry), which had been preincubated with unlabeled MDBK membrane
lysate (prepared as above) and washed with IP buffer, were added and
incubated for 45 min. The suspensions were briefly centrifuged, the
supernatant was removed, and the beads were washed five times for 10 min in IP buffer. The proteins were eluted by boiling in 25 µl of 2×
sample buffer, separated by SDS-PAGE, and visualized by autoradiography
with XAR-5 film (Kodak) overnight.
Localization of GTC-90 by Subcellular Fractionation
Bovine brain lysates were fractionated over sucrose gradients
using a protocol based on that of Taylor et al. (57) with several modifications. Fresh cow brain (25 g) was finely minced and
added to 150 ml of ice-cold lysis buffer (0.5 M sucrose,
0.1 M KPi, pH 6.8, 5 mM
MgCl2, 1 mM DTT, and the same protease
inhibitor mixture used in the preparation of BBC (12)). The tissue was homogenized with a Polytron (Brinkman) with a PT/20S probe for 30 s at a power setting of 3. The homogenate was centrifuged in a SLA-1500
rotor (Sorvall) for 10 min at 3000 rpm (1366 × gavg). The post nuclear supernatant was
decanted, and 18 ml were layered over each of four 0.86 M
sucrose (10 ml) and 1.25 M sucrose (10 ml) step gradients
in SW-28 centrifuge tubes (all sucrose solutions were buffered with 0.1 M KPi, pH 6.8, 5 mM
MgCl2). The gradients were centrifuged at 4 °C in a
SW-28 rotor (Beckman) at 25,000 rpm (82,700 × gavg) for 90 min with slow acceleration and
deceleration. The interfaces of the sucrose steps and the steps
themselves were collected with Pasteur pipettes. The 0.86/1.25
M interface was adjusted to 1.35 M sucrose, and
then 10 ml were loaded into each of two SW-28 tubes. This layer was
overlaid with 1.25 M (7 ml), 1.0 M (7 ml), 0.86 M (7 ml), and 0.5 M (7 ml) sucrose solutions. The gradients were centrifuged at 4 °C in a SW-28 rotor (Beckman) at
25,000 rpm (82,700 × gavg) for 2.5 h
with slow acceleration and deceleration. The interfaces of the sucrose
steps and the steps themselves were collected by side puncture of the
tube with a 19-gauge needle attached to a 10-ml syringe. The fractions
from the first and second gradients were subjected to SDS-8%
polyacrylamide gel electrophoresis, transferred to polyvinylidene
difluoride membrane, and immunoblotted with anti-GTC-90, anti-p115, and
anti-mannosidase II antibodies.
| |
RESULTS |
In Vitro Cis to Medial Intra-Golgi Transport Requires Unidentified
Cytosolic Components--
As indicated above, several cytosolic and
peripheral membrane proteins involved in vesicular transport have been
purified using an in vitro system that reconstitutes protein
trafficking from the cis to medial Golgi compartments (12,
22, 44, 45, 58). The assay entails incubating together two types of
isolated Golgi membranes, termed donor and acceptor, in the presence of cytosol and ATP. The donor membranes are isolated from VSV-infected Chinese hamster ovary (Lec1) cells, which have an inactivating mutation
in the N-acetylglucosaminyltransferase I gene (59) that
encodes a medial Golgi glycosyltransferase (60). The acceptor membranes
are isolated from uninfected wild-type Chinese hamster ovary cells.
Transport is measured by monitoring the addition of
[3H]N-acetylglucosamine to the viral protein
VSV-G, which can only occur upon fusion of VSV-G-containing membranes
from the donor Golgi with the
N-acetylglucosaminyltransferase I-containing acceptor Golgi.
We found that assays performed in the presence of functionally
saturating amounts of NSF, SNAPs, and p115 still required the addition
of cytosol for optimal activity, suggesting that at least one other
factor was required, as had been previously suggested (12).
Specifically, bovine brain cytosol stimulated the assay about 6-fold
over background (Fig. 1A). To
determine whether multiple activities might be present in cytosol, it
was chromatographed on a Mono-Q anion exchange column, and the
fractions were tested for activity in the transport assay. We found
that at least two activities were present in cytosol, one that flowed
through the column and one that bound and could be eluted with a KCl
gradient (Fig. 1B). The simultaneous addition of both the
flow-through and bound activities to the assay resulted in a signal
that was approximately equal to the sum of the signals obtained with
the individual fractions (data not shown). Subsequent size exclusion of
the pooled flow-through fractions revealed that it predominantly contained low molecular weight factors possessing transport activity, one of which is phosphatidylinositol transfer protein (PITP) (58)
and another that may be the recently described protein p16 (22). In
contrast, size exclusion chromatography of the bound activity revealed
that it was composed of high molecular weight factors (data not shown),
indicating that the flow-through and bound activities are distinct.
