(Received for publication, November 14, 1994; and in revised form, April 14, 1995)
From the
Triton X-114 phase partitioning, a procedure used for purifying
integral membrane proteins, was used to study protein components of the
mammalian visual transduction cascade. An integral membrane protein,
rhodopsin, and two isoprenylated protein complexes, cyclic GMP
phosphodiesterase and G Heterotrimeric guanine nucleotide-binding proteins (G
proteins), Domains of
G The carboxyl terminus of
G G proteins appear to interact with the inner
surface of the plasma membrane. Association of G Integral membrane proteins
have been identified and purified based on their phase-partitioning
characteristics(34) . Solutions of Triton X-114 separate into
detergent-rich and detergent-depleted (aqueous) phases at
To prepare G Rhodopsin was prepared by the
method of Hong and Hubbell(41) . Protein concentrations were
determined by Coomassie Blue assay (Bio-Rad) using bovine serum albumin
as a standard.
For
large-scale purification of fusion proteins, E. coli cells
were grown in 500-1000 ml of LB/ampicillin medium at 37 °C to an
absorbance of 0.4 at 600 nm.
Isopropyl-
Figure 1:
Phase partitioning
of retinal proteins. A, purified G
G
Figure 2:
Effect of G
After addition of Gpp(NH)p
in the presence of G
Figure 3:
Effect of Gpp(NH)p on partitioning of
G
To evaluate directly
the role of bound nucleotide in apparent hydrophobicity, samples of
G
Figure 4:
Effect of bound GDP or GTP
Interaction between the PDE complex and
G
Figure 5:
Effect of
G
Figure 6:
Phase partitioning of tryptic fragments of
transducin. G
Figure 7:
Phase partitioning of tryptic fragments of
G
Figure 8:
Phase partitioning of intact recombinant
and carboxyl-terminal truncated G
Figure 9:
Phase
partitioning of myristoylated recombinant and nonmyristoylated
G
Figure 10:
Effect of NAD:arginine
ADP-ribosyltransferase-catalyzed ADP-ribosylation of G
Triton X-114 phase partitioning initially received attention
as a way of purifying integral membrane proteins, which are typically
retained completely within the detergent phase(34) . It also
has been used for the separation of more hydrophobic proteins from
those that are less so, as well as for evaluating the hydrophobicity of
proteins (50, 51, 52, 53, 54, 55, 56) .
Reasons for hydrophobic behavior in a detergent environment include
either the post- or cotranslational addition of hydrophobic moieties
(myristoyl, palmitoyl, isoprenyl, etc.) or intrinsic features of
protein secondary structure. On Triton X-114 phase partitioning, the
integral membrane receptor rhodopsin partitioned completely into the
detergent-rich phase, whereas soluble arrestin was mainly aqueous. The
farnesylated G G protein The partitioning
of G It appears that both the amino and carboxyl termini
contribute to the hydophobicity of G It has been suggested that
the amino and carboxyl termini of G Pertussis toxin-catalyzed
ADP-ribosylation of a cysteine four amino acids from the carboxyl
terminus of G
, partitioned into the
detergent-rich phase. Arrestin, a soluble protein, accumulated in the
aqueous phase. G
distributed about equally between
phases whether GDP (G
GDP) or GTP
(G
GTP) was bound. G
increased recovery of G
GDP but not
G
GTP in the detergent phase. Trypsin-treated
G
, which lacks the fatty acylated amino-terminal 2-kDa
region, accumulated to a greater extent in the aqueous phase than did
intact G
. Trypsinized cGMP phosphodiesterase, which
lacks the isoprenyl group, partitioned into the aqueous phase. A
carboxyl-terminal truncated mutant (Val-331 stop) of G
accumulated more in the aqueous phase then did recombinant
full-length G
, supporting the role of the carboxyl
terminus in increasing its hydrophobicity. N-Myristoylated
recombinant G
was more hydrophobic than recombinant
G
without myristate. ADP-ribosylation of G
catalyzed by NAD:arginine ADP-ribosyltransferase, but not by
pertussis toxin, increased hydrophilicity. Triton X-114 phase
partitioning can thus semiquantify the hydrophobic nature of proteins
and protein domains. It may aid in evaluating changes associated with
post-translational protein modification and protein-protein
interactions in a defined system.
