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J Biol Chem, Vol. 273, Issue 46, 30110-30115, November 13, 1998
From the Division of Biological Science, Graduate School of
Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
The transmembrane domain that connects the
extracellular and intracellular domains of cell-surface receptors must
play a critical role in signal transduction. Here, we report studies of
the interaction between the transmembrane helices (TM1 and TM2) of the
Escherichia coli aspartate chemoreceptor (Tar). Tar exists
as a homodimer regardless of its state of ligand occupancy. A
particular residue substitution in TM1 (A19K) abolishes the signaling
ability of Tar. This signaling defect can be suppressed by single
residue substitutions in TM2 (W192R, A198E, V201E, and V202L). We have found that these suppressors can be divided into two groups. A198E and
V201E (class 1) almost completely suppress the defects caused by A19K,
and this suppression occurs between two subunits of the Tar dimer. In
contrast, W192R and V202L (class 2) fail to suppress some signaling
defects, and their suppression does not occur between subunits. Because
disulfide-crosslinking studies predict that residues 198 and 201 point
toward residue 19 of the partner subunit, we propose that the class 1 suppressors form an intersubunit salt bridge with Lys-19. Indeed, A19K
was suppressed by several additional aspartate or glutamate
substitutions on the same face of TM2 occupied by residues 198 and 201. None of these intersubunit salt bridges perturb signaling function,
suggesting that the mechanism of transmembrane signal propagation does
not involve large displacements (such as extensive rotation) of the TM1
and TM2 helices relative to each other.
Cell-surface receptors detect extracellular signals and convert
them into intracellular signals. Their extracellular and intracellular domains are connected by transmembrane
(TM)1 domains, which
typically consist of The aspartate chemoreceptor (Tar) of enteric bacteria is well suited
for studying the function of TM domains (for reviews, see Refs. 1-4).
Escherichia coli Tar also mediates responses to maltose by
interacting with liganded maltose-binding protein. Tar is a homodimeric
protein (5) with a subunit molecular mass of about 60 kDa. It has two
TM helices, TM1 and TM2. Unlike some homodimeric eukaryotic receptors
with tyrosine-kinase activity, Tar apparently does not undergo
monomer-dimer transitions during signaling, because some
disulfide-crosslinked dimers are fully active (5, 6). Therefore,
binding of ligands to Tar is thought to trigger a conformational change
within the receptor dimer, which must include some displacement of the
TM domains with respect to each other.
The Tar dimer forms a stable ternary complex with a homodimer of the
autophosphorylating histidine kinase CheA and two molecules of the
coupling protein CheW (7, 8). When aspartate or another attractant
ligand binds to Tar, CheA activity is inhibited. Otherwise, phosphorylated CheA transfers the phosphoryl group of the response regulator CheY, and phospho-CheY promotes clockwise rotation of the
flagellar motor and thereby causes the cell to tumble. When phospho-CheY is not bound to it, the motor rotates counterclockwise, and the cell swims smoothly.
The periplasmic, ligand-binding domain of Tar has been crystallized
both in the presence and absence of aspartate (9). Each monomer
contains four In this context, it is relevant to note that a single amino acid
substitution (A19K) in TM1 abolishes the signaling ability of Tar
without impairing the aspartate-binding ability of the receptor (16).
Many intragenic suppressors of A19K were isolated, and four of them
caused residue substitutions in TM2 (W192R, A198E, V201E, and V202L).
None of these suppressors abolish receptor function in the absence of
the original A19K mutation. Consistent with this observation, TM2
(17-19) is relatively tolerant for substitutions. TM2 of Tar can be
replaced by that of the related serine chemoreceptor (Tsr), or vice
versa, without destroying receptor function (17). On the other hand,
certain substitutions at position 204 in TM2 (I204F, I204Y, and I204W)
impair the signaling ability of Tar (18), and several other mutations
causing defects in signaling were identified in TM2 of the related
ribose-galactose chemoreceptor (Trg) (19).
In this study, we examined how mutations in TM2 suppress the TM1
mutation A19K. The original TM2 suppressors could be divided into two
groups: A198E and V201E almost completely suppress the defects caused
by A19K, whereas W192R and V202L fail to suppress some of the defects.
Moreover, the former two mutations, but not the latter two, can
suppress A19K even if they are present in the partner subunit of the
dimer. A19K can also be suppressed by the introduction of negatively
charged residues (Asp or Glu) at position 205 or Asp at position 201, suggesting that intersubunit suppression by A198E or A201E results from
formation of a salt bridge between TM1 and TM2'. This finding places
some clear constraints on the possible mechanisms of TM signaling.
