From the
Mutations in the ETR1 gene of the higher plant
Arabidopsis confer insensitivity to ethylene, indicating a
role for the gene product in ethylene signal perception and
transduction. The ETR1 gene product has an amino-terminal
hydrophobic domain and a carboxyl-terminal domain showing homology to
the two-component signal transduction proteins of bacteria. We report
here that in both its native Arabidopsis and when
transgenically expressed in yeast, the ETR1 protein is isolated from
membranes as a dimer of 147 kDa. Treatment with the reducing agent
dithiothreitol converted the dimer to a monomer of 79 kDa, indicative
of a disulfide linkage between monomers. Expression of truncated
versions of ETR1 in yeast confirmed that the high molecular mass form
is a homodimer and demonstrated that the amino-terminal region of ETR1
is necessary and sufficient for this dimerization. Site-directed
mutagenesis of two cysteines near the amino terminus of ETR1 prevented
formation of the covalently linked dimer in yeast, consistent with a
role in disulfide bond formation. These data indicate that ETR1 may use
a dimeric mechanism of signal transduction in a manner similar to its
bacterial counterparts but with the additional feature of a disulfide
bond between monomers.
Ethylene serves as a gaseous hormone in plants. As a plant
growth regulator, it is involved in seed germination, seedling growth,
leaf abscission, fruit ripening, and organ senescence. In addition,
ethylene can mediate the induction of specific defense proteins
(reviewed by Abeles et al.(1992)). Although the ethylene
biosynthetic pathway is now well characterized (reviewed by
Kende(1993)), the means by which cells recognize and transduce the
ethylene signal have not been established. An important contribution to
our understanding of ethylene signal transduction has come about
through the generation of mutants in the plant Arabidopsis thaliana with altered ethylene sensitivity. At least four separate genes in
the ethylene signal transduction pathway have been identified using
this approach (Bleecker et al., 1988; Guzman and Ecker, 1990;
Kieber et al., 1993; Van der Straeten et al., 1993).
The cloning of two of these genes, CTR1 (Kieber et
al., 1993) and ETR1 (Chang et al., 1993), has
recently been reported.
Mutations in ETR1 result in an
ethylene-insensitive phenotype in Arabidopsis (Bleecker et
al., 1988; Chang et al., 1993). All four of the mutant
alleles of ETR1 that have been isolated are dominant to the
wild-type gene and affect all ethylene responses examined. Plants
containing the mutant etr1-1 allele display only
one-fifth of the saturable ethylene binding of that found in wild-type
plants, indicating that ETR1 may be directly involved in ethylene
perception (Bleecker et al., 1988). Based on this work and the
epistatic relationship of ETR1 with other ethylene response
mutants (Kieber et al., 1993), mutations of ETR1 affect an
early step in the ethylene signal transduction pathway. These findings
are consistent with the possibility that ETR1 may be the ethylene
receptor or a regulator of the pathway.
The ETR1 gene was
cloned and found to encode a polypeptide with homology to the bacterial
two-component signal transduction proteins (Chang et al.,
1993). In bacteria, these signal transduction systems contain a sensor
and a response regulator and mediate responses to a wide variety of
environmental stimuli (reviewed in Parkinson(1993)). The sensor protein
is typically membrane-localized and contains a histidine kinase domain
that autophosphorylates on a conserved histidine residue in response to
an environmental stimulus. The phosphate is then transferred to an
aspartic acid residue on the response regulator protein, which mediates
downstream events. The ETR1 protein of Arabidopsis has both
histidine kinase and response regulator domains with the conserved
residues required for activity of the bacterial counterparts. It also
contains a hydrophobic domain indicative of membrane localization
(Chang et al., 1993).
The finding that the ETR1 gene of Arabidopsis (Chang et al., 1993) as well
as the SLN1 gene of yeast (Ota and Varshavsky, 1993) encode
proteins with homology to the histidine kinases and response regulators
of bacteria provides evidence that the two-component signal
transduction pathways of bacteria are present in eukaryotes. However,
questions remain as to how this system has been adapted for signal
transduction in eukaryotes and, more specifically, ethylene signal
transduction in plants. In this paper, we report that the product of
the ETR1 gene is a dimer in both its native Arabidopsis and when transgenically expressed in yeast. The yeast system has
been used to resolve which residues of ETR1 are required for
dimerization. The finding that ETR1 forms a dimer has implications as
to the manner by which the ethylene signal is transduced in plants.
