(Received for publication, October 27, 1994)
From the
A method is described for purification of G protein and
subunits from Sf9 cells infected with recombinant
baculoviruses. The subunit to be purified is coexpressed with an
associated subunit bearing a hexahistidine tag. After adsorption of the
oligomer to a Ni
-containing column, the subunit to be
purified is eluted specifically by promoting subunit dissociation with
AlF
. The
subunits of
G
, G
, G
, and G
and
the
subunit complex were easily and
efficiently purified by this method. Results were superior to
established procedures in all cases.
Purified was
characterized for the first time. The protein has a slow rate of
guanine nucleotide exchange (k
=
0.01 min
) and a very slow k
for hydrolysis of GTP (0.1-0.2 min
).
GTP
S (guanosine
5`-3-O-(thio)triphosphate)
does not
influence the activity of several adenylyl cyclases or phospholipases.
Activated
inhibits the activity of type I and type V
adenylyl cyclases. It is a somewhat more potent inhibitor of type V
adenylyl cyclase than is activated
.
Heterotrimeric guanine nucleotide-binding proteins (G proteins) ()transduce regulatory signals from a large number of
cell-surface receptors to effectors such as adenylyl cyclases,
phosphodiesterases, phospholipases, and ion
channels(1, 2, 3, 4, 5) .
Each G protein oligomer contains a guanine nucleotide-binding
subunit, which can be palmitoylated and, in some cases, myristoylated,
and a high affinity dimer of
and
subunits;
is
prenylated. Sixteen genes are known to encode G protein
subunits,
while four
and six
subunits have been described to date.
The
subunits are commonly classified as members of four
subfamilies:
and
(stimulators of
adenylyl cyclases);
,
,
,
,
,
,
, and
(a
functionally diverse group; pertussis toxin substrates, with the
exception of
);
,
,
, and
/
(activators of phospholipase C-
s); and
and
. The
subfamily remains
poorly characterized.
cDNAs that encode and
were isolated from a mouse brain library using a
homology-based, polymerase chain reaction strategy(6) . The two
proteins appear to be expressed
ubiquitously(6, 7, 8) . Although little is
known about the identity of the receptors that activate these G
proteins or the effectors that they regulate, it has been reported
recently that overexpression of wild type or constitutively activated
causes transformation of NIH 3T3
cells(9, 10, 11) . In addition, the Drosophila gene concertina, whose product is highly
homologous to both
and
, is
involved in ventral furrow formation and posterior midgut invagination
during gastrulation(12) .
Purification of G proteins from
natural sources is problematic because of limiting quantities (in most
cases) and difficulty of resolution from closely related family
members. Expression of certain subunits (
,
, and
) in Escherichia coli yields large amounts of protein that can be myristoylated where
appropriate (
and
) (13) ,
but the proteins are not palmitoylated and may be missing other unknown
modifications (particularly
). We (14, 15, 16, 17) and others (18) have expressed G protein
and
subunits in
Sf9 cells after infection with recombinant baculoviruses, but yields of
appropriately modified protein are often low and purification is
laborious. We describe herein a general and substantially improved
method for purification of several G protein
and
subunits from Sf9 cells. The protein to be purified is coexpressed with
an associated hexahistidine-tagged subunit. The oligomer is adsorbed to
a Ni
-containing column, and the desired protein is
eluted by subunit dissociation (activation with
AlF
). This combination of specific
adsorption and elution yields a highly enriched product that can be
brought to essential homogeneity by one step of ion exchange
chromatography. We have applied this method to permit characterization
of
and further investigation of
.
The resulting plasmid was digested with BglII and XbaI, and the fragment containing the
His-
cDNA was ligated into the BglII and XbaI sites of baculovirus transfer vector
pVL1392. The amino acid sequence of His
-
is MAHHHHHHA-
(3-71).
To construct a
transfer vector for , a 1.4-kilobase pair cDNA
fragment that encodes murine
was isolated from
pG12-5 (6) by digestion with EcoRI and SacI; the SacI site was blunt-ended (Klenow). This
fragment was ligated into the EcoRI and SmaI sites of
pVL1392. To remove two Bsu36I sites from the 5`-noncoding
region of the
cDNA, this plasmid was digested with EcoRI and Bsu36I and self-ligated after both sites
were blunt-ended (Klenow). The sequences of all constructs were
confirmed by DNA sequencing.
pVLHis-
or
pVL
was cotransfected (Lipofectin, Life Technologies,
Inc.) into Sf9 cells with BacPac6 viral DNA (Clontech) linearized with Bsu36I. Recombinant viruses were plaque-purified and amplified
as described (20) . Positive viral clones were identified by
immunoblotting extracts of infected cells with
antiserum J168 or
antiserum X263.