(
)consisting of
,
, and
subunits, couple cell-surface receptors with effectors and may
also participate in signaling involving intracellular
organelles(1) . G proteins are activated by GTP, which upon
binding to the
subunit (G
), causes a
conformational change (2, 3) and promotes its
dissociation from the
complex (G
). GTP
hydrolysis and GTP-GDP exchange, reactions critical for regulation of
G
activity, are dependent on the presence of receptor,
G
, and
effector(4, 5, 6, 7) .
involved in interaction with receptor and
G
have been mapped using a variety of methods. It
was concluded that the amino-terminal region of G
is
important for its interaction with G
. Removal of
the first 18 amino acids from the amino terminus of G
by trypsin abolished G
stimulation of
pertussis toxin-catalyzed ADP-ribosylation, although the
ADP-ribosylation site itself was intact(8) . Limited
proteolysis with staphylococcal V8 protease, which removes 21 amino
acids from the amino terminus, inhibited other
-dependent
reactions including receptor-stimulated GTP hydrolysis, GTP-GDP
exchange, and binding of G
to rhodopsin(4) . A
monoclonal antibody directed against the amino terminus of G
interfered with pertussis toxin-catalyzed
ADP-ribosylation(9) , as did deletions in the amino terminus of
recombinant G
(10) . Amino acids 7-10 in
recombinant G
were deemed critical for interaction
with G
(11) .
subunits is important for receptor interaction.
ADP-ribosylation of cysteine 347, four residues from the
carboxyl-terminal end of G
, functionally uncoupled it
from receptor(12, 13, 14) . Peptides
possessing the carboxyl-terminal sequence of G
inhibited its binding to rhodopsin(15) . The behavior of
chimeric G
/G
recombinant proteins was
consistent with the conclusion that the carboxyl terminus of
G
is critical for coupling the
-adrenergic
receptor to adenylyl cyclase (16) . The carboxyl terminus is
important in the membrane association of
G
(17, 18) , and the carboxyl terminus
of G
was postulated to be involved in anchoring the
G
phosphodiesterase complex to
membranes(19) .
with membranes is facilitated by farnesylation or
geranylgeranylation of a cysteine four amino acids from the carboxyl
terminus of the
subunit(20, 21, 22, 23) . Membrane
association of some G
subunits (e.g. G
and G
) was dependent on the
amino terminus as limited digestion with trypsin, which removes an
amino-terminal 2-kDa fragment, produced a soluble protein(24) .
The amino-terminal glycines of G
and G
are myristoylated, a modification that enhances membrane
association (25, 26, 27, 28) .
Membrane association may occur directly via the myristoyl moiety and
its neighboring amino acids, as has been postulated for the Src
protein(29, 30) , or indirectly if it increases the
association of the
subunit with the prenylated and
membrane-associated G
subunits(31, 32) . In support of the latter
hypothesis, both
-stimulated ADP-ribosylation and the
affinity of G
for
-Sepharose were
significantly greater with the myristoylated than with the
nonmyristoylated protein(11, 31) . G
modified with more hydrophilic myristate analogues was less
likely than native G
to associate with
membranes(25) . In sum, the data are consistent with a role for
a myristoylated amino terminus in membrane association, although with
the more recent recognition that several G
subunits are
also palmitoylated, the contribution of the less hydrophobic myristate
may need to be reassessed(33) .
30
°C. It is generally agreed upon that the more
``hydrophobic'' a protein or peptide, the more likely it will
partition into the detergent-rich phase. This partitioning difference
has been the basis for separating integral from peripheral membrane
proteins(34) . We investigated this partitioning procedure as a
new way of characterizing the hydrophobic and membrane-association
domains of native and recombinant G
and myristoylated
and nonmyristoylated rG
as well as possibly assessing
protein-protein interactions in a purified defined system.
Materials
Gpp(NH)p, GTP, and GTPS were purchased from Boehringer
Mannheim; [
P]NAD (30 Ci/mmol),
[
S]GTP
S (40 Ci/mmol),
[
H]GTP (15 Ci/mmol),
[
H]myristic acid (16 Ci/mmol), GA/1 antibody, and
AS/7 antibody from DuPont NEN; Triton X-114 from Fluka; TPCK-treated
trypsin from Worthington; glutathione-agarose, NAD, ATP, and soybean
trypsin inhibitor from Sigma; blue Sepharose CL-6B and PD-10 columns
from Pharmacia Biochem Inc.; Triton X-100 from Research Products
International Corp.; isopropyl-
-D-thiogalactopyranoside
from United States Biochemical Corp.; goat anti-rabbit IgG horseradish
peroxidase-coupled antibody from Pierce; goat anti-mouse IgG
horseradish peroxidase-coupled antibody from Promega; LB medium from
Digene; ampicillin sodium from Amresco; and molecular weight standards
from Life Technologies, Inc. Densitometry was performed with a
Molecular Dynamics laser densitometer and Image Quant software.
SDS-PAGE and Western blotting were done in a Novex Minicell apparatus
using 12% or 4-20% gradient Tris/glycine gels.