This report describes the purification and characterization of one of
the high molecular weight activities that bound to Mono-Q.
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Fig. 1.
Transport from the cis to medial
Golgi requires at least two cytosolic factors in addition to NSF,
SNAPs, and p115. A, in vitro transport
assays (see "Materials and Methods") were performed in the presence
of functionally saturating amounts of NSF (25 ng), -SNAP (200 ng),
and p115 (125 ng) with either transport assay buffer or a serial
dilution of a 50% ammonium sulfate precipitate of bovine brain cytosol
(BBC-50). B, at least two distinct activities are separable
by anion exchange chromatography. BBC-50 was dialyzed into 25 mM Tris-Cl, pH 8.0, 50 mM KCl, 1 mM
DTT, 10% glycerol (w/v), diluted to 5.0 mg/ml, and chromatographed on
a Mono-Q anion exchange column into flow-through (fractions 4-18) and
bound fractions (fractions 28-45). Fractions (10 µl) were assayed
for transport-stimulating activity. Buffer, the assay
background; Load, the activity of 10 µl of the column
load. Subsequent size exclusion chromatography of the flow-through and
bound activities indicated that they chromatographed at vastly
different molecular sizes and were thus distinct (data not
shown).
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The response of the assay to either of the two activities suggests that
some of the other component(s) are present in the system, perhaps
associated with the Golgi membranes, which were not treated with salt
to strip peripheral membrane proteins as had been done in previous
assay systems (12, 44). This observation also indicates that the assay
conditions are such that there is not one true "rate-limiting
step." Rather, the biochemical flux through the pathway,
i.e. the movement of VSV-G protein through all of the
biochemical intermediates that are required for its ultimate
glycosylation by GlcNAc transferase, can be affected by the
concentration of more than one enzyme, as is often the case in a series
of biochemical reactions (61).
Purification of a High Molecular Weight Complex That Stimulates in
Vitro Intra-Golgi Transport--
In the fist step of the purification
of the high molecular weight Mono-Q bound activity, bovine brain
cytosol, the starting material, was precipitated with 30% ammonium
sulfate. The resuspended precipitate was passed through a SP-Sepharose
cation exchange column, to which the activity did not bind. The
SP-Sepharose flow-through was then fractionated by DEAE-Sepharose anion
exchange, Cibacron Blue-3GA dye affinity, butyl-Sepharose hydrophobic
interaction, ceramic hydroxylapatite, Superose 6 size exclusion, and
Mono-Q anion exchange chromatographic steps, followed by velocity
sedimentation through a glycerol gradient. During the course of the
purification, we found that the activity eluted from the Superose 6 size exclusion column significantly before the largest molecular weight
standard. Extrapolation from the available protein standards provides
an estimate of the Stokes radius of the activity of about 120-130 Å (Fig. 2). At the last step of the
purification, velocity sedimentation, the activity sedimented at about
13 S and coincided with a hetero-oligomeric complex (Fig.
3), which we have termed GTC. It is
evident that small quantities of the complex (estimated to be less than
100 ng of total protein) are capable of stimulating intra-Golgi
transport activity (Fig. 3A). Based on the Stokes radius and
the S value, we estimate (62) that the complex is
approximately 800-850 kDa and has an axial ratio of about 2, suggesting that it is nonglobular. A typical purification yielded
approximately 50 µg of the complex.
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Fig. 2.
Superose 6 size exclusion chromatography of
GTC. Partially purified material that had been subjected to 30%
ammonium sulfate precipitation, SP-Sepharose cation exchange,
DEAE-Sepharose anion exchange, Cibacron Blue-3GA dye affinity,
butyl-Sepharose hydrophobic interaction, and ceramic hydroxylapatite
chromatographic steps was concentrated and sieved through a 24-ml
Superose 6 column. Fractions (10 µl), as well as buffer and the
column load (3 µl), were assayed for activity. Molecular size
standards (bovine thyroid thyroglobulin (669 kDa, 85Å), horse spleen
ferritin (440 kDa, 61Å), and bovine liver catalase (232 kDa, 52.2 Å))
were chromatographed subsequently under the same conditions and used to
estimate the Stokes radius.