For expression of
fusion proteins with glutathione S-transferase in
Escherichia coli, the vector pGEX-2T (Pharmacia Biotech Inc.)
was used. One fusion protein, designated GST-HRR, was designed to
express that portion of ETR1 corresponding to amino acids
401-738. For this, the 1.4-kb EcoRI fragment of
cETR1-1 was cloned into the EcoRI site of pGEX-2T. A
second fusion protein, designated GST-UNK, was designed to express that
portion of ETR1 corresponding to amino acids 165-400. For this, a
DraI-BglII fragment of cETR1-9 was cloned into
the SmaI-EcoRI sites of pGEX-2T, following addition
of an 8-mer EcoRI linker to the end-filled BglII
site.
For expression in yeast, the vector pYcDE-2 was used (Hadfield
et al., 1986). This vector has a constitutive ADC1 promoter, an EcoRI cloning site, and allows for Trp
selection. For expression of full-length ETR1, the BamHI
fragment of cETR1-5.2 was isolated, end-filled, 10-mer
EcoRI linkers were added, and the fragment was cloned into the
EcoRI site of pYcDE-2. For expression of the amino-terminal
region of ETR1 representing amino acids 1-400, a 1.4-kb
EcoRI-BglII fragment of cETR1-9 was cloned into
pYcDE-2, following addition of a 10-mer EcoRI linker to the
end-filled BglII site. For expression of the region of ETR1
representing amino acids 183-738, a 2.1-kb
DraI-EcoRI fragment of cETR1-9 was cloned into
pYcDE-2, following addition of an 8-mer EcoRI linker to the
DraI site. For this construct, Met-183 was the first ATG codon
capable of serving as the initiator for translation.
Site-directed
mutants for expression in yeast were constructed as follows. For the
etr1-1 mutation (C65Y), a PCR product was first
generated from a 4.2-kb EcoRI subclone of the etr1-1 mutant gene (Chang et al., 1993) using the same primers
and subcloning procedure as for cETR1-5.2. A BstXI
restriction fragment containing the etr1-1 mutation from
this subclone was used to replace the corresponding wild-type
BstXI fragment in cETR1-5.2. The etr1-1 mutant was then expressed in yeast in the same manner as the
full-length wild-type sequence. Site-directed mutants in the
cETR1-5.2 clone for C4S and C6S together and for C99S were made
using the Altered Sites Mutagenesis System (Promega) according to the
manufacturer. The double mutant of C65Y and C99S was made by replacing
the 0.9-kb HpaI-BglII wild-type fragment of the
etr1-1 (C65Y) mutant with the corresponding fragment of
the C99S mutant. All mutations were confirmed by sequencing.
For
isolation of soluble and membrane fractions, cultures were grown at 30
°C to mid-log phase. All subsequent steps were performed at 4
°C. Cells were harvested by centrifugation at 1,500
For analysis of yeast
membranes by density gradient centrifugation, total membranes were
fractionated on a 20-53% (w/w) sucrose gradient in 10 mM
Tris (pH 7.5), 1 mM EDTA. After overnight centrifugation at
30,000 rpm (Beckman rotor SW41Ti), 0.75-ml fractions were collected and
stored at -70 °C until use. Activities of mitochondrial
cytochrome-c oxidase (Villalba et al., 1992) and endoplasmic
reticulum NADH-cytochrome c reductase (Lord, 1987) were
measured as described. 1 unit of activity corresponds to the oxidation
or reduction of 1 µmol of cytochrome c/min. Presence of
the plasma membrane H
For extraction of total yeast protein into SDS-PAGE loading buffer,
5-ml yeast cultures were grown and isolated in mid-log phase. Cells
were pelleted, washed with water, and then resuspended in 75 µl of
glucuronidase buffer (50 mM Tris (pH 7.5), 10 mM
MgCl
For Western blotting, protein samples were
fractionated on 8% polyacrylamide gels in the presence of SDS and
transferred to nitrocellulose. Immunodecorated ETR1 was visualized
using a chemoluminescence detection system according to the
manufacturer (Kirkegaard and Perry). Densitometric analysis of
immunodecorated bands was performed using NIH Image (version 1.6) after
first scanning the exposed film using Adobe Photoshop and an Agfa
scanner.
