(Received for publication, February 27, 1997)
From the Department of Molecular and Cell Biology and
the § Department of Chemistry, University of California at
Berkeley, Berkeley, California 94720, the ¶ Department of Plant
Biology, The Royal Veterinary and Agricultural University, DK-1871
Frederiksberg C, Denmark, the
Life Sciences Division of
Lawrence Berkeley National Laboratory, Donner Laboratory, Berkeley,
California 94720, and the ** Department of Chemistry, College Of Staten
Island, Staten Island, New York 10314
A plasmid vector was developed that permitted
high-level expression of a functional form of the Saccharomyces
cerevisiae -factor receptor (the STE2 gene
product) tagged at its C-terminal end with an epitope (FLAG) and a
His6 tract. When expressed in yeast from this plasmid,
Ste2p was produced at a level at least 3-fold higher than that reported
previously for any other 7-transmembrane-segment receptor expressed in
the same cells. For purification, isolated cell membranes containing
the overexpressed receptor were solubilized with detergent under
specific conditions and subjected to immobilized metal affinity
chromatography. Yields as high as 1 mg of nearly homogeneous (95%)
receptor were routinely obtained even from relatively small scale
preparations (60 g of frozen cell paste). The purified receptor was
reconstituted into artificial phospholipid vesicles. Radioligand
binding studies demonstrated that the purified receptor, in the
reconstituted vesicles, bound its tridecapeptide ligand (
-factor)
with a KD (155 nM) consistent with the
affinity expected for this receptor in the absence of its associated G protein. Efficient restoration of ligand binding activity upon reconstitution required the addition of solubilized membranes prepared
from a yeast strain lacking the receptor. Sufficient amounts of active
material can be obtained by this procedure to allow physical studies of
this receptor and other 7-transmembrane-segment receptors expressed in
this system.
The -factor receptor (Ste2p) from Saccharomyces
cerevisiae belongs to the family of G protein-coupled receptors
(GPCRs)1 that, upon the binding of a
ligand, transduce a signal via an associated guanine-nucleotide binding
protein (G protein) (1, 2). GPCRs function in physiological processes
ranging from vision (the rhodopsins), to smell (the olfactory
receptors), to neurotransmission (e.g. muscarinic
acetylcholine, dopamine, and adrenergic receptors) (1, 2). In S. cerevisiae, the binding of the peptide pheromones
(a-factor and
-factor) to their receptors initiates the
cascade of events that lead to the mating of haploid yeast cells (3,
4).
GPCRs are integral membrane proteins ranging in size from 400 to 1000 amino acids. Hydrophobicity analysis of the GPCRs reveals the presence
of 7 hydrophobic regions predicted to form membrane-spanning -helices, suggesting a similar structural arrangement of these proteins in the membrane (1). Several amino acids in the hydrophobic domains are conserved among all GPCRs, suggesting their importance in
the proper folding of the protein within the membrane. Other amino
acids are conserved only within a particular GPCR subclass and are
therefore thought to confer binding specificity for a certain class of
ligands (5-8).
The hydrophilic domains of GPCRs (the extracellular and intracellular loops and the cytosolic tails) exhibit a large variation in size and amino acid composition. Site-directed mutagenesis and biochemical characterization of the GPCRs has demonstrated that for the smaller ligands (like catecholamines) the transmembrane helices 3, 4, 5, 6, and 7 carry determinants for ligand recognition (6, 8). The binding of larger ligands, such as peptides or glycoproteins, is less well characterized, but the extracellular hydrophilic domains of the receptor appear to be involved in ligand binding to some extent (8, 9). The third cytoplasmic loop of the receptors is implicated in the interaction with and activation of the associated heterotrimeric G protein (10, 11).
Detailed biophysical studies of GPCRs (such as structure determination
by x-ray or electron crystallography) have been hampered by the
difficulties in obtaining large quantities of purified receptors from
natural sources. One exception is bovine rhodopsin, available in large
quantities from bovine rod cells. Recently, Schertler and Hargrave (12)
succeeded in forming well diffracting two-dimensional crystals of
rhodopsin. The electron diffraction data permitted the production of a
projection map of rhodopsin at 9 Å resolution, showing high density
areas corresponding to the seven putative membrane-spanning
-helices. The successful determination of a high resolution
structure of bacteriorhodopsin (13) and light-harvesting complex II
(14) by electron diffraction of two-dimensional crystals suggests that
a high resolution structure of rhodopsin may be available in the
future.
Overexpression in heterologous expression systems (such as insect
cells, yeast, or E. coli) has been used to
produce GPCRs. Several GPCRs have been expressed in a functional form
using the baculovirus system, including the D4 and
D2 dopamine receptors (15, 16), the M1 and M2 muscarinic
acetylcholine receptors (17, 18), and the 2-adrenergic
receptor (17). The D2S dopamine receptor has been
functionally expressed in both S. cerevisiae and
Schizosaccharomyces pombe (19, 20), and an E. coli expression system has also been successfully used for
expression of GPCRs (21, 22).