Bacterial Strains and Plasmids--
All strains used in this
study are derivatives of Escherichia coli K-12. Strains
RP4372recA (F Mutagenesis of Tar--
DNA manipulations were carried out using
standard methods. Site-directed mutagenesis was performed by a two-step
polymerase chain reaction (24) using plasmid pAK101 as the template and primers synthesized by Sawadi Technology (Tokyo). Polymerase chain reaction was carried out using ExTaq polymerase (Takara Shuzo, Kyoto)
with 25 cycles of denaturing at 96 °C for 5 min, annealing at
58 °C for 30 s, and extension at 72 °C for 1 min. The
polymerase chain reaction products were cloned between the
XbaI and KpnI sites of pAK101. EcoRV
fragments (1.2 kilobases) containing the desired mutations were
introduced into pAK101 or pIT6. The DNA sequences were verified by the
dideoxy chain termination method.
Swarm Assay of Chemotaxis--
Chemotaxis was examined in
tryptone semisolid agar (TSA) (1% tryptone, 0.5% NaCl, 0.25% agar)
or minimal semisolid agar (MSA) (0.05 M potassium phosphate
buffer (pH 7.0), 1 mM MgSO4, 1 mM glycerol, 0.1 mM each of threonine, leucine, histidine,
methionine, 1 µg/ml thiamine, 1 mM
(NH4)2SO4, and 0.25% agar),
supplemented with 0.1 mM aspartic acid or maltose as
needed. Ampicillin and/or chloramphenicol were added as required.
Semisolid agar was inoculated with aliquots of exponential-phase
cultures (about 4 × 106 cells) and incubated at
30 °C.
Temporal Assay of Chemotactic Response--
Temporal-stimulation
assays were carried out essentially as described previously (25). Cells
were grown at 30 °C in TG broth (1% tryptone, 0.5% NaCl, 0.5%
(w/v) glycerol) with ampicillin and/or chloramphenicol. When necessary,
0.2% maltose was added. Cells were harvested in late exponential
phase, washed twice with motility medium (10 mM potassium
phosphate buffer (pH7.0), 0.1 mM EDTA, 0.1 mM
methionine, 10 mM sodium DL-lactate), and
resuspended in motility medium at room temperature. The swimming
pattern of the cells was observed with a dark-field microscope and
recorded on videotape. For time-course assays, the fraction of
smooth-swimming cells was determined every 30 s after the addition
of an attractant. Video images were analyzed using an Argus-10 image
processor (Hamamatsu Photonics K. K., Shizuoka). In an image
integrated for 1 s, smooth-swimming and tumbling cells gave linear
and blurred-dot traces, respectively. The smooth-swimming fraction was
defined as the percent fraction of smooth-swimming cells per total
swimming cells.
Analysis of Methylation Patterns--
Receptor methylation was
assayed as described previously (26). Cells expressing wild-type or
mutant Tar proteins were grown, harvested, and washed as described
above. A chemoeffector was added to cells suspended in motility medium,
and the suspension was then incubated at room temperature for 30 min.
The cells were collected by centrifugation and suspended in SDS-loading
buffer (67 mM Tris-HCl (pH 6.8), 8% glycerol, 1% SDS,
0.003% bromphenol blue) supplemented with 7.7% 2-mercaptoethanol.
Samples were boiled for 3 min and subjected to SDS-polyacrylamide gel
electrophoresis. Proteins were transferred onto a polyvinylidene
difluoride membrane (Millipore Japan, Tokyo) using a semidry blotting
apparatus (Biocraft, Tokyo). Anti-Tsr-T156C serum (27), which
cross-reacts with Tar, and alkaline phosphatase-conjugated goat
anti-rabbit IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD)
were used as the first and the second antibodies, respectively.
Protein-antibody complexes were visualized in 5 ml of AP buffer (0.1 M Tris-HCl (pH 9.5), 1 M NaCl, 5 mM
MgCl2) supplemented with 33 µl of nitroblue tetrazolium solution (50 mg/ml in 70% (v/v) dimethylformamide) and 16.5 µl of
5-bromo-4-chloro-3-indolylphosphate solution (50 mg/ml).
