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Microsome-B, ased Assay for Analysis of Endoplasmic Reticulum to Golgi Transport in Mammalian Cells
|Microsome-B, ased Assay for Analysis of
Endoplasmic Reticulum to Golgi
Transport in Mammalian Cells
The trafficking of proteins along the first stage of the
secretory pathway is mediated by small vesicles that
bud from the endoplasmic reticulum (ER) and subsequently
fuse with the cis
-Golgi compartment. This
article describes a biochemical assay using mammalian
microsomes that can be used to measure these events
independently. The microsomes are prepared from cells
infected at the restrictive temperature (39.5°C) with the
ts045 strain of vesicular stomatitis virus (VSV) (Lafay,
1974). As a reporter molecule the assay utilizes ts045
VSV-glycoprotein (VSV-G), which is retained in the ER
during infection due to a thermoreversible folding
defect; incubation in vitro
at the permissive temperature
(32°C) results in the synchronous folding and transport
of VSV-G to the Golgi complex. To follow vesicle formation,
a differential centrifugation procedure is employed
to separate the more rapidly sedimenting ER and Golgi
membranes from the slowly sedimenting vesicles. Consumption
is analyzed using a two-stage assay in which vesicles
isolated by differential centrifugation during stage
1 are subsequently added to stage 2 (fusion) reactions
containing acceptor Golgi membranes. Transport to
the Golgi is measured by following the oligosaccharide
processing of VSV-G from the high mannose ER form,
which is sensitive to endoglycosidase H (endo H), to
form, which is endo H resistant
(Schwaninger et al.
, 1992). The biochemical characteristics
of the overall ER to Golgi transport reaction and the
vesicle formation and consumption assays are described
elsewhere (Aridor et al.
, 1995,1996b, 1998,1999a,b, 2000,
2001; Rowe et al.
II. MATERIALS AND
Culture medium (α-MEM; Cat. No. 11900-099) is
from Life Technologies. The medium is supplemented
with penicillin/streptomycin from a 100× stock solution
(Cat. No P0781; Sigma). Fetal bovine serum (FBS;
Cat. No FB-01) is from Omega Scientific. D-Sorbitol
(Cat. No. S-1876), leupeptin (Cat. No L-2884), chymostatin
(Cat. No C-7268), pepstatin (Cat. No P-4265),
phenylmethylsulfonyl fluoride (PMSF; Cat. No P-
7626), actinomycin D (Cat. No A-1410), uridine 5'-
-acetylglucosamine (UDP-GlcNAc; Cat.
No. U-4375), and dimethyl sulfoxide (DMSO; Cat. No.
D-2650), are from Sigma. The nitrocellulose membrane
(Cat. No. 68260) is from Schleicher & Schuell.
Horseradish peroxidase-conjugated goat anti-rabbit
IgG (Cat. No. 31460) is from Pierce. Chemiluminescence
reagent (Cat. No. NEL-101) and autoradiography
film ("Reflection") are from NEN. Polyallomer
microfuge tubes (Cat. No. 357448) are supplied by
Beckman Instruments Inc. A polyclonal antibody to
VSV-G is generated in rabbits immunized with the
C-terminal 16 amino acids of VSV-G (Indiana serotype)
coupled to KLH (Plutner et al.
, 1991). Centrifugation at
20,000 or 100,000g
is performed using an Optima TL
ultracentrifuge (Beckman) equipped with a TLA 100.3
rotor. A laser-scanning densitometer (personal densitometer;
Molecular Dynamics) is used to quantitate
The following procedures are performed on ice
unless otherwise stated.
A. Preparation of Cytosol
The following procedure for the preparation of rat
liver cytosol is based on that described by Davidson et
Phosphate-buffered saline (PBS) (10× stock): 90mM phosphate and 1.5M NaCl (pH 7.4). To make 1 liter,
add 80g NaCl, 2g KCl, 2g KH2PO4, and 21.6g
Na2HPO4·7H2O to distilled water. Store at room
- 25/125: 0.125M KOAc, 25mM HEPES (pH 7.4).