Yeast-based expression systems for GPCRs have several advantages over other systems. First, the cost of growing yeast in large fermentors is small compared with the costs of insect cell maintenance and growth. Second, a yeast-based system is genetically flexible. It is possible to easily construct, express, and purify mutant versions of receptors without the complications of recombinant virus selection and viral amplification encountered in the baculovirus system. Third, certain vertebrate GPCRs heterologously expressed in S. cerevisiae cells are able to elicit growth arrest in response to their natural agonists (23, 24). As a result, mutants of these GPCRs (truncations or point mutants useful in biophysical studies) can be functionally characterized in S. cerevisiae cells.
Here we describe overexpression, purification, and initial in
vitro characterization of Ste2p, the -factor receptor from S. cerevisiae. Because the purification requires only a
single column and yields approximately 1 mg of receptor protein,
detailed biophysical studies of the
-factor receptor are now
possible. Other results using this system indicate that this expression and purification protocol is applicable to the overexpression and
purification of other GPCRs, making future structural work on this
class of molecules less expensive and more tractable.
S. cerevisiae
strains used were DK102 (MATa ura3-52
lys2-801am
ade2-101oc trp1-63
his3-
200 leu2-
1
ste2::HIS3 sst1-
5) (25), RC629 (MATa ura3-52 trp1-
63
his3-
200 sst1-
2) (26), and BJ2168 (MATa prc1-407 prb1-1122
pep4-3 leu2 trp1 ura3-52) (27). The pNED1 vector
(see Fig. 1) was constructed using the progenitor plasmid pG3 (28). The
STE2 gene was amplified by polymerase chain reaction (PCR).
Two sequences functioning as affinity tags, encoding DYKDDDDK (the FLAG
tag (FT)) and HHHHHH (His6 tag (HT)), were introduced at
the C terminus of Ste2p. Once constructed, the nucleotide sequence of
the entire STE2 gene open reading frame was confirmed by
dideoxy sequencing (29). Radioligand binding medium (YM1 + i medium)
contained 5 g/liter yeast extract, 10 g/liter peptone, 6.7 g/liter
yeast nitrogen base without amino acids, 10 g/liter succinic acid, 6 g/liter sodium hydroxide, 10 g/liter glucose, 10 mM
NaN3, 10 mM KF, and 10 mM
p-tosyl-L-arginine methyl ester (30).
-Factor is a tridecapeptide with the
sequence Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr.
[Nle12]
-Factor has an affinity for Ste2p that is
indistinguishable from wild-type
-Factor (30).
[3H,Nle12]
-Factor was produced by
catalytic tritiation of a dehydroproline residue as described
previously (30) and purified by reversed-phase high-pressure liquid
chromatography. The peptide was then diluted to 0.01 mg/ml with a
storage buffer (20% ethanol, 1 mM methionine, 0.01%
trifluoroacetic acid), flash-frozen in liquid nitrogen, and stored at
80 °C.
[D-Ala9,Nle12]
-Factor and
[L-Ala9,Nle12]
-factor were
synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on an Applied Biosystems synthesizer and purified to near
homogeneity by high-pressure liquid chromatography. Details of the
synthesis and the biological activity of these peptides against whole
cells will be described elsewhere. The masses of all peptides used in
these studies were confirmed by mass spectrometry. Because all of the
-factor peptides used in this work contained the norleucine
substitution at position 12 (which makes the peptides more resistant to
oxidation) instead of the naturally occurring methionine, subsequent
references to these peptides in the text shall lack the
Nle12 denotation for the sake of clarity.
The nucleotide sequence encoding the
final 131 residues of Ste2p was inserted in frame into the pET15b
expression vector (Novagen, Madison, WI) and introduced into the BL21
DE3 E. coli strain by transformation. The pET15b expression
vector allows for the rapid purification of expressed proteins,
primarily due to the fact that cloning into the vector introduces an
N-terminal polyhistidine tag. The His-tagged 131-amino acid Ste2p
subdomain was purified to homogeneity by immobilized metal affinity
chromatography. Rabbits were immunized essentially as described by
Harlow and Lane (31). Bleeds were tested for immunoreactivity by
immunoblot analysis and visualized by enhanced chemiluminescence (ECL).
Antibodies generated in this work showed no cross-reactivity with whole
cell extracts from strain DK102, which carries a deletion mutation (ste2) of the
-factor receptor gene.
All manipulations with membranes and all purification steps were carried out in the presence of the following protease inhibitor mixture: 1.0 µg/ml leupeptin, 1.0 µg/ml pepstatin A, and 17.4 µg/ml phenylmethylsulfonyl fluoride.
Growth of Yeast CellsDK102, RC629, DK102 [pNED1], and
BJ2168 [pNED1] were grown to midexponential phase
(A600 1). DK102 and RC629 were grown at
30 °C in YPD medium (32), while the strains transformed with pNED1
were grown at 30 °C in a synthetic medium without tryptophan (32) to
maintain selection for the plasmid. For the large-scale growth of cells
for the purification of Ste2p.FT.HT, BJ2168 cells carrying the pNED1
plasmid were grown at 26 °C to A600
3 in medium lacking tryptophan using a 200 liter fermentor. Cell paste was
harvested, frozen as cakes (60 g), and stored at
80 °C until use.