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Fig. 3.
Velocity sedimentation of GTC. As a
final step in the purification, the active fractions were sedimented
through 10-25% glycerol gradients (see "Materials and Methods").
After centrifugation, the gradients were fractionated, and 2.5% of the
load and fractions were analyzed by SDS-10% polyacrylamide gel
electrophoresis and silver staining. S value standards
(indicated across the top) from a parallel
gradient were visualized by Coomassie staining. Undialyzed fractions
(7.5 µl) were assayed to determine the peak of activity. The use of
undialyzed fractions results in an increase in the glycerol
concentration in the assay by 3.0% to 7.5%. Assay background
(bkg., the signal obtained with buffer rather than a
gradient fraction) was 200 cpm and has been subtracted. Two independent
purifications are shown. The purity of the complex obtained was
variable, often being homogeneous (Fig. 3A) but sometimes
showing minor contamination (Fig. 3B).
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At the end of the purification, the complex is purified to apparent
homogeneity (Fig. 3A), although there is frequently some contamination from other proteins (Fig. 3B). Nevertheless,
transport activity invariably fractionated only with the complex and
not with any of the minor contaminants. Another noteworthy point is that the Superose 6 size exclusion step near the end of the
purification separates the activity from the bulk of the protein,
resulting in a precipitous drop in protein concentration. This
necessitated the addition of an exogenous protein to reduce nonspecific
losses and perhaps to stabilize the activity. For this purpose, we
employed highly purified BSA, which has no affect on transport activity (data not shown) and can be efficiently removed at the last step of the
purification (Figs. 3 and 4).
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Fig. 4.
Protein profiles of fractions through the
purification and subunit composition of GTC. A, a
silver-stained gel showing the protein profiles of fractions from the
GTC purification. The lanes contained 6 µg cytosol (BBC),
30% ammonium sulfate precipitate (BBC-30), S-Sepharose
flow-through (S-Seph), DEAE-Sepharose pool
(DEAE-Seph), Cibacron Blue-3GA (Blue-3GA),
butyl-Sepharose (Butyl-Seph), or ceramic hydroxylapatite
(C-HA) pools or 5-µl Superose 6 (Sup-6),
1.25-µl Mono-Q, or 5-µl glycerol gradient pools. Because of the
addition of BSA at the Superose-6 step to stabilize the activity of the
load, values are given in µl for the last three steps, since protein
concentrations were not determined. B, GTC subunit
composition. Silver-stained SDS-8% polyacrylamide gel electrophoresis
gels of a pool of the active fractions from the glycerol gradient and
an immunoprecipitation from 35S-labeled solubilized MDBK
cell membranes. At the end of the purification, the activity
co-fractionates as a 13 S complex coincident with seven polypeptides
ranging in molecular weight from 71 to 110 kDa (110, 109, 90, 86, 82, 76, and 71 kDa). The 110- and 109-kDa polypeptides run as a tight
doublet (inset), which was resolved on a different gel.  ,
a contaminant, as its fractionation in the gradient did not correspond
to the transport activity;  , a small amount of contaminating BSA.
When the complex is immunoprecipitated from a 35S-labeled
solubilized membrane pool, all of the polypeptides (denoted by an
asterisk) but those of 86 and 76 kDa appear to be present.
In addition, three proteins of approximately 180, 92, and 87 kDa
(denoted by a double asterisk) are specifically
precipitated from solubilized membrane preparations. The "mock"
immunoprecipitation control did not contain antibody.
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Fig. 4A shows a silver-stained gel of fractions of each step
of the purification. Quantitation of activity and protein yields is
shown in Table I. It should be noted that
the activity measurements early in the purification are not indicative
of the specific activity of GTC alone, because several other activities
that also affect the assay were present, as described previously. For
this reason, the "specific" activity does not significantly
increase until the last few steps of the purification when GTC has been
separated from other components that affect the in vitro
system. Thus, the -fold purification of GTC is greatly underestimated.
Another issue that affected the determination of GTC-specific activity
was the addition of BSA at the later stages of the purification, which precluded an accurate determination of GTC protein concentration and
thus specific activity. Examination of the protein yield and protein
profiles of fractions through the purification indicated that only a
few purification steps (DEAE-Sepharose, Superose 6, and the glycerol
gradient) seem to be highly effective (Fig. 4A). Nevertheless, attempts to remove other steps from the purification resulted in a significant loss of purity of the final material.