A
cDNA clone containing the entire coding sequence for ETR1 was expressed
in yeast using a shuttle vector with constitutive promoter. Soluble and
membrane fractions were then isolated from control yeast transformed
with just the vector or from yeast transformed with the ETR1 construct.
An immunodetectable polypeptide of 79 kDa was identified in yeast
expressing ETR1 but not in control yeast transformed with vector alone
(Fig. 2). This 79-kDa polypeptide is close to the molecular mass
of 82 kDa predicted from amino acid sequence of ETR1. The 79-kDa ETR1
polypeptide was predominantly in the pelletable membrane fraction of
yeast, with very little in the soluble fraction (Fig. 2). To
determine if ETR1 was present as a membrane protein rather than an
insoluble aggregate, the pelletable membrane fraction was further
analyzed by sucrose density gradient centrifugation. On the gradient,
the ETR1 polypeptide resolved into a discrete vesicle population with a
peak at 35% (w/w) sucrose (Fig. 3), confirming that ETR1 is
indeed expressed as a membrane protein in yeast, consistent with the
hydropathy analysis. The peak of ETR1 did not align precisely with any
of the membrane markers examined. In this respect, it should be noted
that when the plasma membrane H
Based upon these results, we conclude that ETR1
is present as a high molecular mass species when isolated from either
its native Arabidopsis or the heterologous yeast system and is
capable of being converted to a lower molecular mass species by
reduction. The requirement of reducing agent for conversion of the high
to the lower molecular mass species is indicative of a disulfide
linkage. In addition, the oxidized 147-kDa species has approximately
twice the molecular mass of the 79-kDa reduced species, consistent with
the possibility that it represents a dimer of the reduced species. The
primary difference between ETR1 expressed in its native Arabidopsis and the heterologous yeast system is that ETR1 occurred in about
100-fold lower abundance in Arabidopsis membranes than in
yeast membranes.
ETR1 from both its native Arabidopsis and a
transgenic yeast system has been isolated as a dimer joined by a
covalent disulfide linkage. Since ETR1 is capable of forming a dimer in
yeast and Arabidopsis, dimerization may be a self-assembly
process with a disulfide bond formed following an initial noncovalent
association. Otherwise, one must postulate that additional factors
needed for association are present in both yeast and
Arabidopsis. The results from yeast demonstrate that two ETR1
monomers form a cross-linked homodimer, although this does not preclude
the possibility that in Arabidopsis ETR1 could form a
heterodimer with an isoform of similar molecular mass (Chang and
Meyerowitz, 1995). In any case, the consistency of results observed
with ETR1 expressed in Arabidopsis and the heterologous yeast
system indicates that yeast may be a suitable system for further
analysis of ETR1 structure and function. This finding is of particular
importance given that the ETR1 protein appears to be expressed in
native plant tissue at very low levels.
Mutagenesis of two cysteines
located near the amino terminus of ETR1 prevented formation of the
covalent linkage between monomers of ETR1 when expressed in yeast,
consistent with a role in disulfide bond formation. These cysteines are
located in a portion of ETR1 predicted to be on the extracytoplasmic
face of a membrane (Fig. 1). Disulfide bonds have frequently been
observed in the extracytoplasmic domains of other proteins, notably
dimeric growth factors and the insulin receptor of animals, and a wide
variety of proteins involved in the cell wall of both plants and fungi.
This is consistent with the localization of protein-disulfide
isomerase, an enzyme of the endoplasmic reticulum that catalyzes the
formation of disulfide bonds (Noiva, 1994). While the amino-terminal
cysteines appear to be both necessary and sufficient for covalent
dimerization, we cannot rule out the possibility that formation of
disulfide linkages within the membrane may subsequently occur. In
addition, we cannot entirely preclude the possibility that a disulfide
linkage is formed during protein isolation, although conditions where
this is known to occur were avoided (Kumar and Davidson, 1992). Even in
such a case, the cysteines would function in cross-linking and still
indicate a dimeric structure to ETR1 with the amino termini in close
proximity (Pakula and Simon, 1992).