To make 100ml, add 3.125ml of 4M KOAc stock and
2.5 ml of 1M HEPES-KOH (pH 7.4) stock to distilled
water. Store at 4°C.
- Protease inhibitor cocktail (PIC): 10µg/ml leupeptin,
10µg/ml chymostatin, 0.5µg/ml pepstatin A, and
1.0mM PMSE To supplement 50ml of buffer (e.g.,
25/125) with PIC, add 50µl of 10mg/ml leupeptin
stock (in H2O), 50 µl of 10 mg/ml chymostatin stock (in
DMSO), 5µl of 5mg/ml pepstatin A stock (in DMSO),
and 500µl of 0.1M PMSF stock (in ethanol). Use PIC
buffer immediately or store at -20°C.
B. Preparation of Microsomes
- Decapitate two anesthetized adult Sprague-
Dawley rats (~250g), remove the livers, and place the
tissue in a 250-ml glass beaker. Determine the weight
of the tissue (typically ~20 g) and then wash two times
with -50 ml of Ix PBS and once with ~50 ml of Ix PBS
and once with ~50ml of 25/125.
- Finely mince the tissue using a pair of scissors
and then homogenize in 2-3 volumes (ml/g of tissue)
of 25/125 (PIC) with 20 strokes using a 40-ml Dounce
(Wheaton). Use a "loose-fitting" Dounce for the first 10
strokes followed by a "tight-fitting" one for the second
- Pour the homogenate into a 38-ml polycarbonate
tube (Nalgene; Cat. No. 3117-0380) and centrifuge for
10min at 12,000g (10,000 rpm) in a Beckman JA20 rotor
(Beckman Instruments Inc.). Using a pipette, transfer
~13 ml of the supernatant into each of two 14 x 89-mm
ultraclear centrifuge tubes (Beckman Cat. No. 344059)
and centrifuge at 150,000 g (35,000 rpm) for 90 min in a
Beckman SW41 rotor.
- After centrifugation, remove the overlying lipid
layer by aspiration and then withdraw the remaining supernatants (cytosol) from each tube using a pipette.
- Divide the cytosol into 250-µl aliquots in 0.5-ml
microfuge tubes, freeze in liquid N2, and store at
-80°C. The protein concentration of the cytosol is
~25 mg / ml.
- Actinomycin D (200× stock): Add 10mg actinomycin
D to 10ml of ethanol. Store at -20°C.
- Homogenization buffer: 0.375M sorbitol and 20mM HEPES (pH 7.4). To make 500 ml, add 34.2 g sorbitol
and 10 ml of 1M HEPES-KOH (pH 7.4) stock to distilled
water. Store at 4°C.
- 0.21M KOAc buffer: 0.21M KOAc, 3 mM Mg(OAc)2,
and 20mM HEPES (pH 7.4). To make 100ml, add
5.25 ml of 4M KOAc stock, 0.3 ml of 1M Mg(OAc)2 stock, and 2 ml of 1M HEPES-KOH (pH 7.4) stock
to distilled water. Store at 4°C.
- Transport buffer: 0.25M sorbitol, 70mM KOAc, 1 mM Mg(OAc)2, and 20mM HEPES (pH 7.4). To make
100ml, add 10ml of 2.5M sorbitol stock, 1.75ml of
4M KOAc stock, 0.1 ml of 1M Mg(OAc)2 stock, and
2ml of 1M HEPES-KOH (pH 7.4) stock to distilled
water. Store at -20°C.
C. Preparation of Acceptor Golgi Membranes
- Prepare vesicular stomatitis virus (VSV; Indiana
serotype) strain ts045 according to Schwaninger et al. (1992). Store the virus in 1-ml aliquots in screw-capped
tubes at -80°C.