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Table I
Quantitation of the GTC purification
A 3-fold dilution series was made of fractions from the GTC
purification, and 15 µl of each dilution was assayed. Specific
activities were determined from the slope (cpm/µg) in the linear
region of the curve, with the exception of the last three steps where
the protein concentration was not determined. The glycerol gradient
pool was estimated to have a protein yield of <0.05 mg based on
Coomassie- and silver-stained SDS-PAGE gels of the activity pool.
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GTC purified from cytosol appears to be composed of seven polypeptide
subunits with molecular masses of approximately 110, 109, 90, 86, 82, 76, and 71 kDa (Fig. 4B, Standard
Purification). Although the staining intensities of the
individual components differ with respect to each other, the profile is
highly reproducible using several staining techniques (silver,
Coomassie, and copper) as well as in multiple purifications. It is
possible, however, that some of the subunits could be proteolytic
fragments that remain associated with the complex and resist further
degradation as has been observed with other multisubunit complexes such
as coatomer (63, 64) and the "exocyst" complex in the yeast
Saccharomyces cerevisiae (65).
The 90-kDa Subunit (GTC-90) Is Encoded by an Alternatively Spliced
mRNA--
The amino acid sequences of three tryptic peptides from
the 90-kDa subunit (GTC-90) were determined by either Edman degradation or mass spectroscopy. None of the sequences showed significant homology
to known proteins; however, they were homologous to a set of
overlapping human cDNA clones (52) in the EST data base (dBEST),
which together contained all three peptides and had identical 3'-ends
(Fig. 5A). Sequencing of the
ESTs revealed that the largest cDNA (EST 626427, derived from a
HeLa cell library) encodes approximately 71% of the predicted open
reading frame (see below), whereas a second cDNA (EST 53376, derived from a human infant brain library) encodes approximately 43%
of the predicted open reading frame. The overlapping sequence of the
two ESTs are identical except for one region (encoding amino acids
557-593), which is absent in EST 626427, suggesting that GTC-90 may be
alternatively spliced. Further searches in the dBEST data base revealed
several ESTs from various tissues that encode human and mouse homologs
of GTC-90 and have different splicing patterns over this putative
alternatively spliced region (Fig. 5C).
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Fig. 5.
GTC-90 is alternatively spliced.
A, one of the GTC-90 ESTs (number 626427) does not contain a
region (the area enclosed by the dashed lines)
that encodes one of the GTC-90 tryptic peptides (Pep. 1),
which is present in another GTC-90 EST (number 53376). B, a
schematic representation of the intron/exon structure of a region of
human chromosome seven (7q31.1) encompassing the alternatively spliced
region, exons 14-19 (contained in bacterial artificial chromosome
RG363E19). C, BLAST searches of the dBEST data base revealed
four human ESTs and one mouse EST whose nucleotide sequences differs in
the GTC-90 alternatively spliced region. For one EST, clone 727321, the
5'-end of the cDNA is within the alternatively spliced region. The
clones were from the following cDNA libraries: G3204, human fetal
heart, GenBankTM accession number R58366; 53376, human
infant brain, GenBankTM no. R15430 (I.M.A.G.E. Consortium);
626427, HeLa cell, GenBankTM no. AA188905 (Stratagene, La
Jolla CA); 727321, human testis, GenBankTM no. AA292919
(I.M.A.G.E. consortium); 1247363, mouse mammary gland,
GenBankTM no. AA839785 (I.M.A.G.E. Consortium).
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The Human Genome Sequencing Project recently sequenced a region of
human chromosome seven (7q22 and 7q31.1) that encodes GTC-90. Analysis
of the intron/exon boundaries (54) for each of the 24 putative exons
confirmed that all exons, including the alternatively spliced exons
deduced from analysis of the ESTs, are present in the genomic sequence
(Fig. 5B). Interestingly, two of the GTC-90 ESTs (G3204 and
727231) are predicted to encode truncated versions of the protein due
to inclusion of an exon (number 15 or 18) containing a stop codon.
These mRNAs would encode proteins with predicted molecular masses
of 63 and 68 kDa, respectively.