The finding that ETR1 is a dimer
indicates that it may function similarly to the bacterial histidine
kinases in which the dimer plays a vital role in the signaling process
(Parkinson, 1993; Pan et al., 1993). With the bacterial
histidine kinases, perception of the environmental signal results in an
intermolecular phosphorylation event in which one monomer
phosphorylates its neighboring monomer upon a histidine residue. While
this dimeric form is apparently in place prior to perception of the
signal, in all known cases it is a noncovalent association of monomers
(Parkinson, 1993; Pan et al., 1993). The finding that the
dimeric form of ETR1 involves a covalent disulfide linkage represents
an intriguing variation upon the bacterial paradigm.
The
relationship between the biochemical properties of the ETR1 protein and
the mechanism of ethylene signal transduction is not yet clear.
Evidence presented here that the amino-terminal half of the ETR1
protein is necessary and sufficient for dimerization, in addition to
the previous finding that all of the dominant mutations of ETR1 result from single amino acid changes in the hydrophobic domain
(Chang et al., 1993), compel us to consider the importance of
this dimerization domain in the transduction of the ethylene signal.
This region may be particularly important in determining the quaternary
structure of the dimer, with ethylene-mediated changes in the structure
necessary for propagation of the ethylene signal. Dimerization of ETR1
may help explain the dominant effect of mutations in the gene. If both
the mutant homodimer and the mutant/wild-type heterodimer were
inactive, then only 25% of the dimers would be expected to be
functional in a heterozygous plant. This could be below the limit
needed to induce measurable ethylene responses. Alternatively, the
dominant ETR1 mutant proteins could be locked in an active state and be
unable to respond to changes in the ethylene signal. Both mechanisms
for dominance, either inactivation or locking of the dimeric form in
one active state, have been previously observed with the bacterial
histidine kinases (Pazour et al., 1991; Pan et al.,
1993). Interestingly, a mutation that apparently locks the VirA sensor
protein into an active conformation also lies within a transmembrane
domain of the protein (Pazour et al., 1991). While the
bacterial paradigm is useful in developing models for ETR1 function, it
should be noted that it has not yet been proven that mutations in ETR1
are of the dominant loss-of-function type as defined by
Herskowitz(1987). It is conceivable that these mutations arise due to a
gain of function, in which case the true function of ETR1 could be
obscured. Further biochemical characterization of ETR1 with regard to
kinase activity and direct interactions of the protein with ethylene
should help to resolve this issue.
We thank Joseph Ecker, Ronald Davis, Ramon Serrano,
Lucy Robinson, and Caren Chang for generously providing materials and
information, James Siedow for helpful discussions, and Richard Vierstra
and Michael Sussman for critical reading of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Plasmid Constructions
Standard
molecular cloning techniques were used (Sambrook et al., 1989;
Ausubel et al., 1994). A 4.2-kb(
)EcoRI fragment of the genomic ETR1 clone
(Chang et al., 1993) was used as probe in screening
Arabidopsis cDNA libraries. A 2.85-kb cDNA clone
(cETR1-9) in pBlueScript containing the complete coding sequence
for ETR1 was isolated from an Arabidopsis cDNA library cloned
into the EcoRI site of
ZAPII (Kieber et al.,
1993; library provided by J. Ecker, University of Pennsylvania,
Philadelphia). To remove noncoding sequence at the 5`- and 3`-ends of
cETR1-9, the clone was used as template DNA for a PCR reaction
(Perkin-Elmer Corp. PCR kit) using a 5`-primer
(CCCGGATCCATAGTGTAAAAAATTCATAATGG) and a 3`-primer
(CCCGGATCCTTACATGCCCTCGTACAGTACCCGG). The PCR product (cETR1-5.2)
was digested with BamHI and cloned into the BamHI
site of pBlueScript KS-, and the product was confirmed by
sequencing (Sequenase kit, U. S. Biochemical Corp.). In addition, a
1.4-kb cDNA clone (cETR1-1) containing partial coding sequence
and flanked by EcoRI sites was isolated from an
Arabidopsis cDNA library cloned in
YES (library provided
by R. Davis, Stanford University, Stanford, CA).