Methods
Preparation of PDE
PDE was essentially prepared
as described previously(35, 36) . Briefly, fresh
bovine eyes were obtained from a local abattoir; retinas were
dissected, frozen on dry ice, and stored at -70 °C. After
thawing, retinas were suspended in buffer A (60 mM KCl, 30
mM NaCl, 1 mM dithiothreitol, 10 mM MOPS, pH
7.5, 1 mM NaN, 2 mM MgCl
, 0.1
mM EGTA) containing 45% sucrose, vortexed, and centrifuged
(27,750
g, 20 min). After diluting the supernatant
with an equal volume of buffer A without sucrose, rod outer segments
(ROS) were pelleted by centrifugation (27,750
g, 20
min). Pellets were washed twice by suspending in buffer A and
centrifuging (27,750
g, 20 min). Washed ROS were then
suspended in buffer B (5 mM Tris-HCl, pH 7.5, 0.5 mM MgCl
, 0.05 mM EGTA, 0.5 mM
NaN
, 1 mM dithiothreitol, 1 µg/ml soybean
trypsin inhibitor, 1 µg/ml leupeptin) and centrifuged (45,000
g, 30 min). This supernatant, which contained mainly
PDE (35) and arrestin, was concentrated in a Macrosep 100
concentrator (Filtron Technology Corp.) and used as such or further
purified by molecular sizing on an Ultrogel AcA-34 column.
Preparation of Transducin and Subunits
ROS, after
extraction of PDE, were washed three additional times by centrifugation
in buffer B without soybean trypsin inhibitor or leupeptin (45,000
g, 30 min). Transducin was then eluted with buffer B
containing 100 µM GTP with centrifugation (45,000
g, 30 min). G
and G
subunits were resolved by blue Sepharose
chromatography(39) .
with
radiolabeled GDP or GTP, PDE-depleted ROS were washed three times in
buffer A, and G
was eluted by two extractions with
buffer A containing 50 µM [
S]GTP
S (2-5 mCi/mmol) or 100
µM [
H]GTP (4-10
mCi/mmol)(38) . G
was concentrated with a
Centriprep-10 (Amicon, Inc.), and free nucleotide was removed using
PD-10 columns. Samples were stored at 4 °C and used within 3 days.
Bound nucleotide was identified by heating G
at 95
°C and analyzing the released nucleotide on
polyethyleneimine-cellulose TLC plates developed with 1.6 M LiCl(40) , which verified that G
eluted
with [
S]GTP
S retained this nucleotide.
[
H]GTP bound to G
was, however,
hydrolyzed to [
H]GDP. To obtain
G
in GDP-bound
(G
GDP) and GTP
S-bound
(G
GTP
S) states, procedures were
identical except that hypotonic conditions were used for elution with
labeled nucleotide(38) .
PDE Assay
PDE activity was assayed for 10 min at
30 °C in 0.3 ml of 50 mM HEPES, pH 7.5, 0.1 mM EGTA, 8.3 mM MgCl, 1 µM [
H]cGMP (
20,000 cpm/assay). Reactions
were terminated by addition of 100 µl of 11.25 mM cGMP and
5 mM 5`-GMP in 250 mM HCl; neutralized with 100
µl of 250 mM NaOH in 250 mM Tris-HCl, pH 8.0; and
then incubated with 5`-nucleotidase (Crotalus atrox venom) at
30 °C for 20 min. The
H-labeled nucleoside produced was
collected for radioassay(42) .
Triton X-114 Phase Partitioning
In a modification
of the method of Bordier(34) , Triton X-114 was added (final
concentration of 1%) to protein samples in buffer C (20 mM
Tris-HCl, pH 7.5, 0.5 mM MgCl, 0.05 mM EGTA, 0.5 mM NaN
, 1 mM dithiothreitol, 150 mM NaCl) in a final volume of
40-75 µl. Mixtures were gently mixed and kept on ice for
3-5 min. After warming to 37 °C for 5 min, the cloudy
solution was centrifuged (300
g, 5 min) in a swinging
bucket rotor prewarmed to 30-37 °C. The upper aqueous layer
was aspirated. Buffer C was added to the detergent phase, which
contained no visible particulates, and Triton X-114 was added to the
aqueous phase to equalize the compositions and volumes of the two
fractions. Equal volumes of each phase were subjected to SDS-PAGE.
Proteins were stained with Coomassie Brilliant Blue and quantified by
densitometry. In experiments in which G
contained
radiolabeled nucleotide, samples of the two phases were subjected to
PD-10 column chromatography to separate unbound from protein-bound
nucleotide, which was quantified by liquid scintillation counting.
Nucleotide that became unbound during partitioning accumulated
primarily (>85%) in the aqueous phase.
Trypsinization
Transducin
(G; 0.5 mg/ml), G
(0.2
mg/ml), or PDE (1 mg/ml) in hypotonic buffer was incubated with
TPCK-treated trypsin (1.2 µg/ml) at 30 °C for 2-10 min.
Reactions were stopped by adding phenylmethylsulfonyl fluoride or
soybean trypsin inhibitor (final concentrations of 1 mM and
0.1 mg/ml, respectively).