All steps were performed at 4 °C,
and all buffers were supplemented with protease inhibitors. For
small-scale experiments, cells equivalent to
A600 = 5 were resuspended in 50 mM
Tris-HCl (pH 7.9) and lysed by vortexing with glass beads. Unlysed
cells were removed by centrifugation at 700 × g, and
the membrane fraction was collected by centrifugation at 150,000 × g. For large scale preparation of membranes for
purification of Ste2p.FT.HT, approximately 60 g of yeast cell
paste (1 frozen cake) were thawed and resuspended in 90 ml of 10%
(w/v) sucrose in buffer A (50 mM HEPES (pH 7.5), 5 mM EDTA, protease inhibitors, and 500 nM
-factor). The cells were lysed by vigorous shaking with glass beads
for three 2-min pulses in a Braun Scientific (Allentown, PA) cell
homogenizer. Unlysed cells were removed by centrifugation at 700 × g, and the membrane fraction was collected by
centrifugation at 186,000 × g for at least 1 h.
Cell lysate corresponding to a suspension of A600 = 5 (in 100 µl) was solubilized in sample buffer (50 mM Na2CO3, 50 mM dithiothreitol, 15% (w/v) sucrose, 2.5% SDS). A volume corresponding to 3% of this starting material was subjected to electrophoresis on a 12% SDS-polyacrylamide gel. For immunoblot analysis, the separated proteins were transferred to nitrocellulose and analyzed using a rabbit polyclonal antibody directed against the C-terminal 131 amino acids of wild-type Ste2p. The dilution factors were chosen so as to give comparable signals when the immune complexes were visualized by ECL.
Halo Assay for Growth ArrestThe response of cells carrying
pNED1 to -factor was analyzed by halo assay (33). Different amounts
of
-factor (100 ng, 500 ng, and 1000 ng) were spotted on filter
paper disks and placed on agar plates containing DK102, RC629, or DK102
[pNED1]. Plates were grown at 30 °C for 48 h, and the sizes
of the halos were determined.
All steps were performed at
4 °C, and all buffers were supplemented with protease inhibitors and
500 nM -factor. Yeast membranes from 60-g cells were
homogenized in 90 ml of 10% (w/v) sucrose in the buffer A described
under "Membrane Preparation." Discontinuous sucrose gradients were
made from 14 ml of 43% (w/v) and 6 ml of 53% (w/v) sucrose in buffer
A. Aliquots (15 ml) of the homogenized membranes were layered on the
gradients and centrifuged overnight at 27,000 rpm in a Beckman SW28
rotor. The band between 10-43% (w/v) sucrose was collected, diluted
with dH2O, and centrifuged at 186,000 × g
for 1 h. The pellet was resuspended in 25 ml of 50% glycerol and
added to 25 ml of 2% n-dodecyl-
-D-maltoside (DBM) (Anatrace, Maumee, OH), 50% glycerol, 50 mM HEPES
(pH 7.5), and 150 mM NaCl. After 1 h of incubation,
unsolubilized material was removed by centrifugation at 186,000 × g for 30 min. The soluble fraction was diluted with 12.5 ml
of 99% glycerol and 200 ml of buffer B (0.075% DBM, 15% glycerol,
500 mM NaCl, 50 mM imidazole, 50 mM
HEPES (pH 7.5), 0.4 mg/ml soybean L-
-lecithin (type II-S from Sigma)). The sample was loaded at 12 ml/h onto a 4-ml
Ni-NTA-agarose column (Qiagen, Chatsworth, CA) pre-equilibrated with
buffer B. The column was then washed with 80 ml of buffer B. The
concentrations of NaCl and glycerol were reduced by washing with 20 ml
of buffer C (buffer B with 150 mM NaCl) and 60 ml of buffer
D (buffer C with 7.5% glycerol). Ste2p.FT.HT was eluted with buffer D
adjusted to 200 mM imidazole (pH 6).
Protein composition was analyzed by SDS-PAGE on high Tris 12% gels according to Fling and Gregerson (34). The separated proteins were stained with Coomassie Brilliant Blue R-250. Deglycosylation of Ste2p.FT.HT was carried out using PNGase F (New England Biolabs, Beverly, MA). Purified Ste2p.FT.HT (0.17 µg in 20 µl) was incubated with 30 units of PNGase F at room temperature for 1 h. The deglycosylation products were analyzed by immunoblot using an anti-FLAG M2 monoclonal antibody (Sigma).
Reconstitution of Purified Ste2p.FT.HT into Phospholipid VesiclesThe method used was a modification of the octyl
glucoside-mediated reconstitution of the sarcoplasmic reticulum
Ca2+-ATPase (35). Phospholipids were from Avanti Polar
Lipids, Inc. (Alabaster, AL). 1-Palmitoyl-2-oleoylphosphatidylcholine
(POPC) and 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) were mixed 60:40 (w:w) in chloroform and dried under a stream of nitrogen gas.
