(Received for publication, July 8, 1996, and in revised form, October 7, 1996)
From the Department of Molecular Genetics and Microbiology,
University of Massachusetts Medical School,
Worcester, Massachusetts 01655-0122 and the MDB/NIDDK,
National Institutes of Health,
Bethesda, Maryland 20892-1752
Genetic evidence suggests that the yeast
STE4 and STE18 genes encode G and G
subunits, respectively, that the G
complex plays a positive role
in the pheromone response pathway, and that its activity is subject to
negative regulation by the G
subunit (product of the
GPA1 gene) and to positive regulation by cell-surface pheromone receptors. However, as yet there is no direct biochemical evidence for a G
protein complex associated with the plasma membrane. We found that the products of the STE4 and
STE18 genes are stably associated with plasma membrane as
well as with internal membranes and that 30% of the protein pool is
not tightly associated with either membrane fraction. A
slower-migrating, presumably phosphorylated, form of Ste4p is enriched
in the non-membrane fraction. The Ste4p and Ste18p proteins that had
been extracted from plasma membranes with detergent were found to
cosediment as an 8 S particle under low salt conditions and as a 6 S
particle in the presence of 0.25 M NaCl; the Ste18p in
these fractions was precipitated with anti-Ste4p antiserum. Under the
conditions of our assay, Gpa1p was not associated with either particle.
The levels of Ste4p and Ste18p accumulation in mutant cells provided additional evidence for a G
complex. Ste18p failed to accumulate in ste4 mutant cells, and Ste4p showed reduced levels of
accumulation and an increased rate of turnover in ste18
mutant cells. The gpa1 mutant blocked stable association of
Ste4p with the plasma membrane, and the ste18 mutant
blocked stable association of Ste4p with both plasma membranes and
internal membranes. The membrane distribution of Ste4p was unaffected
by the ste2 mutation or by down-regulation of the
cell-surface receptors. These results indicate that at least 40% of
Ste4p and Ste18p are part of a G
complex at the plasma membrane
and that stable association of this complex with the plasma membrane
requires the presence of G
.
The pheromone response pathway of Saccharomyces
cerevisiae provides a microbial model for studying function of
heterotrimeric G proteins. -Factor and a-factor
pheromones are peptides synthesized by haploid cells of
and a
mating type, respectively. Each pheromone binds specific receptors
located on cells of the opposite mating type; receptor activation
causes cell division arrest at G1 and expression of genes
controlling the conjugation of the two mating types. Both
-factor
receptors (encoded by the STE2 gene) and a-factor
receptors (encoded by STE3) are members of the
rhodopsin/
-adrenergic receptor family in that they contain seven
transmembrane segments, and they require homologs of mammalian G
,
G
, and G
protein subunits (encoded by GPA1, STE4, and STE18, respectively) for signal
transduction. The receptor appears to form a direct physical
association with the G-protein subunits since
-factor binds
ste4 mutant cells more weakly (1) and since it dissociates
more rapidly from membranes assayed in the presence of GTP analogs or
when the membranes are prepared from mutants with defects in
GPA1, STE4, or STE18 (2). Genetic evidence indicates that G
, rather than G
, activates subsequent events in the response pathway, that is loss of function mutations in
STE4 and STE18 block the signal (3, 4, 5) whereas loss of function mutations in GPA1 activate signaling
(6, 7, 8). Moreover, gain of function mutations affecting STE4
(designated STE4Hpl) (9, 10) and overexpression
of wild-type Ste4p (11, 12, 13) cause constitutive activation of the
pathway. Gpa1p and Ste18p are modified covalently with myristoyl and
farnesyl moieties, respectively (14, 15).
Genetic studies also suggest that G associates with the G
and
G
homologs in vivo as well as with other proteins thought to play roles in the pheromone response pathway. However, biochemical characterization of these potential interactions has not been reported.
Interactions between Gpa1p and Ste4p were inferred from two-hybrid
genetic analysis (10, 16). Moreover, overexpression of Gpa1p
compensates for overexpression of Ste4p (11, 12, 13);
STE4Hpl mutations are suppressed by mutations in
GPA1 (10), and one allele is suppressed by overexpression of
the wild-type Gpa1p (17). Two-hybrid genetic analysis also predicts
that Ste4p binds to Ste18p (16). Mutations in the STE18 gene
have been identified that either suppress or enhance partial defects in
STE4 (16, 18). Some ste4 alleles are suppressed
by overexpression of Ste18p (16, 19), while certain ste18
alleles are suppressed by overexpression of Ste4p (20). Genetic tests
also suggest that Ste4p interacts with the products of the
STE5 (21, 22), STE20 (22, 23, 24, 25), CDC24
(26), SYG1 (27), and AKR1 (28, 29) genes; the
interaction with Ste5p has been confirmed by demonstrating that
epitope-tagged Ste4p coimmunoprecipitated with Ste5p (21). Thus, these
proteins may function as either effectors or regulators of the G
complex.