Preparation of Recombinant G
DNA
inserts for G and a carboxyl-truncated form of
G
were prepared by polymerase chain reaction using
G
cDNA as template. The 5`-primer for both transducin
molecules was 5`-GCAGCAGCATGATCAGGGGCCAGCGCTGAGGAGAAG-3`, which
includes a BclI restriction site enabling excision of the
polymerase chain reaction product with BclI and cloning into
the BamHI site of the pGEX-2T vector (Pharmacia Biotech Inc.)
without cleaving the BamHI site within the G
coding sequence. The 3`-primers
(5`-GCGCCACGGGAATTCTCAGAAGAGCCCGCAGTCTT-3` and
5`-GCGCCAGCGGAATTCTCAAAACTTGACGTTCTGCGTGTC-3`) included an EcoRI site after the natural stop codon or after a stop codon
introduced to replace codon 331 (valine) to yield a truncated protein.
Following polymerase chain reaction, Taq polymerase (Perkin
Elmer) and unincorporated nucleotides were removed using a QIAGEN
polymerase chain reaction purification kit. Products were digested with BclI and EcoRI. G
inserts were
gel-purified with Qiaex resin (QIAGEN) and ligated into the BamHI/EcoRI-restricted pGEX-2T vector using T4 DNA
ligase. Escherichia coli DH5
competent cells (Life
Technologies, Inc.) were transformed using heat shock and selected on
ampicillin-containing LB agar plates. After
isopropyl-
-D-thiogalactopyranoside induction, positive
clones were screened for protein production using SDS-PAGE.
-D-thiogalactopyranoside was added (final
concentration of 100 µM), and incubation (with shaking)
was continued for 3-4 h. After centrifugation, cell pellets were
washed once with phosphate-buffered saline, suspended in 10 ml of
phosphate-buffered saline, and lysed using nitrogen cavitation.
Sarcosine (0.5%) was added to solubilize the fusion proteins, and cell
debris was removed by centrifugation (15,000
g, 4
°C, 10 min). Triton X-100 (final concentration of 1%) and
glutathione-agarose beads were added to the supernatant, and the
mixture was rocked for 2-3 h at 4 °C. Binding of fusion
protein to the beads in the presence of sarcosine required Triton
X-100. Bound fusion protein (0.25-1.0 mg) was eluted with
3-5 ml of 10 mM reduced glutathione, 200 mM NaCl, 50 mM Tris, pH 8.0, 0.5% Triton X-100 and cleaved
with thrombin (2-4 µg/ml). Intact G
was
quantified by immunoblot using AS/7, a polyclonal antipeptide antibody
directed against the carboxyl terminus of
G
(43) . GA/1, an amino terminus-directed G
protein-specific polyclonal antibody, reacted similarly with intact and
carboxyl-truncated forms of recombinant G
. Intact
G
was ADP-ribosylated by pertussis toxin; as expected,
the truncated form, which lacks the ADP-ribose acceptor site (Cys-347),
was not.
Preparation of rG
The coding
region of bovine G cDNA was inserted into the pT7/Nde
expression vector(44) . This was transfected into E. coli BL21(DE3) cells (Novagen), which had or had not been previously
transfected with the pACYC177/ET3d/yNMT vector(45) . Bacteria
cotransfected with both plasmids overexpressed the yeast N-myristoyltransferase and G
, with the
resultant production of myristoylated rG
(46) ,
whereas those transfected only with pT7/Nde containing G
sequence overexpressed nonmyristoylated rG
. An
overnight culture (0.2 ml) of the cotransfected bacteria was added to 2
ml of LB medium containing the appropriate antibiotics, grown at 37
°C for 1 h, and induced with 0.2 mM
isopropyl-
-D-thiogalactopyranoside in the presence of
[
H]myristic acid (100 µCi, 16 Ci/mmol).
Growth was continued for an additional 3 h before centrifugation
(15,000
g, 7 min). Cells were then resuspended in
phosphate-buffered saline, pH 7.4; lysed by sonification; and
centrifuged (15,000
g, 45 min). The supernatant, which
contained crude
H-labeled myristoylated
rG
, was used in phase-partitioning experiments. The
same procedure was used for the singly transfected cells, which
produced nonmyristoylated rG
. Following partitioning,
proteins were detected by autoradiography in experiments using
H-labeled myristoylated rG
and with an
anti-G
antibody (47) after blotting to
nitrocellulose for both
H-labeled myristoylated
rG
and nonmyristoylated rG
.
Pertussis Toxin-catalyzed
ADP-ribosylation
G (1 µg) with or without
the indicated amounts of G
was incubated at 30
°C for 20 min (total volume of 50 µl), in 50 mM
potassium phosphate, pH 7.5, 10 mM thymidine, 10 µM [
P]NAD (1 µCi/assay), 40 µM ATP with 0.1 µg of pertussis toxin-activated with 20 mM dithiothreitol immediately before use(13) .
[
P]ADP-ribosylated proteins were subjected to
Triton X-114 phase partitioning, separated by SDS-PAGE, and exposed to
Kodak XAR film at -70 °C for 1-4 h.