Based on the sequence of the human brain EST through the alternatively
spliced region and the fact that GTC-90 from purified bovine brain GTC
contains a peptide encoded by exon 16, the most likely brain cDNA
was constructed (see "Materials and Methods"). The putative
cDNA contains exons 1-14, exon 16, and exons 19-24. Analysis of
the first ATG in the open reading frame indicates that it is in the
appropriate context for translation initiation (66). The cDNA
encodes a protein of 839 amino acids (Fig.
6), which encompasses the three tryptic
peptides obtained from purified bovine brain GTC-90 and has a predicted
molecular mass of 92,700 Da and a pI of 6.57. The predicted molecular
mass is in close agreement with the apparent molecular mass of GTC-90
on SDS-PAGE.
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Fig. 6.
Predicted amino acid sequence of GTC-90.
Predicted amino acid sequence of the putative GTC-90 full-length brain
cDNA. The thick underlines indicate bovine
GTC-90 tryptic peptide sequences that are identical to the predicted
human protein sequence. The thin underline
indicates a bovine GTC-90 tryptic peptide sequence (VLTQPTQSIVR) that
was 81% identical and 100% similar to the predicted human protein
sequence. The two mismatches are with amino acid residues that could
not be determined with a high degree of confidence during peptide
sequencing, although the difference could be accounted for by
conservative substitutions between human and bovine proteins.
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Data base searches indicate that GTC-90 is a novel protein that has yet
to be described. There are putative homologs in Drosophila melanogaster, Caenorhabditis elegans, and
Arabidopsis thaliana EST data bases. However, we were
surprised to find no convincing homolog in the S. cerevisiae
genome, which has been completely sequenced, since most vesicular
transport factors have functional homologs in yeast that are
recognizable at the primary sequence level.
GTC-90 Is Enriched during the Purification and Exists Exclusively
in a Complex--
While the enrichment of the complex through the
purification was difficult to assess based on activity (see above and
Table I), immunoblot analysis of the fractions over the course of the purification with an affinity-purified anti-GTC-90 antibody showed that
GTC-90 was enriched as the purification proceeded (Fig.
7A). The antibody also
recognized a second protein in cytosol (Fig. 7A,
lane 3×-BBC), but, only one of these
cross-reactive proteins is present in GTC, since the lower molecular
weight protein is removed early in the purification.
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Fig. 7.
GTC-90 is enriched during the purification
and the cytosolic pool exists exclusively in a 13 S complex.
A, an immunoblot of fractions from the GTC-90 purification.
The same fractions as employed in Fig. 4A were immunoblotted
with affinity-purified anti-GTC-90 antibodies. Load amounts were the
same except for 3× BBC, for which 18 µg rather than 6 µg were
loaded in order to detect GTC-90. The anti-GTC-90 antibody recognizes
an additional protein in cytosol; however, only one of these
cross-reactive proteins is enriched during the course of the GTC
purification. B, Superose 6 size exclusion chromatography of
BBC-30. 30% ammonium sulfate quantitatively precipitates GTC-90 from
BBC (data not shown) and was used to concentrate BBC for loading onto a
24-ml Superose 6 size exclusion column. The buffer and run parameters
were the same as those used for the GTC purification (Fig. 2) except
that no BSA was added to the column buffer. Fractions (0.5 ml) were
analyzed by SDS-8% polyacrylamide gel electrophoresis and
immunoblotting with affinity-purified anti-GTC-90 antibodies.
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Although analysis of the purification fractions indicated that GTC-90
was enriched as the purification proceeded, we investigated whether
GTC-90 is entirely associated with GTC, or if a noncomplexed pool is
also present. Superose 6 size exclusion chromatography followed by
immunoblot analysis for GTC-90 was utilized for this purpose. GTC-90
exists exclusively in a complex that behaves identically to GTC (Fig. 2
and Fig. 7B). The cross-reactive protein in cytosol also
appears to exist in a complex, since it has a Stokes radius of
approximately 60 Å (Fig. 7B). The nature of this complex
and its potential relationship to GTC remain to be explored.
In Vitro Transport Is Inhibited in the Presence of Anti-GTC-90
Antibodies--
To confirm that GTC was the factor responsible for the
activity in our purified fractions, we tested whether affinity-purified antibodies raised against GTC-90 would inhibit the transport assay. Antibody was added to the assay in the presence of varying amounts of
either the starting material of the purification (cytosol) or partially
purified GTC (hydroxylapatite pool, CHT2-I). Inhibition of the assay
was evident when 300 ng of anti-GTC-90 was added to an assay driven by
cytosol; the signal was reduced approximately 40% (at 100 µg of
BBC-30) (Fig. 8A). The
antibody was also an effective inhibitor of the assay driven by
partially purified GTC, reducing the signal by about 40% (at 6 µg of
hydroxylapatite pool) (Fig. 8B). The addition of more
than 300 ng of antibody to the assay did not result in further
inhibition of transport (data not shown). In general, the signal could
be reduced 40-60% upon the addition of antibody to the assay.