Identification of Two Classes of TM2 Suppressors of A19K--
We
characterized the Tar proteins having the A19K substitution and a TM2
suppressor on the same subunit (collectively referred to as
Tar-A19K·Sup) (Table I). These proteins
were expressed in strain KO607, which lacks all of the methyl-accepting
chemoreceptors except Aer, which mediates aerotactic responses (28,
29). The resulting transformants were tested for their swarming
ability. In TSA and MSA-aspartate (MSA-Asp), cells expressing any
Tar-A19K·Sup protein formed swarms like those of cells expressing
wild-type Tar. In MSA-maltose (MSA-Mal), however, cells expressing
Tar-A19K·W192R or V202L did not swarm well, whereas the swarms made
by cells expressing Tar-A19K·A198E or V201E were comparable with
those of cells expressing wild-type Tar.
In temporal-stimulation assays, responses to aspartate or maltose of
cells expressing any Tar-A19K·Sup were similar to those of cells
expressing wild-type Tar, and the threshold attractant concentrations
(apparent sensitivities to the attractants) were similar. However,
adaptation to maltose was impaired in cells expressing Tar-A19K·W192R
or V202L, whereas cells expressing Tar-A19K·A198E or V201E adapted
normally to maltose. In contrast, cells expressing any Tar-A19K·Sup
adapted to aspartate indistinguishably from cells expressing wild-type Tar.
These results suggest that the TM2 suppressors can be classified into
two groups. Class 1 suppressors (A198E and V201E) almost completely
suppressed the signaling and adaptation defects caused by A19K. Class 2 suppressors (W192R and V202L) did not suppress some of the defects,
notably those in adaptation to maltose.
Trans Suppression of A19K by the Class 1 Suppressors--
To
address how the TM2 substitutions suppress defects caused by A19K, we
tested their ability to suppress an A19K substitution in the partner
subunit. We reported previously that A198E suppresses A19K in
trans and used this system to ask whether a receptor dimer with
only one intact signaling domain can mediate an attractant response to
aspartate (30). Subsequently, we constructed a series of mutant Tar
proteins having one of the TM2 suppressors and the aspartate-binding
site alteration T154P. These mutant proteins (collectively referred to
as Tar-T154P·Sup) were co-expressed with Tar-A19K in strain
RP4372recA, which lacks Tar and the related chemoreceptors
Tsr and Tap (Fig. 1). Immunoblots
verified that all of the mutant proteins were expressed (data not
shown). Essentially similar results were obtained when the mutant
proteins were expressed in strain KO607.
These cells were then tested for their swarming ability in MSA-Asp
(Fig. 2) and TSA (not shown). In TSA or
MSA-Asp, neither of these Tar derivatives alone supported formation of
swarm rings, indicating that the homodimers of these Tar proteins do
not mediate an attractant response to aspartate, as expected. However,
cells expressing both Tar-A19K and Tar-T154P·A198E or V201E swarmed well in both TSA and MSA-Asp. In contrast, neither W192R nor V202L in trans could suppress the defect in swarming caused by
A19K.
We then examined the attractant responses of these cells to aspartate
directly, using the temporal-stimulation assay (Fig. 3). Again, cells expressing Tar-A19K or
any Tar-T154P·Sup protein alone did not respond to aspartate (data
not shown). However, cells expressing Tar-T154P·A198E or V201E with
Tar-A19K, did respond to aspartate. The concentration of aspartate
required for a half-maximal response was similar to that of cells
expressing wild-type Tar. However, cells co-expressing Tar-A19K and
Tar-T154P·W192R or V202L did not respond, even to 0.01 M
aspartate. These results indicate that the class 1 suppressors, but not
the class 2 suppressors, can suppress the defect in signaling caused by
the A19K substitution on the partner subunit (intersubunit
suppression).
Identification of Suppressors of A19K among Asp or Glu
Substitutions at Residues Near A198 and V201--
The class 1 (intersubunit) suppressors A198E and V201E introduce a negatively
charged residue into TM2, whereas the other suppressors introduce a
positively charged or uncharged residue. Furthermore,
disulfide-crosslinking studies of TM1 and TM2 (10, 11, 31, 32) predict
that residues 198 and 201 of one subunit face residue 19 of the partner
subunit (Fig. 4). Therefore, we propose
that intersubunit suppression results from formation of a salt bridge
between the
To test this hypothesis, we introduced Asp or Glu into positions on the
face of TM2 helix predicted to be facing the partner subunit (Fig. 4).
Immunoblots demonstrated that the mutant proteins were expressed (data
not shown). Cells expressing these proteins were tested for their
swarming ability (Fig. 5). In MSA-Asp,
they produced three types of swarms (Fig. 5). (i) Cells expressing Tar-A19K·A198E, V201D, V201E, L205D, or L205E produced swarm rings that were as sharp as although smaller than that of cells expressing wild-type Tar. (ii) Cells expressing Tar-A19K·I204D or I204E produced small and diffuse swarms. (iii) Cells expressing Tar-A19K·A198D, A208D, or A208E produced no swarm ring. Essentially, similar swarming patterns were observed in TSA (data not shown).