- Grow normal rat kidney (NRK) cells on 150-mm
tissue culture dishes (Cat. No. 3025; Falcon) in α-MEM
medium supplemented with 5% FBS at 37°C and 5%
CO2. At confluency, infect the cells with a 5-ml cocktail
(per dish) containing 0.1-0.25 ml (2-10 pfu/cell) of
ts045 VSV (thawed at 32°C) and 25µg of actinomycin
D in serum-free α-MEM as described by Schwaninger et al. (1992). Rock the dishes for 45min to ensure an
even spread of the infection cocktail. After infection,
add 20ml of α-MEM medium supplemented with 5%
FBS to each dish and incubate in the presence of
5% CO2 for 3 h and 40min to 4h at the restrictive temperature
(39.5°C) (see Comment 1). The method
detailed later is based on a typical 12-dish microsome
- Following incubation at 39.5°C, transfer each
dish to ice, aspirate the medium immediately, and add
12ml of ice-cold Ix PBS to cool the cells as quickly as
- Remove the PBS by aspiration, add 5ml of
homogenization buffer, and scrape the cells from the dishes using a rubber policeman. Use a pipette to
transfer the cells to 50-ml plastic tubes (Cat. No. 25325-
50: Corning Inc.) and then repeat the scraping procedure
to ensure that all the cells are collected.
Centrifuge at 720g for 3min and remove the supernatant
- Resuspend each cell pellet (from four dishes) in
0.9ml of homogenization buffer supplemented with
PIC and homogenize by three complete passes (three
downward strokes with both plungers) through a 1-ml
ball-bearing homogenizer (Balch and Rothman, 1985).
- Combine the cell homogenates and dilute with
an equal volume (~3 ml) of homogenization buffer +
PIC. Divide the diluted homogenate into six 1.0-ml
aliquots in 1.5-ml microfuge tubes and centrifuge at
720g for 5 min.
- Carefully remove the postnuclear supernatant
(PNS) fractions and combine in a plastic 15-ml tube
(Cat. No. 25319-15: Corning Inc.). Add 0.5 volume
(~2.5 ml) of 0.21M KOAc buffer to the PNS and mix.
Divide the mixture into 0.8- to 1.0-ml aliquots in
1.5-ml microfuge tubes and centrifuge at 12,000g (12,200rpm) for 2min in an Eppendorf Model 5402
refrigerated microfuge using the soft spin function.
- Remove the supernatants by aspiration and
resuspend the pellets (including any membranes on
the sides of the tubes) using a P1000 Gilson tip in a
total volume of 1.0ml of transport buffer + PIC. Dispense
0.5-ml aliquots into two 1.5-ml microfuge tubes
and recentrifuge the microsomes at 12,000g for 2min
as described earlier.
- Resuspend the membrane pellets by repeated
trituration using a Pl000 Gilson tip in 6-8 volumes
(~1 ml per 75-µl membrane pellet) of transport buffer
containing PIC. In a typical 12-dish preparation,
1.0-1.5ml of resuspended microsomes (at a protein
concentration of 3-4mg/ml) is obtained depending on
the starting cell density. Pool the membranes, divide
into 50- or 100-µl aliquots in 0.5-ml microfuge tubes,
freeze in liquid N2, and store at -80°C. The microsomes
can be stored for several months with no loss of transport
The following procedure for the preparation of
Golgi membranes by flotation on a sucrose density
gradient is a modification of that originally described
by Balch et al.
57.5mM KOAc buffer
: 87.5 mM
KOAc, 1.25 mM
, and 20mM
HEPES (pH 7.4). To make
100ml, add 2.18ml of 4M
KOAc stock, 0.125ml of
stock, and 2ml of 1M
(pH 7.4) stock to distilled water. Store at 4°C.
D. Reconstitution of ER to Golgi Transport
- Prepare an enriched Golgi membrane fraction
from noninfected wild-type Chinese hamster ovary
cells by flotation in sucrose density gradients as
described by Beckers and Rothman (1992). Recover the
membranes at the 29-35% sucrose interface, mix thoroughly
with 4 volumes of 87.5 mM KOAc buffer, and
divide into 0.5- to 1.0-ml aliquots in microfuge tubes.
Centrifuge at 16,000 g (soft spin) for 10 min.
- Remove the supernatants by aspiration, wash the
pellets with 2 ml (final volume) of transport buffer, and
combine the membranes into two 1.5-ml microfuge
tubes. Centrifuge at 16,000 g (soft spin) for 10 min.