Comparable levels of preimmune IgG added to the assay failed to inhibit
either the cytosol- or hydroxylapatite pool-driven assays, indicating
that the inhibition is specific to the anti-GTC-90 antibody (Fig. 8). Because the antibody recognizes a single protein in the hydroxylapatite pool by immunoblot (Fig. 7A) and all of the detectable
GTC-90 is in a complex (Fig. 7B), these results are
consistent with GTC being responsible for part of the transport
activity present in cytosol. We also attempted to inhibit the transport
assay using monovalent Fab fragments generated from the
affinity-purified antibodies, but the Fab fragments were not inhibitory
(data not shown). Since the Fab fragments could still recognize GTC-90
on immunoblots (data not shown), the lack of inhibition suggests that inhibition of GTC activity by the anti-GTC-90 antibodies requires
more than simply interaction of the antigen with the antigen-binding
site of the antibody. For example, the bivalent nature of the antibody
may be critical to effect GTC-90 cross-linking, or the larger size of
the whole antibody relative to the Fab fragment may cause inhibition
for steric reasons.
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Fig. 8.
Inhibition of transport activity with
affinity-purified anti GTC-90 antibodies. Affinity-purified
antibodies or preimmune IgG were dialyzed into transport assay buffer
without DTT, and serial dilutions of the antibodies were added to
serial dilutions of either BBC-30 (A) or partially purified
GTC (a hydroxylapatite pool, CHT2-I) (B) and
preincubated on ice for 10 min before the addition of the remaining
assay components. Standard assay protocol was followed after the
preincubation step (see "Materials and Methods").
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GTC Affinity-purified from Solubilized 35S-Labeled
Membranes Has a Subunit Composition Similar to the Chromatographically
Purified Complex--
The composition of GTC was examined by
immunoprecipitation in order to determine if the subunits we observe in
the purified material co-fractionate using a different method. Because
most of the GTC-90 is membrane-associated (data not shown), we
affinity-purified GTC from a [35S]cysteine and methionine
metabolically labeled MDBK cell membrane fraction solubilized in
nonionic detergent. The affinity-purified material contained eight
subunits (180, 110, 109, 92, 90, 87, 82, and 71 kDa), while the
chromatographically purified material contained seven subunits (Fig.
4B), as mentioned above. Five of these subunits (110, 109, 90, 82, and 71 kDa) are common between the two.
The two additional subunits that are present in the chromatographically
purified GTC (86 and 76 kDa) and absent in the affinity-purified complex may represent 1) polypeptides that dissociated from the immunopurified complex due to the presence of detergent or antibody, 2)
partially proteolyzed subunits that remain associated during the
standard purification, or 3) proteins that happen to co-fractionate with GTC. The three additional polypeptides that are present in the
affinity-purified GTC (180, 92, and 87 kDa) may be a result of 1) the
complex being isolated from a different source (MDBK cells rather than
bovine brain), 2) a GTC membrane receptor co-precipitating with the
complex, since a different subcellular fraction (membranes rather than
cytosol) was used for the affinity-purification, or 3) components of
GTC that are proteolyzed during the chromatographic purification. The
92- and 87-kDa polypeptides could also be alternatively spliced
isoforms of GTC-90 that are not expressed in brain.
GTC Co-localizes with Golgi Markers by Subcellular
Fractionation--
The intracellular localization of GTC was examined
by subcellular fractionation of bovine brain postnuclear supernatant on equilibrium density sucrose gradients (Fig.
9A). Fractions were analyzed
by immunoblotting with affinity-purified anti-GTC-90 and with
antibodies that recognize either p115, a marker of the cis
Golgi apparatus (12, 67), or the medial Golgi marker protein mannosidase II (Fig. 9B). In a first gradient, membranes
that collected at the 0.86/1.25 M interface were harvested,
adjusted to 1.35 M sucrose, and loaded onto the bottom of a
second gradient (Fig. 9A). Immunoblot analysis of the second
gradient showed that GTC-90, p115, and mannosidase II floated with the
membranes to the 1.0/1.25 M interface (Fig. 9B).