NAD:Arginine ADP-ribosyltransferase-catalyzed
ADP-ribosylation
G (45 µg) was incubated at
30 °C for 45 min with or without the NAD:arginine
ADP-ribosyltransferase (4.5 µg), which had been purified from
turkey erythrocytes (48) in 200 µl of buffer containing 50
mM KPO
, pH 7.5, 200 mM NaCl, 100
µM NAD (10-15 µCi). Following labeling, samples
were applied to NAP-5 columns (Pharmacia Biotech Inc.) to separate
protein-bound from unbound radioactivity (enabling an estimation of
extent of labeling) before use in phase-partitioning experiments.
Phase Partitioning of Native Rod Outer Segment
Proteins
Arrestin, a soluble 48-kDa protein that copurified with
G, remained primarily in the detergent-depleted phase (Fig. 1A and Table 1), whereas G
(which contains farnesylated G
) and PDE (the
subunit of which is prenylated) (49) were found mainly in
the detergent-rich phase (Fig. 1A and Table 1).
Rhodopsin, an integral membrane protein, partitioned, as expected,
almost exclusively into the detergent-rich phase (Fig. 1B and Table 1).
(8
µg), G
(4 µg), and purified
G
(20 µg) in 40 µl of buffer C were
subjected to Triton X-114 phase partitioning. C lanes, sample
before partitioning; A lanes, aqueous phase; D lanes,
detergent phase. The positions of arrestin (Arr; present in
G
and G
preparations),
G
, G
, and G
are
indicated on the left. B, rhodopsin (24 µg) in
phosphatidylcholine vesicles underwent phase partitioning and SDS-PAGE.
The position of rhodopsin (R) is indicated on the left. Lanes
are labeled as described for A. Molecular weight markers
(
10
) are indicated on the right. After
exposure to Triton X-114, rhodopsin appeared as multiple aggregated
species on SDS-PAGE.
, in the absence of
G
, partitioned about equally between the
detergent-rich and detergent-depleted phases (Fig. 1A and Table 1). On repeated partitioning of G
from either the aqueous or detergent phase, distribution between
detergent and aqueous phases was similar regardless of the phase from
which G
originated (data not shown). In the presence
of G
, G
was recovered to a
greater extent in the detergent-rich phase. Recovery of G
in the detergent-rich phase increased with increasing amounts of
G
(Fig. 2, A and B). In
contrast, G
appeared not to affect the partitioning of
G
(Table 1).
on
partitioning of G
. A, G
(1.0
µg) was incubated with the indicated amounts of G
at 23 °C for 30 min before partitioning. Lanes are labeled as
described for Fig. 1A. B, mean ± S.E. (n = 4) of percentage of G
in the
detergent phase as a function of the amount of G
added.
and rhodopsin, G
was recovered to a greater extent in the aqueous phase presumably
as a result of dissociation of G
Gpp(NH)p from
G
(Fig. 3). In the absence of rhodopsin,
Gpp(NH)p had no effect on the partitioning of G
(data
not shown). Rhodopsin, in the absence of G
, did
not significantly influence the phase partitioning of G
(data not shown), consistent with other evidence that
G
is important for functional association of
G
and receptor(5) .
and rhodopsin. G
(20 µg) and rhodopsin (12 µg) were incubated at 30 °C
for 30 min with or without 100 µM Gpp(NH)p (GN)
as indicated before phase separation. Lanes are labeled as described
for Fig. 1A. Similar results were obtained with 100
µM GTP
S (data not shown). The experiment was
replicated twice with similar results. Arr,
arrestin.
GDP, G
GTP
S,
G
GDP, and
G
GTP
S underwent phase
partitioning. PD-10 columns were used to separate protein-bound from
unbound nucleotide. Ratios of protein-bound nucleotide in aqueous and
detergent phases concurred with ratios of total protein on Coomassie
Blue-stained SDS-polyacrylamide gels quantified by densitometry (Table 2), indicating that the total protein reflected the
fraction containing bound nucleotide, i.e. G
retained nucleotide during partitioning. Whether GTP
S or GDP
was bound made no difference to the overall hydrophobicity of
G
in the absence of G
(Fig. 4A and Table 2). In the presence of
G
, however, G
GDP was
recovered to a much greater extent in the detergent phase, likely as a
result of its tight association with the more hydrophobic
G
complex containing the farnesylated
subunit. In contrast, G
GTP
S partitioning
was independent of G
(Fig. 4B and Table 2).
S on
partitioning of G
with and without
G
. A,
G
[
H]GDP (8 µg) and
G
[
S]GTP
S (8 µg)
were subjected to partitioning. B,
G
[
H]GDP (30
µg) and
G
[
S]GTP
S
(30 µg) were subjected to phase partitioning. The positions of
G
and G
are indicated on the left.
Nucleotide bound is indicated at the bottom; A and D indicate aqueous and detergent phases,
respectively.