Although heterotrimeric G proteins are present among plant, animal, and
fungal kingdoms, our current understanding of G protein structure and
subcellular localization is based largely on studies of mammalian
proteins (reviewed in Refs. 30, 31, 32). Mammals are known to contain over
20 different isoforms of G representing four different classes;
there are 5 and 11 known isoforms of G
and G
, respectively. X-ray
crystallographic structures have been solved for two G protein
heterotrimers (33, 34), as well as for the transducin
-subunit bound
to GDP and to GTP analogs (35) and for the transducin
dimer
(36). G proteins are bound to the plasma membrane where they mediate a
variety of hormonal responses; they also associate with internal
membrane compartments where they play a role in membrane trafficking.
Stable association of G proteins with membranes involves cooperative
interactions among the three subunits as well as lipid modifications of
G
and G
(see Ref. 37). Tight binding of G
to the plasma
membrane is thought to occur only when it contains myristoyl and
palmitoyl modifications and when it is complexed with G
(37). It
is unknown whether G
requires G
for membrane association.
The object of the present study was to initiate biochemical
characterization of G from an organism that is amenable to
genetic analysis. We sought to determine whether the products of the
STE4 and STE18 genes form a G
protein
complex that is associated with plasma membranes. We found that
approximately 40% of both proteins was bound tightly to plasma
membranes, whereas the remainder was associated with internal membranes
or was not tightly bound to membranes. Stable association of the
G
complex with plasma membranes required G
but did not require
the receptor. After detergent extraction of plasma membranes, Ste4p and
Ste18p were found to cosediment and to coimmunoprecipitate. The size of
the resulting protein complex depended on salt concentration,
suggesting that Ste4p and Ste18p interact with other proteins in a
salt-dependent manner.
Strains used in this study are described in Table I. All strains are congenic to strain 381G except for strain JH59. Strain JH59 was constructed by transforming strain W303 (6) with three plasmids (pPgk-Scg, pEL37, and pBH21) that lead to high level expression of all three G protein subunits. High copy plasmid pPgk-Scg (38) contains the URA3 gene; the PGK1 promoter directs synthesis of GPA1 mRNA. The single copy YCp plasmid pEL37 (provided by E. Leberer) contains the HIS3 gene; the divergent GAL1,10 promoter directs synthesis of both GPAl and STE4 mRNA. High copy plasmids pBH21 and M91p1 contain the LEU2 and URA3 genes, respectively; the ADH1 promoter on both plasmids directs synthesis of STE18 mRNA. Plasmid pBH21 was constructed by replacing a 1.2-kilobase BglII fragment of plasmid M91p1 (provided by M. Whiteway) with a 3.5-kilobase BglII fragment from YEp13 carrying LEU2.
|
YM-1 medium is a rich liquid medium (39).
Minimal galactose medium is yeast nitrogen base medium (Difco) buffered
to pH 5.8 with 85 mM sodium succinate and supplemented with
ammonium sulfate (5 mg/ml) as the nitrogen source, galactose (2%) as
the carbon source, and adenine (20 µg/ml) and tryptophan (40 µg/ml). Ura + CAA medium (40) is a supplemented minimal medium
that contains 0.1% casamino acids (Difco) and lacks uracil.
Dodecyl--D-maltoside and
cholesterol hemisuccinate were from Sigma.
Escherichia coli aspartate transcarbamoylase
(ATCase)1 was a gift from Y. R. Yang and H. K. Schachman. Mouse gamma globulin was a monoclonal antibody
preparation provided by V. Yuschenkoff. Renografin-76 (76% Renografin)
was from Squibb.
Polyclonal rabbit antisera were anti-Ste2p
(provided by J. B. Konopka), specific for the C-terminal domain of the
-factor receptor (41); anti-Ost1p (provided by R. Gilmore), specific for the yeast oligosaccharyltransferase
-subunit (42); anti-ATCase (provided by Y. R. Yang and H. K. Schachman); anti-Gda1p (provided by
C. Hirschberg), specific for the yeast Golgi guanosine diphosphatase; and anti-Gpa1p (provided by H. Dohlman), specific for the C terminus of
Gpa1p (43). Mouse monoclonal anti-Pma1p (provided by J. Aris), C56
(44), is specific for the yeast plasma membrane ATPase (45). Mouse
monoclonal anti-Vph1p (obtained from Molecular Probes, Eugene, OR) is
specific for the 100-kDa subunit of the yeast vacuolar ATPase.
Peroxidase-labeled goat anti-mouse IgG was from Life Technologies, Inc.; peroxidase-labeled goat anti-rabbit IgG was from Kirkegaard and
Perry.
Anti-Ste4p antiserum was prepared by immunizing rabbits with full-length Ste4p expressed from plasmid pBH19 in E. coli strain BL21(DE3) (46). Plasmid pBH19 was constructed by cloning the 1.3-kilobase NcoI/SalI fragment (containing the STE4 gene) from plasmid pL19 (11) into plasmid pET-8c (46). Antibodies were affinity purified by using a resin that had been coupled to bacterially expressed Ste4p. Anti-Ste18p was prepared by injecting rabbits with the peptide, TSVQNSPRLQQPQEamide, conjugated with glutaraldehyde to keyhole limpet hemocyanin.