This verifies that GTC-90, and therefore GTC, like the peripheral
membrane protein p115 (12), has both a cytosolic and a membrane-bound
pool. In addition, the results suggest that GTC may be associated with
the Golgi. Another observation of interest is that the
affinity-purified antibody recognizes an additional protein of
approximately 70 kDa. This cross-reactive protein, which is different
from the one observed in cytosol (compare Fig. 7B to Fig.
9B), is membrane-associated and also fractionates with the
Golgi markers. This protein could be a unique species, a degradation
product of GTC-90, or the ~68-kDa alternatively spliced form of
GTC-90.
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Fig. 9.
Localization of GTC-90 determined by
subcellular fractionation. A, schematic representation
of the gradients used for the fractionation. Bovine brain postnuclear
supernatant in 0.5 M sucrose was overlaid on top of a
sucrose step gradient composed of 0.86 and 1.25 M sucrose
layers. The gradients were centrifuged, and the interfaces and layers
were collected (see "Materials and Methods"). The 0.86/1.25
M sucrose interface was adjusted to 1.35 M
sucrose and loaded at the bottom of a second step gradient with 1.25, 1.0, 0.86, and 0.5 M sucrose overlaying the pool.
B, immunoblots of the fractionation. After centrifugation
and fractionation (see "Materials and Methods"), the interfaces and
layers (see Fig. 9A for lane explanations) were analyzed by
SDS-8% polyacrylamide gel electrophoresis and immunoblotting with
affinity-purified anti-GTC-90 antibodies or with anti-p115 or
anti-mannosidase II monoclonal antibodies. The band labeled Man
II represents the intact mannosidase II, whereas the multiple
lower relative molecular weight bands are presumably degradation
products that have been previously described (73, 74).
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GTC-90 Localizes to the Golgi Apparatus in NRK Cells--
To
examine further whether GTC-90 (and GTC) is localized to the Golgi,
NRK-52E cells were examined by double label indirect immunofluorescence. In order to obtain an immunofluorescent signal with
the affinity-purified anti-GTC-90 antibodies, it was necessary to treat
the cells with 6 M urea after fixation. This manipulation may expose an immunoreactive epitope on GTC-90 or enhance accessibility of the antibody to GTC-90 (68). Fig.
10A shows that GTC-90 is localized in the perinuclear region in a pattern very similar to that
of p115. To confirm that the urea treatment did not have an adverse
affect on immunofluorescent analysis of the Golgi, we also examined
the colocalization of p115 with the medial Golgi marker
mannosidase II. As expected from previous reports, p115 was localized
adjacent to, but not precisely co-localized with, mannosidase II (67),
indicating that the urea treatment does not interfere with the
analysis.
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Fig. 10.
GTC-90 localizes to the Golgi by indirect
immunofluorescence microscopy. A, low magnification
confocal image of NRK cells incubated with anti-p115 monoclonal
antibody (3A10, 1:1000) and either anti-mannosidase II polyclonal
antibody (1:5000) or affinity-purified anti-GTC-90 polyclonal antibody
(1:2). B, higher magnification confocal images using the
same conditions as A.
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Close examination of the GTC-90/p115 double labeling indicated that
although the two proteins are both present in the perinuclear region,
they do not precisely co-localize (Fig. 10B). Whereas a reticular pattern was obtained for p115, the staining pattern of GTC-90
appears to be more punctate in nature, raising the possibility that
p115 and GTC-90 reside within closely apposed but distinct regions of
the Golgi stack. The absence of a clearly evident cytoplasmic pool of
GTC-90 by immunofluorescence may be due to either a higher concentration of GTC-90 on the Golgi relative to cytosol, as is found
with other Golgi peripheral membrane proteins, or depletion of
cytosolic GTC-90 by the somewhat stringent washing conditions required
to obtain the immunofluorescent signal.
Since the anti-GTC-90 recognizes more than one membrane-associated
protein by immunoblot (Fig. 9B), immunofluorescence was also
performed using a second anti-GTC-90 antibody that does not recognize
this additional 70-kDa band (data not shown). The localization of
GTC-90 using the second antibody was indistinguishable from the first
(data not shown). These results, taken together with the size exclusion
and fractionation results, indicate that that GTC exists in both
cytosolic and Golgi-associated pools.