Recombinant baculoviruses encoding ,
, and
subunits have been
described(14, 16, 21) . A virus encoding
hexahistidine-tagged
was constructed and kindly
provided by Christiane Kleuss (this laboratory). A recombinant
baculovirus encoding
was generously provided by
Patrick Casey and Tim Fields (Duke University).
Sf9 cells (4-liter
culture; 1.5 10
cells/ml) were infected with
amplified recombinant baculoviruses encoding the desired G protein
subunit, the
subunit, and the
His
-
subunit (1 plaque-forming unit/cell
for each virus). Cells were harvested 48 h later by centrifugation at
1000
g for 10 min and suspended in 600 ml of ice-cold
lysis buffer (50 mM NaHepes (pH 8.0), 0.1 mM EDTA, 3
mM MgCl
, 10 mM
-mercaptoethanol, 100
mM NaCl, 10 µM GDP, 0.02 mg/ml
phenylmethylsulfonyl fluoride, 0.03 mg/ml leupeptin, 0.02 mg/ml
1-chloro-3-tosylamido-7-amino-2-heptanone, 0.02 mg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), and
0.03 mg/ml lima bean trypsin inhibitor). Remaining procedures were
carried out at 4 °C unless otherwise specified. Cells were lysed by
nitrogen cavitation (Parr bomb) at 500 p.s.i. for 30 min. Cell lysates
were centrifuged at 750
g for 10 min to remove intact
cells and nuclei. The supernatants were centrifuged at 100,000
g for 30 min, and the resultant pellets were suspended in 300
ml of wash buffer (50 mM NaHepes (pH 8.0), 3 mM MgCl
, 10 mM
-mercaptoethanol, 50 mM NaCl, 10 µM GDP, and proteinase inhibitors as above)
using a Potter-Elvehjem homogenizer and centrifuged again as above. The
pellets (1.6-2.4 g of protein suspended in 160 ml of wash buffer)
were frozen in liquid nitrogen and stored at -80 °C.
For purification of , the concentration of
GDP in the buffers was raised to 50 µM. The Ni-NTA column
was washed with buffer A containing 300 mM NaCl and 5 mM imidazole. The column was then incubated at room temperature with
buffer F (20 mM NaHepes (pH 8.0), 100 mM NaCl, 10
mM
-mercaptoethanol, 0.2 mM MgCl
, 5
µM GTP
S, 0.2% sodium cholate, and 5 mM imidazole) and washed with 32 ml of the same buffer. This step is
utilized to elute an endogenous (Sf9 cell)
-like
protein.
was eluted with buffer E. The peak fractions
from the Ni-NTA column were diluted 3-fold with buffer D and loaded
onto a Mono Q HR 5/5 anion exchange column that had been equilibrated
with buffer D.
was eluted with a linear gradient of
NaCl (0-400 mM; 20 ml). Fractions were assayed by
immunoblotting with
/
antiserum Z811
and
antiserum B825. Recombinant
is
recognized by both antisera and was eluted in fractions containing
about 220 mM NaCl. An endogenous Sf9 cell
-like protein is recognized by antiserum Z811 but not
by antiserum B825 and eluted later in the gradient (about 280 mM NaCl)(14) . The peak fractions that contained only
recombinant
were pooled and concentrated, and the
buffer was changed to buffer D containing 100 mM NaCl and 0.5
µM GDP.
For purification of , the
Ni-NTA column was processed as described for
. The
peak fractions from the Ni-NTA column were diluted 3-fold with buffer D
and loaded onto a Mono Q HR 5/5 column that had been equilibrated with
buffer D.
was eluted with a NaCl gradient
(0-400 mM; 20 ml). Fractions were assayed for
[
S]GTP
S binding activity and by
immunoblotting using
/
antiserum
P960.
was eluted in fractions containing about 200
mM NaCl. The peak fractions were processed as described for
. This
preparation contained a very
small amount of an Sf9 cell
-like protein (about 1%).
Steady state GTPase activity was
measured as described in the figure legends. Reactions were terminated
by addition of 20-µl aliquots to 780 µl of ice-cold 5% (w/v)
Norit in 50 mM NaHPO
, and
P
was determined as described
previously(23) .