Immunoblotting Methods and QuantitationProtein samples
were diluted 1:3 with sample buffer (1 g of urea dissolved in 1 ml of
17.5 mM Tris-HCl, pH 6.8, 1.75% sodium dodecyl sulfate,
1% -mercaptoethanol, bromphenol blue) and heated to 37 °C for 10 min. Proteins were resolved by using SDS-PAGE (47) and transferred (48)
to an Immobilon membrane (Millipore Corp., Bedford, MA). Membranes were
blocked with 20 mM Tris-Cl, pH 7.5, 0.5 M NaCl,
0.05% Tween 20 containing 5% nonfat dried milk; they were probed with
primary antiserum and secondary antibodies diluted in blocking buffer.
Secondary antibodies were either goat anti-rabbit or goat anti-mouse
immunoglobulins conjugated with horseradish peroxidase (Kirkegaard and
Perry Laboratories, Inc., Gaithersburg, MD). Conjugates were visualized
by using chemiluminescence reagents (Renaissance kit, DuPont NEN; or
SuperSignal kit, Pierce). Autoradiographic results were quantified by
using a densitometer (Molecular Dynamics Corp.).
Unless otherwise indicated, a 150-ml culture was grown overnight at 30 °C in YM1 medium (39) to 107 cells/ml. Cells were collected by centrifugation, washed twice with ice-cold membrane buffer (10 mM Tris acetate, pH 7.6, 1 mM magnesium acetate, 0.1 mM EDTA, 8% glycerol) containing 1 × protease inhibitors (100 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A), and resuspended in 0.5 ml of the same buffer. Glass beads were added, and cells were lysed by mechanical disruption. Unbroken cells were removed from the lysate by centrifugation for 5 min at 330 × g.
Renografin Density GradientsMembranes from the cleared lysates were resolved on Renografin gradients as described (45).
Plasma Membrane PurificationStrain JH59 was cultured to
approximately 5 × 107 cells/ml in minimal galactose
medium. 12.5 g (wet weight) of cells were rinsed twice with
membrane buffer containing 0.1 mM dithiothreitol and 1 × protease inhibitors, suspended in 7.5 ml of the same buffer, and
lysed by mechanical disruption with glass beads. Unbroken cells were
removed by centrifuging twice at 330 × g for 5 min. 1.1 ml of cleared lysate was mixed with an equal volume of
Renografin-76 (to give 38% Renografin). A gradient of Renografin was
formed by layering successively 8 ml each of 34, 30, 26, and 22%
Renografin solutions (prepared by diluting Renografin-76 with 50 mM Tris-Cl, pH 7.5, 1 mM EDTA). Gradients were
centrifuged in an SW28 rotor at 27,000 rpm for 20 h at 4 °C.
Fractions (2.6 ml) were collected into tubes containing 1 × protease inhibitors and stored at 70 °C. Pooled fractions
containing plasma membrane marker, Pma1p, were identified by using
immunoblotting methods.
Purified plasma membranes from
strain JH59 were collected by centrifugation (2.5 h at 250,000 × g) and washed with either buffer A (20 mM Tris
acetate, pH 7.6, 1 mM magnesium acetate, 1 mM
dithiothreitol, and 0.5 × protease inhibitors) or with buffer B
(buffer A containing 250 mM NaCl). Washed membranes were
resuspended in 180 µl of the same buffer containing 0.2 × protease inhibitors, 2 mg/ml dodecyl--D-maltoside, and
0.4 mg/ml cholesterol hemisuccinate. After 2.5 h on ice, the
insoluble material was removed by centrifuging for 3 min in a Beckman
airfuge. Bovine serum albumin (3 µg), mouse gamma globulin (5 µg),
and bacterial ATCase (1 µg) were added as sedimentation markers. 150 µl was loaded onto each of two 8-30% glycerol gradients containing
buffer A and buffer B, respectively, and centrifuged in an SW50.1 rotor
at 48,000 rpm for 15 h at 4 ° C. Twenty-three 220-µl
fractions were collected into tubes containing protease inhibitors
(1 × final concentration); fraction 23 contained the resuspended
pellet. Proteins were assayed by using immunoblotting methods.
Fractions 10, 11, and 12 from the
glycerol gradient in Fig. 3A were pooled and centrifuged
(16,000 × g) 10 min at 4 °C to remove insoluble
material. 30 µl of supernatant was mixed with 5 µl of protein
A-Sepharose beads that had been coated with either preimmune or immune
serum. After incubating overnight at 4 °C, the mixture was
centrifuged briefly. Supernatants were removed, and pellets were washed
in the same buffer. Supernatants and washed pellets were mixed with
sample buffer (47), and Ste18p was detected by immunoblotting
methods.