GTP
S was studied by partitioning the
proteins separately and together, which yielded similar results whether
the amounts of G
and PDE were equimolar or one or the
other was in excess (data not shown). To verify that the proteins were
not denatured during partitioning, PDE activity and
G
GTP
S-activated PDE activity were
determined. The distribution of PDE activity in detergent and aqueous
phases was similar whether or not G
GTP
S was
present during partitioning (Fig. 5) and also when PDE
partitioned in the absence of G
GTP
S was
assayed in its presence (data not shown). The partitioning of
G
GTP
S was not affected by PDE whether
measured by SDS-PAGE and densitometry or by quantifying protein-bound
radioactive nucleotide (data not shown).
GTP
S on phase partitioning of PDE
activity. PDE (0.1 µg) was subjected to phase partitioning with or
without G
GTP
S (2.0 µg). Triton X-114
and buffer A were then added back to equalize detergent and buffer
concentrations in the two phases, and an additional 100 µl of
buffer A were added to each. Samples (100 µl) of these mixtures
were assayed for PDE activity. Emptybars represent
activity recovered in the aqueous phase, and hatchedbars in the detergent phase. Errorbars represent
mean ± S.E. (n = 3). Bars 1 and 2, PDE partitioned alone; bars3 and 4, PDE partitioned with
G
GTP
S.
Phase Partitioning of Tryptic Fragments of
G
To define better the
structural domains responsible for partitioning, Gand PDE
,
G
, and G
were
subjected to limited proteolysis with trypsin. Cleavage products of
G
were recovered in the detergent-rich phase to a
greater extent than were those from G
(Fig. 6).
In the presence of G
, the fraction of intact
39-kDa G
recovered in that phase was greater than that
of the 37-kDa fragment lacking the amino terminus (Fig. 6).
Since the amino terminus of G
is involved in
interaction with G
, it was unclear whether the
difference in behavior of intact G
and the 37-kDa
protein resulted from disruption of the G
-binding
site with loss of ability of G
to associate with and
be influenced by G
or whether the amino terminus
itself contributed significantly to the hydrophobicity of
G
. To evaluate this, G
was
trypsinized without G
, and in some experiments,
after stopping trypsinolysis with phenylmethylsulfonyl fluoride or
soybean trypsin inhibitor, intact G
was added back
before partitioning to ensure that sufficient intact G
would be recovered for densitometric measurement. Even in the
absence of G
, intact G
was
recovered to a greater extent in the detergent-rich phase than was the
37-kDa fragment (and other major tryptic products), although amounts of
both were less than when G
was present (Fig. 7; data not shown), consistent with the conclusion that
the amino terminus is primarily responsible for the hydrophobic nature
of G
and that this hydrophobicity is not a consequence
solely of interaction with G
. After
trypsinolysis, PDE accumulated mostly in the detergent-depleted phase (Table 1).
(20 µg) in hypotonic
buffer was subjected to limited proteolysis with trypsin (Try)
before partitioning. The positions of protein standards,
G
, G
, G
, and
arrestin (Arr) are indicated on the left, and those of major
proteolysis fragments on the right. Lanes are labeled as described for Fig. 1A. 33% of the intact 39-kDa G
was aqueous, whereas 70% of the 37-kDa fragment was aqueous. The
experiment was repeated four times with similar
results.
. G
(8 µg) in hypotonic buffer
was subjected to limited proteolysis with trypsin before partitioning.
The positions of arrestin (Arr), intact G
and
the 37-kDa fragment are indicated on the right. Lanes are labeled as
described for Fig. 1A. Of intact G
, 56
± 2.3% was in the aqueous phase versus 88.3 ±
9.0% of the 37-kDa product. The mean ± S.E. was derived from
four separate assays.
Effect of Carboxyl-terminal Truncation of
G
Recombinant G and
G
lacking 20 amino acids at the carboxyl terminus were
prepared as described under ``Methods.'' When partitioned in
the same tube, carboxyl-terminal truncated G
was
consistently recovered to a greater extent in the detergent-depleted
phase than was intact recombinant G
(Fig. 8).
. Intact (4 µg)
and carboxyl-truncated (3 µg; Val-331 stop) G
were
added to the same microcentrifuge tube for partitioning. The positions
of glutathione S-transferase (G S-T), intact
recombinant G
(rG), and carboxyl-truncated
G
(Val 331 stop) are indicated on the right.
Molecular weight standards are indicated on the left. Lanes are labeled
as described for Fig. 1A. The experiment was repeated
four times with different preparations of intact and carboxyl-truncated
G
. Consistently, a higher percentage of the truncated
than the intact form was recovered in the aqueous phase. The mean
± S.E. of differences in the percentage of G
and truncated G
recovered in the aqueous phase
was 18.7 ± 5.2% (n =
4).
Effect of Myristoylation of the Amino Terminus of
rG
When subjected to phase partitioning,
nonmyristoylated rG was recovered primarily in the
aqueous phase, whereas myristoylated rG
was totally
recovered in the detergent-rich phase (Fig. 9, A and B).
. Crude supernatants of bacterially expressed
myristoylated and nonmyristoylated G
, prepared as
described under ``Methods,'' were subjected to phase
partitioning and SDS-PAGE (12% acrylamide). Detection of
H-labeled myristoylated rG
was after
treatment of the gel with Promote (Integrated Separation Systems) and
exposure to Kodak X-Omat film at -70 °C for 1-4 days.