We have previously described the purification of relatively
small amounts of ,
, and
from Sf9 cells following their coexpression with G
protein
and
subunits(14, 15) . Expression
of the heterotrimer increases the amount of active
subunit in
membrane fractions and prevents the aggregation of
and
. However, difficulty with purification of
prompted the present effort to improve and
generalize this technology. The resulting method for purification of
G
subunits takes advantage of the high specificity,
affinity, and capacity of Ni-NTA resin (33, 34) for
hexahistidine-tagged
and specific elution of a coexpressed
subunit following activation with
AlF
.
Figure 1:
Purification of
on Ni-NTA column. A cholate extract of membranes
(600 mg of membrane protein) from Sf9 cells expressing
and
His
-
was
diluted, loaded onto a 4-ml Ni-NTA column, and chromatographed as
described under ``Materials and Methods.'' Fractions (4
µl) were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (9% gels) and stained with silver nitrate. Lane
1, load; lane2, flow-through; lanes
3-5, wash with equilibration buffer containing 5 mM imidazole; lanes 6-10, elution with AMF plus GDP; lanes11 and 12, elution with 150 mM imidazole. The arrows at the right indicate the
positions of
and
.
is not well stained with silver nitrate;
His
-
runs at the dye
front.
was further purified by Mono
S cation exchange chromatography using CHAPS as the detergent (Fig. 2). The final sample of
was visualized
as a homogeneous silver-stained band in polyacrylamide gels with an
apparent molecular weight of 43,000 (Fig. 3, lane 1).
The yield of purified protein was 600 µg from 600 mg of Sf9 cell
membrane protein, which is obtained from a 1-1.5-liter culture (Table 1). The relatively low enrichment of GTP
S binding
activity is undoubtedly due to the presence of other GTP-binding
proteins in the membrane extract. The actual stoichiometry of binding
of GTP
S to
is approximately 0.5 mol/mol
(approximately 10 nmol/mg protein) (see below).
Figure 2:
Mono
S chromatography of . The peak fractions of
from the Ni-NTA column were loaded onto a Mono S
column and chromatographed as described under ``Materials and
Methods.'' Fractions (3 µl) were assayed for GTP
S binding
activity with 5 µM GTP
S and 10 mM MgSO
for 90 min at 30 °C.
Figure 3:
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and silver staining of purified ,
,
,
, and
.
subunits (50 ng) purified as
described in the text were subjected to electrophoresis through 9%
polyacrylamide gels and were stained with silver nitrate. Lane1,
; lane2,
; lane3,
; lane4,
; lane5,
.
The fractions from
the Ni-NTA column that were eluted with AlF also contained Sf9 cell proteins that were detected with
/
antiserum P960 (Sf9
) or
/
antiserum
Z811 (Sf9
). However, both immunoreactive proteins
flow through the Mono S column. When the purification procedure was
followed using membranes from Sf9 cells expressing only
His
-
, immunoreactive
proteins were not detected with
antiserum J168 or
broadly reactive antisera in the fractions corresponding to those where
is normally found. We conclude that the final
preparation of
is essentially free of other G
protein subunits.
To purify , the Ni-NTA column was
also washed with buffer containing 5 µM GTP
S to
remove Sf9
. This wash takes advantage of the
relatively poor affinity of
for GTP
S in order to
activate and remove endogenous G
subunits with high
affinity for the nucleotide. Sf9
was resolved from
recombinant
by Mono Q chromatography (Fig. 3, lane2). To purify
(Fig. 3, lane4), endogenous
was removed by
Mono Q chromatography, but it was not possible to resolve Sf9
from recombinant
. However,
performance of the purification protocol for
in the
absence of expression of this subunit suggests that the level of
contamination by Sf9
is only about 1%. The yields of
and
were 110 and 1400 µg,
respectively, from 600 mg of Sf9 cell membrane (Table 1).
Figure 4:
Immunoblot analysis of purified
subunits. Each purified
subunit (50 ng each of
,
,
,
,
,
, and
) was subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and stained with silver
nitrate (A) or immunoblotted (B) with
antisera J168 and J169,
/
antiserum Z811,
/
antiserum
P960, and
antiserum P961.
,
,
, and
were
purified as described under ``Materials and Methods.''
was purified as described previously (15) .
was purified from E. coli.
was purified from Sf9 cells and was a generous gift from Dr.
William Singer(7) .