Ste4p Turnover Assay
Cells were cultured at 30 °C in YM-1 medium to 107 cells/ml. Cycloheximide was added to 10 µg/ml. At the various time points, a 25-ml sample was removed to a chilled flask containing NaN3 and KF (final concentration, 10 mM). Cells were collected by centrifugation, washed with 1 ml of membrane buffer, and resuspended in 0.25 ml of ice-cold membrane buffer. Cleared lysates were prepared as for membrane fractionation. Protein concentrations were determined by using the bicinchoninic acid assay (Pierce), and samples were diluted to 2 mg of protein/ml. Two volumes of 3 × sample buffer (47) was added, and the samples were boiled for 10 min. Proteins were resolved by using SDS-PAGE and detected by using immunoblotting methods. Linearity of the detection was verified by analyzing 2-fold dilutions of the zero time point.
Heterotrimeric G proteins are peripheral
membrane proteins that are stably associated with membranes. We wished
to determine whether the yeast G and G
homologs (Ste4p and
Ste18p, respectively) share this property. Membranes, as well as
ribosomes and other large macromolecular aggregates, are found in the
pellet fraction (particulate fraction) when cleared cell lysates are
subjected to centrifugation at 100,000 × g. The
solvent conditions that promote dissociation of a specific protein from
this particulate fraction provide a criterion that is commonly used to
define a peripheral or integral membrane protein. We used
immunoblotting procedures to test for the presence of yeast G
and
G
homologs in the soluble and particulate fractions of cleared yeast
lysates. Table II shows that Ste4p and Ste18p were
extracted from the particulate fraction under similar conditions. When
the lysates were prepared in membrane buffer alone, nearly all of Ste4p
and Ste18p was associated with the particulate fraction (Table II).
Greater than 75% of the Ste4p and Ste18p was tightly associated with
the particulate fraction, that is this portion was resistant to
extraction with either 1.0 M NaCl or 4 M urea.
Both proteins were efficiently extracted from the particulate fraction
by exposing the lysates either to harsh alkaline conditions
(Na2CO3, pH 11) or to a combination of 0.25 M NaCl and the nonionic detergent,
dodecyl-
-D-maltoside. We also found that the G
homolog, Gpa1p, was released efficiently with this combination of
detergent and NaCl (data not shown). These results suggest that up to
75% of the yeast G
and G
homologs form a tight peripheral
association with membranes in that they were released from the
particulate fraction with Na2CO3, pH 11, but not with
1.0 M NaCl. However, with this technique, it is not possible to distinguish whether the Ste4p and Ste18p in the particulate fraction are associated with membranes or with other non-membranous particles.
|
We tested whether plasma membranes contain tightly bound G protein subunits by using density gradient centrifugation to fractionate the crude cellular membranes. A gradient of Renografin was layered over a cleared cell lysate that had been adjusted to 38% Renografin (ionic strength, roughly equivalent to 0.5 M NaCl), and the membranes were then allowed to float in a centrifugal field until they reached their buoyant density. We had previously shown that this technique separates plasma membranes from non-membrane proteins and from the more buoyant internal membrane species, i.e. endoplasmic reticulum, Golgi complex, and vacuole (45). The advantages of this fractionation technique are 2-fold. First, by lysing cells rapidly with glass beads and analyzing the crude extracts directly, we minimized potential problems with protein degradation and redistribution of proteins among the cellular membranes. Second, the high ionic strength of Renografin apparently strips the loosely associated proteins from the membranes, maximizing the difference in density between plasma membranes and the internal membranes and thus limiting our analysis to the G protein subunits that are tightly associated with membranes.
As shown in Fig. 1A, the Ste4p was contained
in three different portions of the Renografin gradient. Approximately
40% of the Ste4p (Fig. 1, A and B)
cofractionated with plasma membrane ATPase (Pma1p) and with the bulk of
the -factor receptor (Fig. 1C). Roughly 30% was found
among the more buoyant membranes; the remaining 30% was in the denser
fractions that contain none of our membrane markers (Fig.
1B). From a total of eight independent analyses representing
three different wild-type strains, we found that a range of 20-40% of
the Ste4p was present among the buoyant membranes, 30-50% was in the
plasma membrane fraction, and 20-45% in the denser fraction
containing nonmembrane proteins. The amount of Ste4p associated with
the combined membrane fractions (70%) was somewhat less than the
amount of Ste4p found in the particulate fraction (85%) at comparable
ionic strength (Table II); thus, it is likely that the particulate
fraction contains Ste4p that is not associated with membranes.
Consistent with this interpretation, two-thirds of the Ste4p in
non-membrane fractions from the Renografin gradient contained Ste4p
that was particulate after the fractions had been dialyzed against
buffer containing 0.25 M NaCl (not shown). The association
of Ste4p with at least two different membrane species (i.e.
plasma membranes and the more buoyant internal membranes) apparently
does not reflect redistribution of the Ste4p during cell lysis or
sample preparation. Each pooled membrane fraction was added to cells
containing a deletion of the STE4 gene and then processed as
in Fig. 1. In both cases, the membranes migrated to their original
position in the second Renografin gradient (not shown).
Previous workers (49) have resolved electrophoretically distinct forms
of Ste4p that differ by their state of phosphorylation. We found that
about 10% of Ste4p migrates more slowly during SDS electrophoretic
analysis, although we have no direct evidence that this species is in
fact phosphorylated. In our Renografin density gradient analysis (Fig.