- Resuspend the membranes using a P1000 Gilson
tip in transport buffer (total volume of 1.0ml per
1 × 106 cells). Divide the Golgi membrane fraction into
50- to 100-µl aliquots in 0.5-ml microfuge tubes, freeze
in liquid N2, and store at -80°C.
- Set up 40-µl transport reactions containing the
components indicated in Table I in 1.5-ml microfuge
tubes (see Comment 2). A reaction cocktail consisting
of the salts, ATP-regenerating system, and water is
added first, followed by the cytosol and finally the
microsomes. Mix by pipetting up and down four times
using a P20 Gilson tip.
- Transfer the reactions to a 32°C water bath and
incubate for 75-90 min.
- Terminate the reactions on ice, harvest the membranes
by centrifugation at 20,000g (27,000rpm) in a
Beckman TLA 100.3 rotor, and remove the supernatant
by aspiration (see Comment 3).
- Solubilize the membranes in one-third volume of
0.3% SDS in 1M Na acetate, pH5.6, and freshly added
30mM BME. Boil 5 min, cool, and two-third volume
1M Na acetate containing 3mU of endo H. Incubate
overnight, then terminate the reactions by adding
Laemmli sample buffer as described by Schwaninger et al. (1992).
- Separate the endo H-sensitive and -resistant
forms of VSV-G on 6.75% (w/v) SDS-polyacrylamide
gels as described by Schwaninger et al. (1992).
- Transfer the proteins to a nitrocellulose membrane
and perform Western blotting using anti-VSV-G
polyclonal primary antibody (1:10,000) and peroxidase-
conjugated anti-rabbit IgG secondary antibody
(1:10,000) according to Rowe et al. (1996).
- Develop the blots using enhanced chemiluminescence
and expose to autoradiography film. Quantitate
the relative band intensities of the endo
H-sensitive and -resistant forms of VSV-G in each lane
by densitometry (see Comments 4 and 5).
E. Vesicle Formation Assay
sucrose and 20 mM
(pH 7.4). To make 100 ml, add 8.55 g of sucrose and
2 ml of 1M
HEPES-KOH (pH 7.4) stock to distilled
water. Store at 4°C.
F. Two-Stage Fusion Assay
0.25 M sorbitol buffer
- Set up 40-µl reactions as described in Section
III,D, step 1, containing 5-10µl of microsomes, 5-12µl of cytosol, salts, and an ATP-regenerating system (see
- Incubate at 32°C for 0-60min and harvest the
membranes as described in Section III,D, step 3. The
membrane pellets can be stored for several hours at
this stage prior to the differential centrifugation procedure
- Add 40 µl of resuspension buffer and disperse the
membrane pellets by pipetting up and down 10 times
using a P200 Gilson tip (see Comments 3 and 7). Incubate
the membranes for 10min on ice and repeat the
trituration procedure to resuspend the membranes
completely. Add 8.5 µl of a salt mix [7.3 µl of 1M KOAc
+ 1.2µl of 0.1M Mg(OAc)2] to the resupended membranes
and mix by pipetting up and down 5 times with
a P20 Gilson tip. Perform the differential centrifugation
step at 16,000g (14,000 rpm) for 3 min in an Eppendorf
Model 5402 refrigerated microfuge using the soft
- Using a P200 Gilson tip, carefully take the top
34-µl supernatant fraction from the side of the tube opposite the pellet. Transfer to a 1.5-ml polyallomer
microfuge tube and centrifuge at 100,000g (60,000 rpm)
for 20min. Carefully aspirate the remaining supernatant
fraction from the 16,000-g (medium speed)
pellet and the entire supernatant from the 100,000-g (high speed) pellet (see Comment 8).
- Add 50 and 35µl of IX Laemmli sample buffer
(Laemmli, 1970) to the medium-speed pellet (MSP)
and high-speed pellet (HSP) fractions, respectively,
and boil at 95°C for 5min. Determine the relative
amounts of VSV-G in the MSP and HSP from each reaction
by SDS-PAGE and quantitative immunoblotting
as described by Rowe et al. (1996).