Yeast Transformation, Growth, and Protein
Isolation
Constructs were transformed (Schiestl and Gietz,
1989) into yeast strain LRB520 (MAThis3leu2trp1
ura3-52yck2-1::HIS3), and standard media and
procedures were used for growth (Ausubel et al., 1994).
g for 5 min, washed in 10 mM Tris (pH 7.5), 0.5 M
sucrose, 2.5 mM EDTA, and then resuspended in homogenization
buffer (100 mM Tris (pH 7.5), 0.3 M sucrose, 5
mM EDTA) containing 1 mM phenylmethylsulfonyl
fluoride and 10 µg/ml leupeptin as protease inhibitors at 1 ml of
buffer/g of cells. Cells were broken open by vortexing with chilled
glass beads (Ausubel et al., 1994), and the homogenate was
centrifuged at 10,000
g for 10 min to remove debris
and intact organelles. This supernatant was centrifuged at 100,000
g for 30 min to separate the soluble protein fraction
from the pelletable membrane fraction. The membrane pellet was
resuspended in 10 mM Tris (pH 7.5), 10% (w/w) sucrose, 1
mM EDTA with protease inhibitors.
-ATPase was determined by Western
blot analysis, using a polyclonal antibody generated against the
carboxyl terminus of the enzyme (Monk et al., 1991; antibody
provided by R. Serrano, Universidad Politecnica, Valencia, Spain).
, 1 M sorbitol). After the addition of 10
µl of
-glucuronidase (Sigma, G7017), cells were incubated 90
min at 30 °C. Cells were washed with glucuronidase buffer and then
extracted directly into 75 µl of SDS-PAGE loading buffer.
Isolation of Arabidopsis
Membranes
Arabidopsis was grown in liquid culture
as described (Chang et al., 1992). Leaves were homogenized at
4 °C in extraction buffer (30 mM Tris (pH 8), 20%
glycerol, 5 mM EDTA) containing 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, and 10 µg/ml
aprotinin as protease inhibitors. The homogenate was strained through
Miracloth (Calbiochem) and centrifuged at 8,000 g for
15 min. The supernatant was centrifuged at 100,000
g for 30 min, and the membrane pellet was resuspended in 10
mM Tris (pH 7.5), 10% (w/w) sucrose, 5 mM EDTA with
protease inhibitors.
Treatment with Dithiothreitol
(DTT)
Protein samples were mixed with SDS-PAGE loading
buffer (125 mM Tris (pH 6.8), 20% glycerol, 4% SDS, 0.01%
bromphenol blue, 1 mM phenylmethylsulfonyl fluoride)
containing 100 mM DTT. Controls contained no DTT. Samples were
incubated at 37 °C for 1 h and then subjected to SDS-PAGE (Laemmli,
1970). Boiling samples caused aggregation of the ETR1 protein.
Antibody Production and Western
Blotting
Fusion proteins were expressed in E. coli and affinity purified on glutathione-Sepharose (Smith and Johnson,
1988). Antisera were prepared from recombinant proteins by the
University of Wisconsin Medical Facility, cleared against E. coli proteins as described (Chang et al., 1992), and used at a
1:1000 dilution.
Expression of the ETR1 Gene in
Yeast
Hydropathy analysis of ETR1 identifies three
potential transmembrane segments at the amino terminus
(Fig. 1A). Although ETR1 does not contain a canonical
signal sequence for membrane insertion, a topological analysis based on
the model recognition approach of Jones et al.(1994) predicts
that the amino terminus of ETR1 would lie on the extracytoplasmic side
of a membrane while the large carboxyl-terminal region containing
histidine kinase and response regulator domains would lie on the
cytoplasmic side of a membrane (Fig. 1B). The sequence
analysis does not provide any information as to which membrane system
in the cell the ETR1 protein is localized. Since ethylene is diffusable
in both aqueous and lipid environments (Abeles et al., 1992),
ETR1 could theoretically be present in any membrane.