1A), we found that nearly all of this slower-migrating form
was associated with the non-membrane fractions (fractions 13-15).
Moreover, it was also largely excluded from the particulate fraction in
the analysis described in Table II (not shown). A lesser amount of the
slower-migrating species was found among the internal membranes (Fig.
1A, fractions 1-6), whereas it was undetectable
in the plasma membranes (fractions 7-11). In the presence
of -factor, slower migrating species were found in all three regions
of the gradient (data not shown).
The fractions from the Renografin gradient depicted in Fig.
1A were assayed for the presence of Ste18p (Fig.
2A). In order to distinguish Ste18p from
other cross-reacting proteins, we included control strains that either
contained a deletion of the STE18 gene (Fig. 2B)
or contained an over-producing plasmid (Fig. 2C). Like
Ste4p, a portion of Ste18p was found to be tightly associated with
plasma membranes (fractions 7-10) in addition to its
association with internal membranes (fractions 2-6) and to
the non-membrane fractions (fractions 13 and 14). We also
examined Renografin gradients for Gpa1p; it was present in both
internal membrane and plasma membrane fractions (not shown). We could
not determine whether Gpa1p was in the non-membrane fraction due to the
presence of cross-reacting proteins.
Solubilized Plasma Membranes Contain a Complex of Ste4p and Ste18p
To obtain biochemical evidence for a Ste4p·Ste18p
complex, we extracted these proteins from purified plasma membranes
with detergents and then examined their sedimentation properties under two different solvent conditions. To maximize detection of the proteins
in this experiment, we used a diploid strain containing plasmids that
express STE4, STE18, and GPA1 from
strong promoters. Renografin gradient analysis of Ste4p and Ste18p from
this strain showed a similar profile as observed for the wild-type
haploid strain (see Figs. 1 and 2). Plasma membranes were purified by using Renografin gradient centrifugation and then solubilized in buffer
containing dodecyl--D-maltoside and cholesterol
hemisuccinate in the presence and in the absence of 250 mM
NaCl. The detergent-extracted proteins were resolved on 8-30%
glycerol gradients that contained the same detergent and salt
concentrations. Under both conditions, Ste4p and Ste18p cosedimented
(Fig. 3), despite the large difference in their
molecular masses (51 and 15 kDa, respectively (5)). When compared with
marker proteins, the apparent sedimentation coefficient was 6 S at 250 mM NaCl (Fig. 3A) and 8 S under low salt
conditions (Fig. 3B). The complexes depicted in Fig. 3 did not appear to be a consequence of protein overproduction or expression in diploid cells since identical sedimentation coefficients under both
low and high salt conditions were obtained for the Ste4p that had been
extracted from the plasma membranes of wild-type haploid cells (not
shown); however, in these experiments, the concentration Ste18p was
below the limit of detection. It is presently unclear whether the
larger complex (Fig. 3B) results from dimerization of
G
, from increased association of detergent, or from the binding of additional protein factor(s). Although the Gpa1p peak overlapped with Ste4p and Ste18p in 250 mM NaCl (Fig. 3A),
the sedimentation coefficient of Gpa1p was not influenced by salt (Fig.
3B). Hence, under the conditions of our assay, we could
detect no association of the G
subunit homolog, Gpa1p, with the
G
complex.
To verify that the Ste4p and Ste18p on the glycerol gradient (Fig.
3A) were contained in the same complex, we tested for the ability of anti-Ste4p antiserum to precipitate Ste18p. As shown in Fig.
4, Ste18p was precipitated specifically with either
anti-Ste4p (compare lanes 1 and 2) or with
anti-Ste18p antiserum (compare lanes 3 and 4).
Ste4p was not analyzed as it comigrates with immunoglobulin heavy
chains on SDS-PAGE.
Ste4p Stability Requires Ste18p
As a test for interactions among the three G protein subunits and the receptor in vivo, we tested whether mutations in the genes encoding these components affect steady-state accumulation of Ste4p and Ste18p. The levels of Ste4p and Ste18p observed for the gpa1 and ste2 mutants were essentially the same as those from the wild-type control strain (Table III); however, the steady-state level of Ste4p in a ste18 mutant was reduced 50%. To ascertain whether this decrease was due to an increased rate of Ste4p turnover or to a reduced rate of synthesis, we examined the rate at which Ste4p disappeared from cultures that had been blocked for protein synthesis. Cycloheximide was added to growing cultures of STE+ and ste18 strains, and samples were assayed for the amount of Ste4p that remained in the culture over a 3-h time course (Fig. 5). The kinetics of Ste4p decay in the ste18 mutant were consistent with a first-order reaction and a half-life of 90 min; whereas in the wild-type control, the level of Ste4p (Fig. 5) and Ste18p (not shown) remained undiminished for the duration of the time course. The rate of decay in the ste18 mutant is expected to produce a 2-fold reduction in the steady-state level of Ste4p in growing cultures since Ste4p is also diluted as a result of cell division (90 min doubling time). Ste18p was barely detectable in the ste4 mutant, consistent with rapid decay (Table III). These results suggest that Ste4p and Ste18p form a stable protein complex in vivo.