Figure 5:
Time course of binding of
[S]GTP
S to
. A,
(250 nM) was incubated at 30 °C in HEDL
buffer with 5 µM [
S]GTP
S and
10 mM MgSO
(
), 1 mM MgSO
(
), or 5 mM EDTA (
). Aliquots (20 µl)
were withdrawn at the indicated times, filtered, and counted. Data
shown are the average of duplicate determinations from a single
experiment that is representative of three separate experiments. B,
(250 nM) was incubated at 30
°C in HEDL buffer with 10 mM MgSO
and 1
µM (
), 5 µM (
), 10 µM (
), 50 µM (
), or 100 µM GTP
S (
). Data shown are the average of duplicate
determinations from a single experiment that is representative of three
separate experiments. C, the maximum stoichiometry of binding
calculated from the data of Fig. 5B is plotted against
the concentration of GTP
S.
The
capacity of various nucleotides to compete for GTPS binding to
is shown in Table 2. Only guanine nucleotides
and, to a lesser extent, ITP compete effectively with GTP
S. This
pattern is typical of G protein
subunits(7, 25, 35) .
Most G protein
subunits are substantially protected from tryptic proteolysis when
activated with either nonhydrolyzable analogs of GTP or
AlF
. This is also true of
(Fig. 6). When the protein is incubated with GTP
S or
AlF
(GDP + AMF (30 µM AlCl
, 50 mM MgCl
, and 10 mM NaF)), exposure to trypsin results in the generation of a stable
40-kDa fragment. In the presence of GDP,
is digested
rapidly; one fragment with a mass of about 10 kDa is visualized with
antiserum J168. A small amount of the 10-kDa band is observed after
incubation with GTP
S, consistent with the slow rate of nucleotide
exchange. However, activation with AlF
appears to be complete, indicating that most of the protein in
the preparation is native and capable of undergoing activator-dependent
conformational changes similar to those observed with other G
proteins.
Figure 6:
Protection of from
tryptic proteolysis.
(1 µM) was
incubated with 100 µM GDP (lanes 1-3) or
AMF + 100 µM GDP (lanes 4-6) at 0
°C for 10 min or with 100 µM GTP
S and 10 mM MgSO
(lanes 7-9) at 30 °C for 60
min. TPCK-treated trypsin (1/10 the mass of
) was
added to each tube and incubated at 30 °C. Aliquots were withdrawn
and subjected to gel electrophoresis followed by immunoblotting with
antiserum J168. Lanes 1, 4, and 7, before
addition of trypsin; lanes 2, 3, 5, 6, 8, and 9, after addition of trypsin and
incubation at 30 °C for 10 min (lanes2, 5, and 8) or 30 min (lanes3, 6, and 9).
has intrinsic GTPase activity.
The steady state rate of GTP hydrolysis is 0.006 min
(at 9 mM free Mg
), and the rate is
constant for at least 90 min after an initial lag (Fig. 7A). This rate is approximately equal to that
observed for GTP
S binding (GDP dissociation), and it is reduced
modestly at lower concentrations of Mg
. Although the
slow exchange of nucleotide makes it difficult to load the protein with
substrate and measure the catalytic rate constant (k
) directly, we estimated the value of k
indirectly in two ways. There is a delay in
the release of
P
in the steady state GTPase
assay that is inversely related to the catalytic rate constant and the
rate of substrate binding (Fig. 7A). This lag is equal
to 1/(k
+ k
)(23, 25) . Using a value
for the lag of 5 min and k
of 0.01
min
, k
is estimated at 0.19
min
. The rate at which steady state binding of
[
-
P]GTP is achieved is also related to the
rate of nucleotide binding and k
: k
= k
+ k
, since the label is released upon
hydrolysis. Steady state binding of [
-
P]GTP
is achieved with a rate of approximately 0.1 min
(Fig. 7B). k
is thus equal
to 0.09 min
. Thus, from both measurements we suggest
a value for k
in the range of 0.1-0.2
min
.
Figure 7:
GTPase activity of . A, steady state GTPase.
was incubated in
HEDL buffer at 30 °C for 3 min, and an equal volume of solution was
then added to bring the final concentrations to 250 nM
, 10 µM [
-
P]GTP, and 10 mM MgSO
(
) or 0.5 mM MgSO
(
). Reaction
mixtures were incubated at 30 °C. At the indicated times, aliquots
(20 µl) were withdrawn and release of
P
was determined as described under ``Materials and
Methods.'' Data shown are the average of duplicate determinations
from a single experiment that is representative of five experiments. B, time course of [
-
P]GTP binding.
was incubated in HEDL buffer at 30 °C for 3 min.