Remaining traces of solvent were removed under vacuum for at least
1 h. The dried lipid film was suspended in 50 mM HEPES (pH 7.5), 150 mM NaCl, and 5 mM EDTA at a
concentration of 7.5 mg of lipid/ml. Liposomes were obtained by bath
sonication for 1 h at 10-20 °C under nitrogen gas. The
liposomes were diluted to 3.75 mg of lipid/ml and then saturated with
the detergent n-nonyl--D-glucoside (Anatrace)
as follows. Incorporation of the detergent into vesicles was monitored
by measuring the A540 of the lipid-detergent
suspension until the initial turbidity just started to decrease
rapidly. The pooled Ni-NTA fractions containing purified Ste2p.FT.HT
(~10 ml at a concentration of ~0.1 mg Ste2p.FT.HT/ml) were
incubated with 6.5 ml of the detergent-saturated liposomes for 1 h
at 4 °C. Excess detergent was removed by overnight batch adsorption
to 0.4 ml of moist Bio-Beads SM-2 (Bio-Rad) per ml of total suspension at 4 °C. The resulting suspension of proteoliposomes was separated from the Bio-Beads and then collected by centrifugation at 229,000 × g for 1 h. The pellet was washed with the
HEPES-buffered solution before resuspension and use in radioligand
binding assays.
For reconstitutions in the presence of solubilized yeast membranes, the
above protocol was modified as follows. Total DK102 (ste2) membranes were isolated as described under
"Membrane Preparation" and solubilized at a final protein
concentration of at least 10 mg/ml in 1% DBM, 45% glycerol, 50 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM EDTA, and protease inhibitors. After mixing for 1 h
at 4 °C, unsolubilized material was removed by centrifugation at
229,000 × g for 45 min. The solubilized DK102
(ste2
) extract (2 ml) was incubated with a 1-ml aliquot
of the pooled Ni-NTA fractions containing purified Ste2p.FT.HT for
3 h at 4 °C. The detergent-saturated, 60:40 POPC:POPG liposomes
(3 ml) were then added to this mixture, and the subsequent steps were
carried out as described above.
The concentration of Ste2p.FT.HT present in reconstituted liposomes was determined by quantitative immunoblot. The accuracy of this measurement technique was confirmed by comparing the values determined this way with the values determined by the Bradford protein assay on purified Ste2p.FT.HT solubilized with DBM, the epitope-tagged FLAG-BAP protein standard (Sigma) in DBM, and bovine serum albumin in DBM. In brief, serial dilutions of both the FLAG-BAP protein and the liposome suspension were electrophoresed on the same SDS-polyacrylamide gel. The proteins were then immobilized on a nitrocellulose membrane by electroblotting and probed with the anti-FLAG M2 monoclonal antibody. The dilution factors of the FLAG-BAP protein and of the liposome suspension were chosen so as to give comparable signals when the immune complexes were visualized by ECL. This quantitative immunoblot technique has also been used to measure the amount of Ste2p.FT.HT present in membrane preparations. The values obtained were consistent with the values obtained from radioligand binding measurements.
Ligand Binding MeasurementsThe dissociation constant
(KD) and the number of binding sites per cell
(Bmax) for the binding of
[3H]-factor to whole cells were determined using minor
modifications of the procedure of Raths et al. (30).
Nonspecific binding was measured by adding a 1000-fold molar excess of
competing non-radioactive
-factor. This approach gives similar
values to those obtained for the nonspecific association of
[3H]
-factor with MAT
cells, which do not
produce Ste2p (data not shown). Determination of the
KD and Bmax for the binding of the [3H]
-factor to isolated cell membranes was
performed essentially as described by Blumer and Thorner (3), with two
exceptions. First, the binding buffer was replaced with YM1 + i medium.
Second, the membrane amounts from different strains were normalized
using their A600 rather than their total protein
content assessed via the Bradford method (36). This normalization
method was motivated by the observation that membrane lipids interfered
with accurate assessment of protein concentration. By using
A600 of the membrane suspension, a more
reasonable approximation of bulk membranes could be made as a means to
normalize the total amount of membrane protein from strain to
strain.
For determination of KD and
Bmax for the purified receptor, Ste2p.FT.HT was
reconstituted into lipid vesicles, resuspended in YM1 + i medium, and
then subjected to ligand binding measurements. The reactions were
incubated for 1 h at 25 °C, diluted with 1.5 ml of ice-cold YM1 + i medium, and rapidly filtered through GF/F glass microfibre filters
(Fisher, Pittsburgh, PA) presoaked in 0.3% polyethylenimine (Sigma)
(3). The reaction tubes and filters were washed three times with 1.5 ml
of ice-cold YM1 + i medium. The amount of bound
[3H]-factor was determined by scintillation counting
of the glass filters in ScintiVerse BD scintillation mixture (Fisher)
after overnight solubilization. Nonspecific binding was determined as described above. KD and Bmax
were determined for each experiment using least squares fitting
analysis to the Langmuir isotherm equation [Bound] = Bmax/(1 + KD/[Free]). All manipulations in these studies were carried out using minisorp tubes
(Fisher) and siliconized pipette tips (Fisher) to reduce adsorption of
[3H]
-factor to plasticware.