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Mutations in Ste18p and Gpa1p Affect Localization of Ste4p to the Plasma Membrane
As a second criterion for the presence of
specific protein complexes in vivo, we examined whether G
requires the receptor or the other G protein subunits for its tight
association with plasma membranes. Lysates from ste2,
ste18, and gpa1 mutant cells were subjected to
Renografin gradient analysis, and the fractions were assayed for Ste4p.
In the ste18 mutant, greater than 90% of Ste4p was found in
non-membrane fractions (Fig. 6A), indicating that the G
homolog is required for stable association of G
with both the plasma membrane and the internal membranes. In the
gpa1 mutant, Ste4p was present only in internal membrane and
non-membrane fractions (Fig. 6B), suggesting that G
is
required for stable accumulation of G
at the plasma membrane. In
contrast to ste18 and gpa1, the ste2
mutation did not significantly affect distribution of Ste4p (Fig.
6C), indicating that the receptor is not required for G
to accumulate at either membrane location.
Membrane fractionation and localization of
Ste4p in ste18, gpa1, and ste2 mutants.
Cultures were subjected to Renografin gradient analysis as depicted in
Fig. 1. A, ste18 mutant (DJ1006-17-2, ) and
wild-type control (DJ602-15-1,
). B, gpa1
mutant (DJ803-2-1,
) and wild-type control (DJ803-11-1,
). Both
strains contained the ste5-3 mutation and were cultured at
34 °C; consequently, the gpa1 mutation did not result in constitutive arrest of the cell
division. C, ste2 mutant cells (DJ240-4-1,
)
and wild-type control (DJ147-1-2,
). Amount of Ste4 protein
(solid lines) is the percentage of Ste4p present in each
fraction; for the ste18 mutant (A), this value
was normalized to reflect the reduced level of Ste4p described in Table
III. The plasma membrane marker, Pma1p, is indicated by the
dashed line for each mutant strain and by the dotted
line for each wild-type control.
Effect of Ste18p on the Aggregation State of Ste4p
As an
additional test for interaction between Ste4p and Ste18p, we examined
the sedimentation properties of Ste4p in the ste18 mutant.
When lysates of the ste18 mutant were analyzed as described in Table II, 84% of the Ste4p protein was found in the particulate fraction, and 65% of this particulate fraction was solubilized by
extracting it with a combination of detergent and 0.25 M
NaCl. When the solubilized extract was analyzed by using glycerol
gradient centrifugation (as described in Fig. 3A), we found
a value of 4 S for the sedimentation coefficient of Ste4p from the
ste18 mutant (consistent with a 54-kDa Ste4p monomer),
whereas we obtained a value of 8 S for the
STE18+ control strain under these conditions.
Thus, Ste18p is necessary for assembly of Ste4p into a more rapidly
sedimenting complex. This complex is likely to contain additional
protein components since Ste18p (15 kDa) alone is too small to have an
appreciable influence on sedimentation of Ste4p. The observation that
the sedimentation coefficient for the wild-type control is larger than
the value obtained for purified plasma membranes from diploid cells
(Fig. 3A) or from haploid cells (not shown) implies that protein factors associated with G are removed during plasma membrane purification.
Previous
studies (45, 50) have demonstrated that, when haploid a or
cells are exposed to the pheromone from the opposite cell type, the
receptors exit the plasma membrane within 20 min and accumulate in the
vacuole. If G proteins remain bound to these receptors during the
endocytic process, they should also be eliminated from the plasma
membrane. We tested this possibility by performing Renografin gradient
analysis on cells that had been exposed to
-factor in the absence of
new protein synthesis. As shown previously (45), the
-factor
receptor was quantitatively eliminated from the plasma membrane after
20 min of pheromone exposure (Fig. 7). In contrast,
pheromone treatment resulted in no major change in the distribution of
Ste4p (Fig. 7). Therefore, very little, if any, G
is released from
the plasma membrane as a consequence of receptor endocytosis or G
protein activation.
Consistent with the proposed function of Ste4p and Ste18p in the
pheromone response pathway, we have found that these G and G
homologs form a complex associated with the plasma membrane. First,
approximately 40% of Ste4p and Ste18p fractionated with the plasma
membrane ATPase in Renografin buoyant density gradients, and
association of Ste4p with plasma membranes was dependent upon functional Ste18p. Second, the Ste4p and Ste18p that had been extracted
from plasma membranes were found to cosediment under two different
solvent conditions, and the Ste18p in these fractions was precipitated
in the presence of anti-Ste4p antiserum. Finally, Ste4p and Ste18p were
interdependent for their stability in vivo. Levels of Ste18p
in ste4 mutant cells were barely detectable; in
ste18 mutant cells, Ste4p was reduced in amount and
exhibited a more rapid turnover rate. These results represent the first direct evidence for a G
complex at the plasma membrane. Other biochemical evidence supporting a direct interaction between Ste4p and
Ste18p was provided by Song et al. (51) who found that Ste4p and Ste18p coprecipitate with Gpa1p in a guanine
nucleotide-dependent manner. Other indirect evidence was
provided in a G protein-coupling assay, that is the ability of guanine
nucleotides to influence
-factor binding is disrupted in both
ste18 and STE4Hpl mutants (2).