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DISCUSSION |
We have used an in vitro system that reconstitutes
transport between the cis and medial Golgi cisternae to
identify and purify a novel protein complex that stimulates this
protein trafficking event. The complex, which we have termed GTC,
appears to be composed of at least five subunits ranging from 71 to 110 kDa in molecular mass. Peptide sequence data from the 90-kDa subunit
(GTC-90) allowed identification of a number of putative cDNAs as
well as the human genomic sequence that encodes the protein, which in
turn indicated that GTC-90 can be alternatively spliced. GTC-90 is
found exclusively in the GTC complex, is present in both membrane and
cytoplasmic pools, and localizes to the Golgi by immunofluorescence in
NRK-52E cells. The variety of cDNA isoforms that were observed
might reflect tissue-specific variants of the protein or provide a
means by which a putative heterogeneous population of GTC-related
complexes act at different locations in the secretory pathway within
one cell.
The precise localization of GTC within the Golgi region remains to be
determined. It is noteworthy, however, that confocal microscopy
indicates that it is closely apposed to, but not completely coincident
with, p115, which is thought to reside predominantly on the
cis side of the Golgi (67). It will be interesting to examine whether the alternatively spliced GTC-90 isoforms that are
evident upon cDNA analysis are also present on the Golgi and, if
so, whether their location within the stack is isoform-specific. In
this regard, it is noteworthy that our affinity-purified anti-GTC-90 antiserum detects an approximately 70-kDa protein that cofractionates with the Golgi complex on sucrose gradients. A GTC-90-related protein
of approximately this size has been predicted based on analysis of one
of the alternatively spliced cDNAs, which bears an exon with a stop
codon, resulting in a smaller protein.
The subunit composition of GTC is reminiscent of that of another
multiprotein complex involved in secretion, termed the Exocyst. This
protein complex has seven subunits (Sec3p, Sec5p, Sec6p, Sec8p, Sec10p,
Sec15p, and Exo70p) and is involved in the docking and/or fusion of
post-Golgi secretory vesicles to the plasma membrane in the yeast
S. cerevisiae (65, 69). The mammalian homolog of the exocyst
has also been characterized and found to be localized at the plasma
membrane (70). The molecular masses of the mammalian exocyst subunits
are similar to those of GTC (rsec8, 110 kDa; rsec6, 106 kDa; rsec5, 102 kDa; rsec15, 96 kDa; rsec6, 86 kDa; exo84, 84 kDa; rexo70, 79 kDa;
rsec10, 71 kDa). Given that the subunit composition of GTC is similar
to the mammalian exocyst, we examined whether the two complexes were
related by immunoblotting fractions through the GTC purification (see
Figs. 4A and 7A) with antibodies raised against
the 110-kDa rsec8 protein. The results showed that rsec8 separates from
GTC during the course of purification (data not shown), and thus the
two complexes are distinct. Perhaps more tellingly, the primary
sequence of GTC-90 does not correspond to any of the mammalian exocyst
subunits, most of which have been recently described (71). Moreover,
GTC localizes to the Golgi, whereas the exocyst localizes to the plasma
membrane. It is still possible, however, that GTC might be an
exocyst-like complex that functions in the early secretory pathway.
It is noteworthy that the sequence of GTC-90 is not significantly
homologous to any yeast open reading frame. This is unusual, because
most transport factors are highly evolutionarily conserved (72). It is
possible that GTC is absent in yeast or that there is a functional
homolog to GTC-90 in yeast whose sequence homology is not significant
enough to be clearly evident. Peptide sequencing and cloning of the
remaining GTC subunits may help address this question; while GTC-90 may
not have structural homologs in yeast, the same may not be true for
other subunits.
This report has described the purification and initial characterization
of a novel complex of proteins, termed GTC, that stimulates in
vitro intra-Golgi transport. Although GTC was purified based on
its activity in the in vitro transport assay, its precise
role in facilitating protein traffic remains to be determined. Since the system appears to be the most sensitive to the late steps of
transport, i.e. docking and fusion of VSV-G bearing vesicles and the subsequent glycosylation of the transferred VSV-G protein, it
is likely that GTC affects one of these biochemical events. Further
characterization of GTC-90 and its alternatively spliced isoforms as
well as characterization of the other GTC subunits will help to define
GTC's precise role in intracellular protein traffic.
,
Top
Abstract
Introduction
Supported in part by National Institutes of Health Training Grant
T32-CA9528.