For the
generation of competition binding curves using isolated membranes
containing the overexpressed receptor, a concentrated suspension of
membranes from strain DK102 [pNED1] was diluted to a final
A600 of 0.001 (approximately 10 µg/ml of
membrane protein). This concentration was chosen to minimize ligand
depletion that would invalidate the estimation of KD
from IC50. [3H]-Factor was added to the
diluted membrane suspension to a final concentration of 4 nM. Two peptides were compared for their ability to
displace the bound [3H]
-factor,
[L-Ala9]
-factor and
[D-Ala9]
-factor. Concentrations of the
-factor analogs were determined using the
A280 for each peptide as determined by Raths
et al. (30). The dissociation constants
(KD) for each analog were estimated using the
equation KD, Analog = IC50/(1 + [Tracer]/KD, Tracer), where
IC50 is the concentration of analog required to compete
50% of specifically bound [3H]
-factor. Incubations
were carried out at room temperature for 90 min and then collected on
glass filters as described above.
[3H]-Factor competition studies on the purified
receptor were similar to those for membranes except that the
concentration of [3H]
-factor used in the assays was 40 nM.
The sequence modifications
made to the C terminus of the receptor did not compromise its ability
to mediate growth arrest in response to -factor, as demonstrated in
an in vivo bioassay (see Fig. 2). DK102 (which does not
express Ste2p) showed no response to
-factor, as expected. RC629 and
DK102 [pNED1] (Fig. 1) showed similar halo patterns
(Fig. 2), demonstrating that the FLAG and His6 tags appended to the C terminus of the receptor do not
compromise either ligand binding or signal transduction.
BJ2168 [pNED1] Overproduces Ste2p at Least 80-fold
Expression of Ste2p was monitored using two approaches,
immunoblot analysis (Fig. 3) and
[3H]-factor binding to whole cells
(Fig. 4A) and to isolated cell membranes
(Fig. 4B). As found by immunoblotting (i.e. as
judged by comparing the amounts of membranes required to give
equivalent ECL signals), when the expression plasmid (pNED1) was
propagated in a MATa strain lacking any endogenous
Ste2p (DK102), the receptor was produced at least 50-fold above the
endogenous level, relative to a MATa strain (RC629)
expressing Ste2p from the chromosomal STE2 locus. A
protease-deficient strain (BJ2168) carrying pNED1 produced about
80-fold more receptor than the wild-type MATa strain
(RC629).
Radioligand binding is more quantitative than immunoblotting and has the added advantage of detecting only active receptor molecules. Radioligand binding on whole cells (Fig. 4A) demonstrated that the two overexpression strains have similar levels of cell surface-expressed Ste2p, approximately 20-fold higher than the level on a wild-type cell (RC629). This value was lower than the estimate obtained by immunoblotting, suggesting the possibility that not all of the receptor was present on the cell surface (perhaps contained in an internal membrane compartment) and was thus inaccessible in the whole cell binding assay. Indeed, when cells were lysed and membranes isolated from DK102 [pNED1] were assayed for binding, it was found that they contained 79-fold more binding sites than membranes from RC629. Membranes isolated from BJ2168 [pNED1] cells had a similar number of binding sites as DK102 [pNED1] (Fig. 4B). These results indicate that essentially all of the receptor molecules produced retain their ligand binding ability.
Ste2p.FT.HT and Wild-type Ste2p Have Similar Affinities for [3H]All saturation binding data
performed on whole cells (Fig. 4A) and on cell membranes
(Fig. 4B) were transformed into Scatchard plots and straight
lines were obtained, suggesting that each data set represented a
population of receptors of a single ligand affinity. For whole cells, a
KD of ~6 nM was obtained for all
strains examined (Table I). This value is very similar
to that previously reported by Jenness, et al. (37). The
KD values observed for the membranes from RC629,
DK102 [pNED1], and BJ2168 [pNED1] were 5 nM, 14 nM, and 24 nM, respectively (Table I). These
values are also in agreement with previous reports of the affinity of -factor for Ste2p in cell membranes (37).
|
Due to its extremely high level of expression,
purification of the receptor could be achieved using only two steps (as
described in detail under "Materials and Methods"). Isolated
intracellular membranes were collected on a discontinuous sucrose
gradient, solubilized with detergent, and the solubilized receptor was
then subjected to immobilized metal affinity chromatography
(Fig. 5A, lane 2). The yield of
purified Ste2p.FT.HT was determined using the Bradford protein assay
(36). About 1 mg of purified Ste2p.FT.HT could be routinely obtained
with a purity estimated to be 95% (as judged on an overloaded
Coomassie-stained gel, see Fig. 5A). If one also includes
receptor solubilized from the membrane material that collects between
the 43 and 53% (w/v) sucrose steps, as much as 2 mg of receptor could
be obtained, but at a lower purity (approximately 80%, data not
shown).