Under high salt conditions, the protein complex containing Ste4p and
Ste18p sedimented more rapidly than predicted for a simple complex of
these two proteins, and this discrepancy was greater under low salt
conditions. An increase in the sedimentation rate can be explained by
the binding of additional molecules of protein and/or detergent,
whereas deviations in shape or hydration would both tend to decrease
the rate. It is therefore likely that more protein or detergent binds
under low salt conditions than under high salt conditions. Although we
have not determined the relative contributions of protein and detergent
to the sedimentation behavior, there are a number of proteins predicted
to interact with Ste4p. Candidates include Gpa1p, the pheromone
receptor, Ste5p (21, 22), Ste20p (22, 23, 24, 25), Cdc24p (26), Akr1p (28, 29),
and Syg1p (27). Ste5p and the receptor were not expressed in the
diploid cells analyzed in Fig. 3. Although Gpa1p was expressed in these
cells, its sedimentation rate was not influenced by salt; hence, under
the conditions of our assay, Gpa1p does not appear to bind the G
complex. Ste20p, Cdc24p, Akr1p, and Syg1p are expressed in both haploid
and diploid cells; hence, they are potentially components of the
protein complex that contains Ste4p and Ste18p (Fig. 3). Ste20p encodes
a protein kinase (52); analyses of double mutant strains suggest that
Ste20p acts in the pheromone response pathway at a step that is
executed after Ste4p (23, 24) and before Ste5p (25). Cdc24p is required
for budding and for generating cell polarity (53); recent evidence also suggests that it is involved in pheromone signaling (26, 54). The
ankyrin repeat-containing protein Akr1p is required for normal bud and
projection formation and appears to have a negative effect on signaling
(28, 29). Interestingly, in the two-hybrid system, Akr1p shows an
interaction with free G
but not with the G
heterotrimer
(28). Consistent with this requirement for binding, we do not detect
Gpa1p in the complexes containing Ste4p and Ste18p. Finally, the
truncated form of a putative transmembrane protein, designated Syg1p,
has been shown to interact with Ste4p by using the two-hybrid system,
and it has also been shown to suppress the lethality of a
gpa1 deletion (27). Syg1p is proposed to be a transmembrane
signaling component that can respond to or transduce signals through
G
.
In mammals, G subunits apparently have a broad range of functions
as indicated by the variety of proteins with which they interact. In
addition to interacting with G
, G
subunits appear to bind
receptors (55) and several effector proteins including phospholipase
C
and certain isozymes of adenylyl cyclase (reviewed in Ref. 56). In
other examples, G
complexes are known to promote attenuation of
signaling by binding the
-adrenergic receptor kinase (57) and by
binding phosducin (58). Pumiglia et al. (59) used the
two-hybrid system to show that the protein kinase, Raf-1, binds the
G
2 subunit, thereby implicating G
in regulation of
the mitogen-activated protein kinase pathway. The small GTPase ARF, a
component required for transport of proteins among Golgi compartments,
has also been shown to bind G
(60), supporting the notion that
heterotrimeric G proteins are involved in the control of vesicular
protein traffic.
As judged from our Renografin gradient analysis, a substantial portion
of G was not tightly associated with plasma membranes. Approximately 30% of Ste4p and Ste18p was associated with internal membrane fractions (containing membranes of the endoplasmic reticulum, Golgi, and vacuole), while the remaining 30% was not tightly bound to
membranes. The biological significance of this fractionation pattern is
as yet unclear. Association with different cellular compartments may
reflect independent pools of Ste4p and Ste18p or it may reflect the
presence of intermediates in the assembly, turnover, or activation of
G
. Some, or all, of the Ste4p that was confined to the
non-membrane fractions on Renografin gradients may be associated with
membranes in vivo. Roughly 25% of Ste4p and Ste18p was
released from the particulate fraction in the presence of 1.0 M NaCl (Table II); thus, the G
that was weakly
associated with membranes may be released in the presence of 38%
Renografin (ionic strength roughly equivalent to 0.5 M
NaCl). This material appears, at least in part, to represent a
chemically distinct form of G
since a Ste4p species that migrated
more slowly on SDS-polyacrylamide gels was primarily limited to the
non-membrane fractions of the Renografin gradient (Fig. 1) and since it
was extracted from the particulate fraction with 1.0 M NaCl
(not shown). Interestingly, the
1 subunit of transducin
(Gt) is modified with the C15 farnesyl group
(61), as is Ste18p, and transducin does not require detergent for
extraction from the membrane (62). In contrast, the
2
subunit of Gs, Gi, and Go contains
the C20 geranylgeranyl lipid, and these G proteins
require detergent for extraction (63, 64).