- A quicker alternative method to step 2 is as follows:
After incubation put the tubes on ice and spin at
16,000g (14,000rpm) for 3min. Carefully remove the
top 32µl as described in step 4 and transfer to 1.5-ml
polyallomer tubes. Spin at 100,000g (60,000rpm) for
20min. Aspirate supernatant as in step 4 and add 40
and 32µl 1× Laemmli sample buffer to the mediumspeed
pellet (MSP) and high-speed pellet (HSP) fractions,
respectively. Vortex carefully and boil at 95°C for
: 0.25 M
sorbitol and 20 mM
(pH 7.4). To make 100 ml, add 10 ml of 2.5 M
stock and 2ml of 1M
HEPES-KOH (pH 7.4) stock
to distilled water. Store at 4°C.
- For stage 1 incubations, prepare scaled-up
(100µl) vesicle formation reactions containing 25µl of microsomes and 30B1 of cytosol as described in
Section IIIE, step 1. Incubate for 10min at 32°C.
- Terminate the reactions by transfer to ice and
sediment the membranes as described in Section IIID,
- Resuspend the membranes in 90 µl of resuspension
buffer, add 9.8 µl of salt mix [7.3 µl of 2M KOAc +
2.5 µl of 0.1M Mg(OAc)2], and perform the differential
centrifugation step as described in Section IIIE, step 3.
- Withdraw the top 75-ml medium-speed supernatant
fraction and recover the vesicles by centrifugation
at high speed as described in Section IIIE, step 4.
- Resuspend the HSPs in 25µl of 0.25M sorbitol
buffer by pipetting up and down 10 times with a P200
Gilson tip and then add 1.9µl of 1M KOAc (70mM KOAc final concentration) (see Comment 9).
- Set up 40-µl stage 2 reactions containing 10µl of
resuspended HSP fraction, 4µl of Golgi membranes,
8µl of cytosol, and an ATP-regenerating system and
salts at the final concentrations indicated in Table I (see
- Incubate for 60min at 32°C and terminate the
reactions on ice. Harvest the membranes and quantitate
the conversion of VSV-G to the endo H-resistant
form as described in Section IIID, steps 3-7.
- To reproduce the temperature-sensitive phenotype
of ts045 VSV-G transport in vitro, it is necessary
to supplement the postinfection medium and homogenization
buffer with dithiothreitol as described by
Aridor et al. (1996).
- Preparations of the ATP-regenerating system and
Ca2+/EGTA buffer are described by Schwaninger et al. (1992). Transport inhibitors such as antibodies are dialyzed
against 25/125 prior to addition to the assay, and
the volumes of the salts in the reaction cocktail are
adjusted to achieve the final concentrations described
in Table I. The reactions can be preincubated on ice for
up to 45 min with no loss of transport activity.
- All of the membrane-bound VSV-G is sedimented
at 20,000g following the 32°C incubation. In the
vesicle formation assay, membranes are resuspended
in sucrose buffer prior to differential centrifugation.
- The detection system is linear over the range of
VSV-G concentrations tested.
- As an alternative to densitometry, VSV-G bands
can be detected by direct fluorescence imaging (e.g.,
using a Bio-Rad GS-363 molecular imaging system).
- In the vesicle formation and two-stage assays,
the volumes of salts (see Table I) added to the reaction
cocktail are adjusted to account for the salts present in
the cytosol and membrane preparations in the assay. UDP-GlcNAc is omitted from the vesicle formation
assay and from stage 1 of the two-stage assay.
- Although in the original description of the assay
(Rowe et al., 1996) a 0.25 M sorbitol buffer was used to
resuspend the membranes prior to differential centrifugation,
we have subsequently found that resuspension
in 0.25 M sucrose buffer gives higher yields of
vesicles at this step.
- Because the HSP is small and translucent, care
should be taken to avoid losing it during aspiration of
the high-speed supernatant.
- In a typical experiment, multiple stage 1 reactions
are performed and the HSPs are resuspended
successively in the desired final volume of sorbitol
buffer and are then adjusted to 70 mM KOAc.
This work was supported by postdoctoral fellowships
from the Cystic Fibrosis Foundation to XW and
grants from the National Institutes of Health (GM
42336; GM33301) to WEB.
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