Detection of nonmyristoylated rG
was with an
anti-G
polyclonal antibody (47) after transfer
of proteins from the gel to nitrocellulose. The nonmyristoylated form
was recovered to a greater extent in the aqueous phase (58.8 ±
7.9%, n = 5), while myristoylated rG
was nearly 100% recovered in the detergent phase (n = 6). A, Western blot analysis of
phase-partitioned nonmyristoylated rG
. A
lane, aqueous phase; D lane, detergent phase. B,
H-labeled myristoylated rG
. Lanes are
labeled as described for A.
Effect of ADP-ribosylation of G
Gon
Phase Partitioning
and ADP-ribosylated
G
were evaluated in the same assay by protein staining
and autoradiography. Pertussis toxin-catalyzed ADP-ribosylation did not
significantly alter the partitioning of G
alone. Of
radiolabeled ADP-ribosylated G
, 70.0 ± 11.6% (n = 3) was recovered in the detergent phase compared
with 63.3 ± 3.3% (n = 3) of G
that had been incubated with pertussis toxin without NAD (see
also Table 1). There was a tendency for ADP-ribosylated
G
to associate more than unmodified G
with G
since when it was partitioned with
equimolar G
, 89 ± 5.1% (n = 3) of the ADP-ribosylated G
was
recovered in the detergent phase, whereas only 77.3 ± 1.8% (n = 3) of the protein was recovered in the detergent
phase when no NAD was included (see also Table 1). NAD: arginine
ADP-ribosyltransferase modified G
to the extent of
5 mol of ADP-ribose/mol of G
. Comparing this
G
with G
that had been processed in
an identical fashion without transferase, recovery of ADP-ribosylated
G
in the aqueous phase was greater, i.e. 57.0
± 2.9% (n = 4) versus 46 ± 2.4% (n = 4) as shown in Fig. 10. When equal amounts
of labeled and unlabeled G
were mixed and partitioned,
more of the unlabeled G
was aqueous (55.7 ±
2.5%, n = 3), whereas the partitioning of the labeled
G
was unchanged (56.5 ± 1.7%, n = 3) (Fig. 10). When partitioned in the presence of
excess native G
, the partitioning of ADP-ribosylated
G
was not significantly affected as 58.7 ± 5.9% (n = 3) was recovered in the aqueous phase, whereas
native G
remained 46.0 ± 3.6% (n = 3) aqueous.
on phase partitioning. G
was ADP-ribosylated and
subjected to phase partitioning as described under
``Methods.'' SDS-polyacrylamide gels were stained with
Coomassie Brilliant Blue, and proteins were quantitated by
densitometry; or in the case of
P-labeled ADP-ribosylated
G
, quantitation was also done by phosphoimaging on a
Molecular Dynamics PhosphorImager. Coomassie Blue-stained gel of
2
µg of phased-partitioned arginine-ADP-ribosylated G
(G*), native G
(G), or both (G/G*). The positions of G
* and
G
are indicated on the right. A and D represent aqueous and detergent phases,
respectively.
complex was recovered mostly in
the detergent-rich phase. The PDE complex, which was intact based on
its enzymatic activity, initially partitioned as a highly hydrophobic
complex. After trypsinization,
88% was recovered in the aqueous
phase, consistent with previous findings that trypsin removes a prenyl
moiety from the PDE
subunit, releasing the PDE complex from
membranes(57) . Similarly, addition of a hydrophobic myristate
group to a recombinant heterotrimeric G protein
subunit,
G
, shifted its recovery to the detergent-rich phase,
whereas the nonmyristoylated form was mostly aqueous. Therefore, in
certain instances, the presence of a hydrophobic group may affect the
overall hydrophobicity measured in this assay.
subunits are localized at the cytosolic face of the plasma
membrane(1) . Membrane attachment of at least two of these
proteins, G
and G
, is facilitated by
their myristoylated amino termini, although myristoylation alone may
not be sufficient for membrane attachment(58) .
G
, modified at its amino terminus in a heterogeneous
manner by lauroyl (C
), myristoyl (C
), (cis-
5)-tetradecaenoyl (C
), or (cis,cis-
5,
8)-tetradecadienoyl
(C
) fatty acylation(59, 60) , is mainly
a soluble protein and, unlike other
subunits, does not require
detergents for purification. It is released from ROS by addition of
MgGTP. After binding GTP, G
undergoes a conformational
change, documented in recent crystallographic
studies(2, 3) , although how this change affects
overall hydrophobicity has not been well defined.
was consistent with other evidence that it does
not behave as either a typical integral membrane protein or a soluble
protein. In the absence of G
, G
partitioned approximately equally between the detergent-rich and
detergent-depleted phases. On sequential phase partitioning,
G
derived from either the detergent-rich or
detergent-depleted phase partitioned in much the same way as it did
initially. Thus, there was no evidence of heterogeneity of preparations
isolated in the same way. There was, however, a small difference
between the apparent hydrophobicity of G
preparations
isolated differently. The G
subunits in the
experiments of Table 2were isolated from ROS using a nearly
isotonic wash, which selectively eluted G
, leaving
behind G
, whereas the apparently more hydrophobic
G
used in Table 1was eluted from ROS as a
G
complex using hypotonic conditions and
subsequently separated from G
using blue
Sepharose.