Figure 1:
ETR1 structure and expressed portions
of the sequence. A, hydrophobic, histidine kinase, and
response-regulator (RR) domains of ETR1 are indicated. For
expression of fusion proteins in E. coli, glutathione
S-transferase (GST) is shown as a blackrectangle, with expressed regions of ETR1 (UNK and HRR)
indicated by openrectangles. Regions of ETR1
expressed in yeast are indicated by openrectangles.
B, predicted transmembrane structure of ETR1 constructs
expressed in yeast.
To aid in the
analysis of ETR1, two polyclonal antibodies were raised against
different regions of the ETR1 protein by expressing those regions as
fusion proteins with glutathione S-transferase in E. coli (Fig. 1A). One antibody, designated Ab-UNK, was
generated against amino acids 165-400 of ETR1 and covers a region
of unknown function. A second antibody, designated Ab-HRR, was
generated against amino acids 401-738 of ETR1 and covers most of
the putative histidine kinase domain and the entire response regulator
domain. In an initial analysis of Arabidopsis extracts, both
antibodies were found to recognize several polypeptides in soluble and
membrane fractions. An unambiguous assignment of ETR1 to any of these
proteins was not possible, prompting us to express the full-length cDNA
in yeast. Yeast has proved to be an appropriate system for the
functional expression of eukaryotic proteins, in particular membrane
proteins that can be difficult to express in E. coli (Villalba
et al., 1992). As such, yeast could serve as a model system to
address questions concerning the biochemical properties of ETR1.
-ATPase of plants was
expressed in yeast, the protein was fully functional, but rather than
being exported to the plasma membrane, it was retained in membranes
derived from the endoplasmic reticulum (Villalba et al.,
1992). This vesicle population peaked at 32% (w/w) sucrose when
analyzed by sucrose gradient centrifugation, close to what we observed
with the ETR1 protein.
Figure 2:
Expression of ETR1 in yeast. Soluble
(S) and membrane (M) fractions were isolated from
control yeast transformed with vector alone or from yeast expressing
ETR1. Protein was reduced in 100 mM DTT, subjected to
SDS-PAGE, and ETR1 was visualized by Western blot using two antibodies
(UNK or HRR) generated against different domains of ETR1. Migration
positions of molecular mass markers are indicated in
kilodaltons.
Figure 3:
Sucrose density gradient centrifugation of
membranes from yeast expressing ETR1. Total membranes were loaded on a
linear sucrose gradient, and 0.75-ml fractions were collected after
overnight centrifugation to use for marker analysis. A,
distribution of plasma membrane H-ATPase,
mitochondrial cytochrome c (Cytc) oxidase,
and endoplasmic reticulum cytochrome c reductase as yeast
membrane markers. B, distribution of wild-type (wt)
ETR1 on gradient shown in A and mutant ETR1 (C4S, C6S) from a
second gradient run under similar
conditions.
Presence of a High Molecular Mass Species of ETR1 in
Yeast and Arabidopsis
The ETR1 protein expressed in yeast
migrated at one of two different molecular masses on SDS-PAGE,
depending upon treatment with reducing agent (Fig. 4). In the
presence of the reducing agent DTT, the immunodetectable protein
migrated at 79 kDa, while in the absence of DTT, the protein migrated
at 147 kDa.
Figure 4:
Sensitivity of ETR1 to reductant. Membrane
fractions from yeast or Arabidopsis were incubated in the
absence (-) or presence (+) of 100 mM DTT for 1 h
at 37 °C. Protein was subjected to SDS-PAGE, and ETR1 was
visualized by Western blot using antibodies UNK or HRR. Positions of
the 147- and 79-kDa species of ETR1 are
indicated.
Arabidopsis membranes were examined for ETR1
protein with the same characteristics observed in the heterologous
yeast system. In the absence of reducing agent, both domain-specific
antibodies immunodecorated a protein of 147 kDa in Arabidopsis membranes (Fig. 4). In the presence of reducing agent, the
147-kDa protein was no longer present and a new protein of 79 kDa
appeared. These immunodecorated proteins in Arabidopsis are of
identical mass to the high and low molecular mass species of ETR1
expressed in yeast. Although the UNK and HRR antibodies also
cross-reacted with additional proteins, none were recognized by both
antibodies, and these may represent unrelated proteins or proteolytic
fragments of ETR1.