Cole and Reed (49) found that phosphorylated forms of Ste4p migrate
more slowly on SDS-polyacrylamide gels and that the abundance of these
species increases upon exposure to -factor. Furthermore, these
modifications are likely to regulate the Ste4p activity since
ste4 mutants that block phosphorylation become hypersensitive to pheromones. In our experiments, when a cell cultures were treated with
-factor and the membranes resolved on Renografin gradients (not shown), Ste4p was converted to
slower-migrating species in plasma membrane, internal membrane, and
non-membrane fractions. Assuming that these species represent
phosphorylated forms, then the phosphorylated Ste4p that was tightly
associated with membranes may represent a transient intermediate that
subsequently is either dephosphorylated, degraded, or reduced in its
affinity for membranes. Two models are consistent with the presence of phosphorylated forms of Ste4p in both membrane fractions. Protein kinases that become activated upon
-factor binding may modify Ste4p
molecules both at the plasma membrane and at internal membrane sites;
alternatively, Ste4p may be modified at one site and then transported
to the other site.
While loss of the receptor did not affect localization of Ste4p, loss
of Gpa1p prevented stable association of Ste4p with the plasma
membrane. Gpa1p did not influence association of Ste4p with internal
membranes. These results suggest that G exerts a physical effect on
G
in vivo; Gpa1p may help to anchor G
to the
plasma membrane. Lipid modifications of G
and G
are thought to
mediate (at least in part) the membrane attachment of G proteins (see
Ref. 37). It has been proposed (37) that the G
subunit facilitates
binding of G
to membranes by supplementing the low binding energy
provided by the G
isoprenyl group. Ste18p appears to be farnesylated
since its sequence contains a consensus site for farnesylation and
since mutants affecting that site or the farnesyl transferase enzyme
affect signal transduction (15, 20, 65, 66). Gpa1p is both
myristoylated (14) and palmitoylated.2
In a recent related paper focusing on N-myristoylation of
Gpa1p, Song et al. (51) report on a number of observations
that pertain to subcellular localization of G. Some of their
results differ from ours and provide possible insights for properties of G
and limitations of the two approaches. Using sucrose
gradient fractionation, Song et al. (51) could detect Ste4p
only in fractions containing plasma membranes. Lack of Ste4p in the
non-membrane fractions is likely to reflect the lower salt conditions
used in their assay. In Table II, we found that a portion of Ste4p was
associated with the particulate fraction only under low salt conditions. Perhaps, this species is associated weakly with the plasma
membrane and suggests the presence of at least two populations of
G
species at this location. The inability of Song et
al. (51) to detect Ste4p in other membrane fractions may have
resulted from strain differences, from a failure to resolve these
membrane species from the plasma membrane, or from degradation of Ste4p during spheroplast formation. In gpa1 mutants, Song et
al. (51) found that only a portion of the Ste4p was resolved from
plasma membrane-containing fractions, whereas in the present study, we found that essentially all of the tightly associated Ste4p was resolved
from the plasma membrane. These results may reflect either the presence
of a Ste4p species that is weakly associated with plasma membranes or
insufficient resolution of the sucrose gradient technique. When we
examined the sedimentation behavior of detergent-solubilized G
on
glycerol gradients, we found no evidence for association with Gpa1. In
contrast, Song et al. (51) found that at least a portion of
Ste4p and Ste18p from whole cell extracts coprecipitates with the Gpa1p
that had been tagged with glutathione S-transferase. The
apparent absence of such a complex in our study may reflect the
differences in the assay conditions or the fact that we limited our
analysis to purified plasma membranes.
Although a number of proteins have been proposed to interact with
Ste4p, the mechanism by which G activates subsequent events in
the pheromone response pathway remains unknown. The activity of G
is thought to be stimulated upon its dissociation from G
, that is
when occupied receptors stimulate guanine nucleotide exchange or when
G
is inactivated by a mutation in the GPA1 gene. When
cells were treated with a saturating concentration of
-factor for 20 min, we found that cellular distribution of Ste4p was not altered
discernibly. As this is sufficient time to provide maximal response to
-factor, it appears unlikely that activation of the response pathway
requires movement of G
from the plasma membrane to another
cellular compartment or that removal of G
from the plasma
membrane regulates its activity. However, we cannot rule out the
possibility that the exit of a small quantity of G
in response to
-factor is sufficient for maximal activation. In contrast,
gpa1 mutant cells failed to accumulate G
that was tightly bound to the plasma membrane, thus cells that lack Gpa1p are
not in the same physiological state as wild-type cells treated with
pheromone. G
may retain some contacts with G
during
-factor stimulation; G
may facilitate the assembly of G
into a
structure that remains tightly bound to the plasma membrane when
activated by
-factor, or chronic stimulation of G
in the
gpa1 mutant may disrupt its stable contacts with the plasma
membrane. Even though G
is no longer tightly associated with the
plasma membrane in the gpa1 mutant, the requisite events for
signal transduction may be mediated by G
molecules that are only
weakly associated with the plasma membrane.
We thank Malcolm Whiteway, Ekkehard Leberer, Henrik Dohlman, Victoria Yuschenkoff, David Parker, Susana Silberstein, John Aris, Patricia Berninsone, Ying Yang, and H. K. Schachman for materials used in this investigation. We thank Reid Gilmore, Peter Pryciak, Dan Kelleher, and Gül Bukusoglu for comments on the manuscript and helpful discussions.