. The
phase-partitioning data reported here are consonant with other
observations on the trypsin-cleaved protein (61, 62) that implicate the amino terminus as the
major hydrophobic segment of G
. Unlike
G
, G
, and G
,
G
is not N-terminally myristoylated. The carboxyl
terminus of G
has been implicated in its ability to
associate with membranes(17, 18) . To determine
whether the carboxyl terminus of G
similarly imparts
hydrophobicity, recombinant G
and a carboxyl-truncated
mutant were synthesized. Recombinant G
did not bind
labeled nucleotide in the presence of G
and
rhodopsin, which may have been secondary to its initially insoluble
state and the consequent use of detergent for solubilization. Lack of
N-terminal fatty acylation, resulting in lower affinity of G
for G
, may have been a factor as well.
Although G
did not stimulate the reaction,
recombinant wild-type G
was ADP-ribosylated by
pertussis toxin, from which retention of at least some degree of native
structure in the carboxyl terminus was inferred. ADP-ribosylation did
not occur with G
that had been heat-denatured or, as
expected, with the carboxyl-terminal truncated variant (data not
shown). Nearly 20% (18.7 ± 5.2%, n = 4) more of
the full-length than the truncated G
was recovered in
the detergent-rich phase on partitioning, from which it was inferred
that the carboxyl terminus contributes to its hydrophobicity, as it
does in G
(17, 18) , although to a
lesser extent than the amino terminus.
are in close
proximity and that the amino terminus of G
is critical
for G
binding. The apparent hydrophobicity of
G
was enhanced in a concentration-dependent manner by
G
, but only when G
was in the
inactive GDP-bound form, i.e. the conformational change
induced by GTP binding did not affect the overall hydrophobicity of
G
, but rather influenced its partitioning indirectly
by altering G
interaction with the more hydrophobic
G
. If Triton X-114 does interfere with the
G
GDP-G
interaction,
sufficient interaction apparently persists to permit observation of an
effect. The failure of G
GTP
S or PDE to
alter the partitioning of the other protein could mean that Triton
X-114 interferes with this protein-protein interaction. (The increased
activity recovered upon partitioning PDE in the presence of
G
GTP
S suggests, but does prove, that
interaction occurred during partitioning.) Formation of a ``tight
complex'' between PDE and G
GTP
S that
would partition differently from the individual proteins may require an
additional protein or environment supplied by ROS but lacking in this
simple system, although it is not clear that a tight complex is
required for activation of PDE. In studies reported to demonstrate a
tight complex of PDE and G
, either ROS were the
supporting membrane surface (63, 64, 65) or
the fraction of proteins in the complex was so small (<4%) (66, 67) that it would not have been detected in the
partitioning assay. In one study, phospholipid vesicles were used as a
support for cross-linking PDE and G
(67) .
This, however, requires only an association close enough to enable
cross-linking, and the existence of a stable complex in the artificial
membrane was not established(67) .
did not alter its partitioning in the
absence of G
. However, ADP-ribosylated
G
appeared to have enhanced affinity for
G
, as when partitioned with
G
, more ADP-ribosylated G
than
non-ADP-ribosylated G
was recovered in the detergent
phase. There was, however, a clear difference in the partitioning
behavior of arginine-ADP-ribosylated G
, a difference
likely explained by the greater extent of ADP-ribosylation. At least 5
mol of ADP-ribose/mol of G
were incorporated using
NAD:arginine ADP-ribosyltransferase as catalyst, whereas pertussis
toxin modifies only a single cysteine(12) . It is also possible
that the specific site ADP-ribosylated by pertussis toxin (Cys-347) is
relatively inaccessible to the external milieu and therefore does not
influence partitioning; the ADP-ribosylated arginines may be more
exposed to the medium. In equimolar mixtures of
arginine-ADP-ribosylated G
and native
G
, the latter behaved similarly to the modified
G
, i.e. its recovery in the aqueous phase was
increased. This might be interpreted as a reflection of dimerization of
G
, as has been suggested on the basis of other kinds
of studies(37, 68) .
,
subunit of
transducin; G
,
subunit of transducin;
rG
, recombinant G
; Gpp(NH)p,
guanyl-5`-yl imidodiphosphate; GTP
S, guanosine
5`-O-(3-thiotriphosphate); TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; PAGE,
polyacrylamide gel electrophoresis; PDE, retinal cGMP
phosphodiesterase; MOPS, 4-morpholinepropanesulfonic acid; ROS, rod
outer segments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.