An equal volume of solution was then added to bring the final
concentrations to 1 µM
, 5 µM [
-
P]GTP, and 10 mM MgSO
. At the indicated times, aliquots were withdrawn
and [
-
P]GTP binding was determined as
described under ``Materials and Methods.'' Data shown are the
average of duplicate determinations from a single experiment that is a
representative of three such experiments.
Figure 8:
Inhibition of steady state GTPase activity
of by
. The
indicated concentrations of
were
incubated with or without 50 nM
in 50
µl of HEDL buffer containing 10 µM [
-
P]GTP and 10 mM MgSO
at 30 °C for 120 min. Release of
P
was determined as described under ``Materials and
Methods.'' The difference in release of P
in the
presence or absence of
is plotted. The data shown
are the average of duplicate determinations from a single experiment
that is representative of three such experiments.
P
detected with 50 nM
or 1 µM
at 30 °C for 120 min was
2.1 or 0.67 pmol/50 µl, respectively.
The availability of purified, activated
allowed us to test the effect of the protein on
several known effector targets for G
subunits.
did not stimulate or inhibit the activity of type I
or V (Table 3) or type II (not shown) adenylyl cyclase, nor did
the protein affect the activity of phospholipase C-
1,
2, or
3 or phospholipase C-
1 (data not shown). Similarly,
had no effect on ADP-ribosylation factor-sensitive
phospholipase D activity (36) or phosphatidylinositol 3-kinase
activity (37) (data not shown).
Possible modifications of
by bacterial toxins were also examined.
was not ADP-ribosylated by cholera toxin in the presence of
ADP-ribosylation factor, nor was it ADP-ribosylated by pertussis toxin
(data not shown). The latter is consistent with the fact that the
cysteine residue near the carboxyl terminus that serves as the site of
ADP-ribosylation of
or
by pertussis
toxin is replaced by isoleucine in
.
Figure 9:
Inhibition of type V adenylyl cyclase
activity by and
. The indicated
concentrations of
subunits were reconstituted with 10 µg of
membranes from Sf9 cells expressing type V adenylyl cyclase in the
presence of 50 nM GTP
S
. Adenylyl
cyclase activity was assayed as described under ``Materials and
Methods.'' Data shown are the average of duplicate determinations
from a single experiment that is representative of two such
experiments.
Based on our experience to date,
we suspect that this method will also be useful for purification of
,
,
,
,
,
, and various
species of
. We make no guesses about
,
, and
. We were unable to purify
to homogeneity or
with this
method.
appears to have a lower affinity for
His
-
on the Ni-NTA column
and did not bind adequately.
is not activated with
AlF
(15) and thus cannot be
eluted as described. We can elute
with GTP
S to
purify the irreversibly activated subunit. Attempts to place a
hexahistidine tag at the amino terminus of the
subunit gave adequate results, although the yields of purified
subunits were decreased by about 50%. This is perhaps due to
lower levels of expression of His
-
than of
in Sf9 cells.
and
have significant
differences in the amino acid residues that make up the guanine
nucleotide binding pocket when compared with other G protein
subunits. The crystal structure of
indicates that
Arg
, Cys
, and Thr
contribute
to binding of the guanine and ribose rings of GTP
S(46) . A
similar pattern is seen with G
(47) . These
residues are conserved in all mammalian G
subunits
except
and
, where Leu, Thr, and
Ile, respectively, fill these positions. (
)These sequence
differences in
and
may be
responsible for the reduced affinity of GTP
S binding or slow
nucleotide exchange.
DNA encoding wild type has
been isolated as a sequence capable of highly efficient transformation
of NIH 3T3 cells (9) , and a mutant of
that
is presumed to be GTPase-deficient and therefore constitutively active
can also cause cellular transformation(10, 11) .
However, the effector molecule(s) that are presumably regulated by
have not been identified. Transient expression of
increases serum-activated phospholipase A
activity in NIH 3T3 cells (10) and stimulates a protein
kinase C-dependent Na
/H
exchanger
activity in COS-1 cells(48) . However, there is no evidence for
direct interaction of
with these proteins. Nor is
there evidence for interaction of
with previously
identified targets for G protein
subunits. Expression of
hexahistidine-tagged G protein subunits in appropriate cells may permit
isolation of novel targets for G protein action by approaches analogous
to those presented here for purification of known cohorts of these
proteins.