Receptor Microheterogeneity Is Due in Part to Receptor Glycosylation
Purified Ste2p.FT.HT (as well as wild-type Ste2p) migrates as at least four bands by SDS-PAGE. At least some of this complexity is the result of N-linked oligosaccharides added to the N terminus of the protein (38). Digestion of the purified receptor with PNGase F resulted in a collapse of the four blurry bands into two sharp bands (Fig. 5B), indicating that glycosylation accounts for some of the polydispersity present in the receptor preparation.
Purified Ste2p.FT.HT Has an Affinity for Its Ligand Consistent with the Absence of Its Cognate G ProteinPurified Ste2p.FT.HT was
reconstituted into synthetic liposomes as described under "Materials
and Methods." Steady-state binding of [3H]-factor to
purified, 60:40 POPC:POPG-reconstituted Ste2p indicates that the
purified receptor binds [3H]
-factor with a
KD of 155 nM and a
Bmax of 1064 pmol/mg (Fig. 6).
This affinity is similar to previous reports of the affinity of
-factor for Ste2p in the absence of its associated G protein (150 nM) (3).
Efficient Restoration of Ligand Binding Activity Required the Addition of Solubilized Yeast Membranes
Based on the amount of
purified Ste2p.FT.HT reconstituted into the artificial vesicles, and
assuming all of the receptor molecules were properly oriented in these
liposomes, only a fraction (~6%) of the total expected ligand
binding capacity was observed. We found that ligand binding could be
restored by adding solubilized membranes from DK102
(ste2) before vesicle reconstitution. The resulting
Ste2p.FT.HT-containing proteoliposomes displayed a specific activity of
14,500 pmol/mg Ste2p.FT.HT (Fig. 6), suggesting that most of the
purified receptor (at least 80%) is capable of binding ligand after
vesicle reconstitution.
The
ability of a receptor to discriminate between different ligands is a
measure of the authenticity of ligand binding.
[D-Ala9]-Factor is a pheromone analog that
binds Ste2p with an affinity somewhat greater (>1.5-fold greater) than
that of authentic
-factor.2 It has been
observed additionally that [D-Ala9]
-factor
can bind to Ste2p expressed on the surface of whole cells approximately
300-fold better than
[L-Ala9]
-factor.2 We found
that crude membranes containing Ste2p as well as purified and
reconstituted Ste2p.FT.HT displayed similar differences in affinities
for these stereoisomers. In control studies, authentic
-factor
competed approximately as well as did
[D-Ala9]
-factor (data not shown). For
membranes isolated from DK102 [pNED1], a 433-fold difference in
KD was observed between the
[L-Ala9]
-factor and
[D-Ala9]
-factor (Fig.
7A). Likewise, for purified and 60:40
POPC:POPG-reconstituted Ste2p.FT.HT, a 396-fold difference in
KD was observed between the
[L-Ala9]
-factor and
[D-Ala9]
-factor (Fig. 7B).
Ste2p.FT.HT reconstituted in the presence of solubilized DK102
(ste2
) membranes displayed at least a 200-fold difference
in affinity for the stereoisomers (data not shown). These results
indicate that the purified receptor maintains its correct ligand
recognition characteristics.
The system used to express Ste2p was designed to maximize production of receptor protein and to introduce two different affinity tags on the C terminus of the protein to facilitate receptor purification and detection (Fig. 1). The expression vector was derived from pG3, a high-copy number plasmid designed to constitutively express proteins in S. cerevisiae (28). The vector contains a strong, constitutively active promoter, the glyceraldehyde-3-phosphate dehydrogenase (TDH3) promoter, and an efficient terminator, the phosphoglycerate kinase (PGK1) terminator. The plasmid exists in high copy number due to the presence of the origin of replication derived from the 2-µm DNA plasmid.
Procedures have been described for modifications that can be introduced
at the beginning and the end of open reading frames in S. cerevisiae to increase protein expression (39). These modifications were introduced into the STE2 gene by PCR.
First, the 3,
2, and
1 positions with respect to the initiating
ATG were changed to ATA. Second, the stop codon was changed to TAA (Fig. 1). Procedures have also been described for modifications that
can be introduced into the primary sequence of a GPCR to facilitate its
purification (40), but these procedures result in an expression level
at least one order of magnitude less than that reported here.
To assess the effectiveness of our expression system, the expression
level of the Ste2p.FT.HT receptor was monitored by immunoblotting (Fig.