Dimerization of the ETR1 Polypeptide
The
heterologous yeast system was used as a model system to further analyze
dimerization of ETR1. In addition to the full-length ETR1 protein, two
truncated versions of ETR1 were expressed in yeast (Fig. 1). One
truncated version, ETR1(1-400), covered the first 400 amino acids
of ETR1 and included the amino-terminal hydrophobic domain. The second
truncated version, ETR1(183-738), covered amino acids 183 through
to the carboxyl terminus and included the entire histidine kinase and
response regulator domains. To minimize the possibility that the high
molecular mass species of ETR1 was of artifactual origin, a simplified
extraction procedure was used to isolate protein from transformed
yeast. The cell wall of yeast was digested with -glucuronidase,
and then total protein was immediately extracted into SDS-PAGE sample
buffer. The apparent molecular mass of ETR1 proteins was determined in
the absence and presence of reducing agent (Fig. 5). Both the
full-length ETR1(1-738) and the truncated ETR1(1-400)
versions of ETR1 exhibited high and low molecular mass species,
depending upon the presence of reducing agent. In contrast, the
truncated ETR1(183-738) exhibited only one molecular mass whether
or not reducing agent was present. These results indicate that the
amino-terminal region of ETR1 is necessary and sufficient to form the
high molecular mass species. Furthermore, the difference in mass
between the high and lower molecular mass species of ETR1(1-738)
and ETR1(1-400) was in each case close in value to the mass of
the monomer, a finding consistent with homodimer formation and
inconsistent with the alternative possibility that ETR1 forms a
heterodimer with an unidentified yeast protein.
Figure 5:
Sensitivity of full-length and truncated
forms of ETR1 to reductant. Total protein was isolated from yeast
expressing full-length (amino acids 1-738) and truncated (amino
acids 1-400 and 183-738) forms of ETR1. Protein was
incubated in the absence (-) or presence (+) of 100
mM DTT for 1 h at 37 °C, subjected to SDS-PAGE, and ETR1
was visualized by Western blot using antibody UNK. Molecular masses of
the various ETR1 species are indicated based on
standards.
The sensitivity of
the ETR1 homodimer to reducing agents is indicative of a disulfide
linkage between cysteine residues. In the amino-terminal region of ETR1
there are four candidates for cysteine residues that could be involved
in the formation of disulfide bridges. The possible involvement of
these cysteine residues for homodimer formation was examined by
site-directed mutagenesis. Two cysteine residues (Cys-4 and Cys-6),
located in the amino-terminal domain predicted to lie on the
extracytoplasmic face of a membrane, were changed together to serine
residues. A third cysteine residue (Cys-65) is present in the middle of
the second transmembrane segment. The ethylene-insensitive mutant,
etr1-1, results from a change of this cysteine to a
tyrosine residue and was used for this analysis. A fourth cysteine
residue (Cys-99) is present in the middle of the third transmembrane
segment and was changed to a serine residue. When expressed in yeast,
neither single or double mutants of Cys-65 and Cys-99 abolished the
ability of ETR1 to form a covalently linked homodimer (Fig. 6).
However, a mutant of ETR1, in which the first two cysteines (Cys-4 and
Cys-6) were changed to serine residues, lost its ability to form a
covalently linked homodimer (Fig. 6). This mutation does not
affect the ability of the ETR1 protein to insert in the membrane
because membranes containing the mutant ETR1 still resolved at 35%
(w/w) sucrose when analyzed by density gradient centrifugation
(Fig. 3), like membranes containing wild-type ETR1. These results
indicate that one or both of the initial cysteine residues is necessary
for forming the disulfide-linked dimer of ETR1.
Figure 6:
Sensitivity of site-directed mutants of
ETR1 to reductant. Total yeast protein was isolated, incubated in
loading buffer for SDS-PAGE without reducing agent for 1 h at 37
°C, subjected to SDS-PAGE, and ETR1 was visualized by Western blot
using antibody UNK. Single letter amino acid codes used are C (Cys), S
(Ser), and Y (Tyr).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.