3) and [3H]-factor binding measurements (Fig. 4, Table
I). The specificity of the antibody was demonstrated by the absence of
detectable cross-reaction when DK102 (ste2
) membranes
were analyzed (Fig. 3, lane 1). Comparison of lane
5 (RC629) with 2 (DK102 [pNED1], diluted 1:25) in
Fig. 3 indicates that the pNED1 expression vector transformed into
DK102 gives approximately 50-fold overexpression of Ste2p relative to a
wild-type MATa strain (RC629). Though the dilution
factor chosen was 1:25, the ECL signal was more than 2-fold stronger in
lane 2, suggesting that the apparent 50-fold difference is a
minimum estimate of overexpression. Introducing the same plasmid into
the protease-deficient strain BJ2168 further increased the
overexpression of Ste2p to at least 80-fold relative to RC629 (compare
lanes 4 and 5). Again, this estimate is based on
the comparison of the dilution factors required to give comparable signals. It should be noted that both the wild-type Ste2p and the
overexpressed Ste2p.FT.HT appeared as several closely-spaced bands on
the immunoblot, some of the heterogeneity being due to differing
degrees of glycosylation (Fig. 5B). It should also be noted
that the Ste2p.FT.HT receptor migrates slightly slower than the
wild-type receptor by SDS-PAGE, as expected from its increased molecular mass. This mobility shift is consistent with the presence of
the additional amino acids from the FLAG and His6 tags
appended to the C terminus of the protein.
[3H]-Factor binding measurements (Fig. 4) provided a
similar yet more quantitative estimate of Ste2p overexpression than did the immunoblot results. In the whole cell binding assay, 19-fold (BJ2168 [pNED1]) to 21-fold (DK102 [pNED1]) more binding sites are
observed on the surface of each of the overexpression strains than on a
wild-type MATa strain (RC629). But when total membrane
preparations are assayed for [3H]
-factor binding, many
more sites appear to be available, 79-fold (DK102 [pNED1]) and
80-fold (BJ2168 [pNED1]) more than in RC629. For BJ2168 [pNED1],
the radioligand binding and the immunoblot give comparable assessments
of overexpression, whereas for DK102 [pNED1], the radioligand binding
suggests the presence of more Ste2p than does the immunoblot.
We estimate the expression level of Ste2p.FT.HT in BJ2168 [pNED1] to
be approximately 352 pmol/mg membrane protein. This value is at least
3-fold higher than that reported for any other 7-transmembrane-segment receptor expressed in yeast. The level of expression of the rat M5
muscarinic acetylcholine receptor was approximately 0.1 pmol/mg protein
(41). The level of D2 dopamine receptor expression was reported to be 1-2 pmol/mg protein (20). The highest expression previously reported came from King et al. (23), who observed 115 pmol/mg protein for a fusion protein of the N terminus of Ste2p and
the human 2-adrenergic receptor.
Having determined that our expression system was effective, it was
necessary to demonstrate that the modified receptor was functional.
Cells containing the overexpressed Ste2p.FT.HT responded to -factor
(arrested their growth in a halo assay) with a sensitivity comparable
with that of a wild-type MATa strain (Fig. 2), demonstrating that the introduction of the FLAG and His6
tags at the C terminus of the protein does not compromise receptor function. The halo assay measures the ability of a yeast strain to
undergo growth arrest in response to
-factor. The larger the halo of
non-growth, the more sensitive the strain. All strains used in the halo
assays performed here are disrupted at the SST1 locus (42),
which encodes a protease responsible for
-factor degradation. Thus,
all strains tested in this work should possess the same intrinsic
sensitivities to
-factor. Even though cells carrying pNED1
overproduce Ste2p substantially, it has been observed previously that
halo size is not detectably increased by receptor overexpression (38),
suggesting that components downstream of the receptor are limiting in
the growth arrest response. Thus, it is not surprising that receptor
overexpression in DK102 [pNED1] has no measurable effect on halo
size.
Although the halo assay demonstrated that the modified receptor was
able to mediate growth arrest, it was also necessary to demonstrate
that the receptor had a similar affinity as the wild-type receptor for
-factor. The affinity of [3H]
-factor for all three
yeast strains used in this work (Table I) is in very close agreement
with the value determined by Jenness et al. (37) as measured
on whole cells (6 nM). Additionally, the values measured
for the membranes of each of the strains are similar to previously
observed results (Table I). Blumer et al. (38) observed an
affinity of 2 nM in membranes expressing a wild-type level
of Ste2p (comparable with the value of 5 nM determined for
wild-type cell membranes in this work) and an affinity of 12 nM in membranes derived from a strain overexpressing Ste2p (comparable with the values of 14 nM and 24 nM
determined in this work). Thus, the sequence modifications made to the
receptor did not affect either its signaling functions or ligand
binding properties.
To achieve the purity reported here (approximately 95%), Ste2p.FT.HT was purified only from a sucrose gradient fraction thought to be highly enriched for intracellular membranes (the 10-43% (w/v) interfacial band). This choice, while yielding a slightly purer receptor preparation, recovers only half of the total Ste2p.FT.HT produced by the cells. By purifying the receptor from both sucrose gradient bands (the 10-43% (w/v) and the 43-53% (w/v)), one doubles the yield of receptor, but the preparation is less pure (approximately 80%, data not shown). The yield of Ste2p.FT.HT purified from internal membranes alone is 0.8-1.0 mg from 60 g of cell paste (corresponding to approximately 20 liters of cell culture grown to 3 A600). If one purifies Ste2p.FT.HT from both sucrose gradient bands, the yield is approximately 2 mg of Ste2p.FT.HT. While the FLAG tag is not necessary to purify Ste2p.FT.HT, the epitope tag is useful when this expression and purification system is applied to receptors that express less well or receptors for which one lacks antibodies.3
Digestion of the purified receptor with enzymes that should remove certain types of post-translational modifications helped clarify the nature of the multiple species present in our purified receptor preparation. Deglycosylation of the purified Ste2p.FT.HT with PNGase F collapsed the multiple protein bands around 50 kDa from four diffuse bands to two sharp bands (Fig. 5, A and B). Further treatment of the doublet with endoglycosidase Hf (another deglycosylation enzyme) or alkaline phosphatase had no detectable effect (data not shown). Since deglycosylation of the wild-type Ste2p in solubilized membranes revealed the same shift in mobility as the overexpressed receptor (data not shown), it can be ruled out that the appearance of the multiple protein bands is an artifact of overexpression. The reason for the presence of the two bands even after deglycosylation might be enzyme-resistant glycosylation (O-linked glycosylation), partial proteolytic processing of the receptor, or perhaps post-translational modifications other than glycosylation or phosphorylation.
To demonstrate that the purified, reconstituted receptor in vesicles
had the pharmacological properties of the wild-type receptor, ligand
binding studies were performed. [3H]-Factor binding
studies yielded a KD consistent with the notion that
the receptor has been purified away from its associated G protein (Fig.
6). For many mammalian GPCRs, the physical removal of G proteins from
their cognate receptors (either by receptor purification or through the
addition of a non-hydrolyzable GTP analog) results in a decreased
affinity for ligand. This same phenomenon has been seen previously with
Ste2p in yeast cell membranes (3). If any of the three G protein
subunits is absent, or if the G protein is dissociated from the
receptor by the addition of a non-hydrolyzable GTP analog, the affinity
of the receptor for
-factor is decreased to 150 nM,
which is in agreement with the value of 155 nM that we
found for the purified and reconstituted receptor.
The purified and reconstituted receptor also maintains its ability to
discriminate between atomically identical (but stereochemically distinct) -factor analogs (Fig. 7B), as does the normal
receptor in its native membrane environment (Fig. 7A).
Competition studies using crude membranes showed that Ste2p.FT.HT has a
433-fold greater affinity for
[D-Ala9]
-factor than for
[L-Ala9]
-factor. Likewise, purified and
reconstituted Ste2p.FT.HT has a 396-fold greater affinity for
[D-Ala9]
-factor than for
[L-Ala9]
-factor. The stereoisomers were
chosen because they have identical atomic compositions (and thus
identical hydrophobicities), yet they are reported to vary at least
300-fold in their affinities for Ste2p expressed on the surface of
whole cells.4
Efforts to develop an -factor binding assay using the
detergent-solubilized receptor were hindered by the observation that most detergents interfere with
-factor binding to Ste2p expressed on
the surface of whole cells (data not shown). This phenomenon motivated
the development of a detergent-free binding assay using artificial
vesicles. The specific activity of the purified and 60:40
POPC:POPG-reconstituted receptor (1064 pmol/mg) is, however, notably
less than might be expected for a receptor properly oriented in
liposomes. This specific activity suggests that perhaps only 6% of the
receptor is available for ligand binding.
One possible explanation for the low specific activity is that some
essential membrane component (perhaps a particular lipid or protein) is
required to help Ste2p maintain its proper shape in the membrane. To
test this hypothesis, solubilized membranes from a yeast strain lacking
Ste2p (DK102) were added back to the purified receptor before
reconstitution into artificial phospholipid vesicles. While DK102
(ste2) membranes possessed no ligand binding ability of
their own (data not shown), the addition of the solubilized extract to
purified Ste2p.FT.HT restored most of the expected ligand binding
activity (at least 80%) based on the amount of receptor present. Also,
addition of the DK102 (ste2
) extract had almost no effect
on the affinity of
-factor (or any of the
-factor analogs) for
the purified and reconstituted receptor (Fig. 6). Our laboratory is
currently attempting to identify the co-factors responsible for this
effect.
Despite the hundreds of GPCRs cloned to date, very few are available in the quantities required for physical studies. Using the system described here, as much as 1 mg of Ste2p.FT.HT could be purified using relatively small-scale techniques. These quantities are sufficient for a concerted structural effort. Our laboratory has also successfully used this system to express and purify the human D1A dopamine receptor,5 suggesting that the system may be widely applicable to GPCR expression and purification. The system has also been used to express a catfish olfactory receptor, albeit at lower levels than either Ste2p or the D1A dopamine receptor.6
We thank Dr. David King and Dr. Y. Larry Zhang for peptide synthesis and advice.