In temporal-stimulation assays, the first group of cells showed almost
the same threshold for aspartate as cells expressing wild-type Tar
(Fig. 6A), despite the varied
diameters of the swarms formed by the mutants. The second group of
cells did not give a significant response immediately after the
addition of aspartate (Fig. 6B). However, their
smooth-swimming fractions increased up to 30% within 30 s (data
not shown). In contrast, the third group of cells did not respond to
aspartate at all (Fig. 6C). These results demonstrate that
A19K can be suppressed by Asp or Glu substitutions for Ala-198,
Val-201, and Leu-205 (except A198D), suggesting that the basis of
suppression really is the formation of a salt bridge between Lys-19 and
an introduced negatively charged residue.
We also examined the expression levels and methylation patterns of
these mutant Tar proteins by immunoblotting (Fig.
7). Multiple methylation of a
chemoreceptor by CheR causes stepwise increases in its mobility in
SDS-polyacrylamide gel electrophoresis (33-36). All of the mutant
receptors were detected in whole cell lysates, although their amounts
and levels of methylation varied substantially. Stimulation of
methylation by the addition of aspartate was observed with receptors
that mediated responses to aspartate (Tar-A19K·A198E, V201D, V201E,
L205D, and L205E) but not with those that mediated little or no
responses to aspartate (Tar-A19K·A198D, I204D, I204E, A208D, and
A208E).
Trans Suppression of A19K by Asp or Glu Substitutions in
TM2--
We also tested for trans suppression of A19K by
the Asp or Glu substitutions. In MSA-Asp, RP4372recA cells
co-expressing Tar-A19K with Tar-T154P·V201D, L205D, or L205E produced
swarm rings comparable with those of cells expressing wild-type Tar
(Fig. 8). In contrast, cells
co-expressing Tar-A19K with Tar-T154P·A198D, I204D, I204E, A208D, or
A208E produced little or no swarm ring. Essentially similar swarming
patterns were observed in TSA (data not shown). These results
demonstrate that V201D, L205D, and L205E can suppress A19K in
trans. Thus, V201D, L205D, and L205E are also class 1 suppressors.
In this study, we examined how single amino acid substitutions in
TM2 suppress the detrimental substitution A19K in TM1. A19K does not
affect the ligand binding, but it does abolish signaling ability (16).
Our results divide the four suppressors in TM2 into two groups. The
A198E and V201E substitutions (class 1 suppressors) almost completely
reverse the defects caused by A19K, and this effect can be exerted
between the two subunits of the Tar dimer. In contrast, the W192R and
V202L substitutions (class 2 suppressors) fail to suppress some
signaling defects, and suppressions did not occur between subunits.
This finding supports the validity of the experimental design for
intersubunit suppression, because it excludes the possibility that
apparent intersubunit suppression results from compensation of smooth
swimming biased signaling of the Tar-A19K homodimer by tumbling biased
signaling of the Tar-T154P·Sup homodimer.
Disulfide-crosslinking studies predict that residues 198 and 201 of TM2
point toward residue 19 of TM1' (Lys-19') (Fig. 4). Therefore, the
The possibility of salt bridge formation might be tested by changing
ionic strength. However, such in vivo experiments should be
difficult, because E. coli cells show abnormal responses to higher salt concentrations without chemoreceptors or any other chemotactic signaling proteins (termed pseudotumbling) (37). The
hypothesis might also be examined by introducing a negative charge in
TM1 and a positive charge in TM2. Although either of these mutations
might be detrimental and suppressed by a positive charge in TM2 or a
negative charge in TM1, respectively, there is no obvious candidate for
such mutation (it should be noted that W192R is harmless).
Perhaps the most realistic way to test the salt bridge formation is to
systematically introduce Asp or Glu substitutions on the face of the
TM2 helix on which residues 198 and 201 are located. Indeed, some of
these mutations (V201D, L205D, and L205E) reversed the defects caused
by A19K, whereas others (A198D, I204D, I204E, A208D, and A208E)
produced little or no suppression. The effective suppressors result
from substitutions for residues 198, 201, and 205 that are predicted to
face Lys-19' (Fig. 4). On the other hand, substitutions that do not
suppress, with the exception of A198D, are predicted to be located
further from Lys-19'. The positively charged side chain of Lys-19
probably disturbs normal packing of the four TM helices (TM1, TM1',
TM2, and TM2'), and introduction of a negatively charged residue in
TM2' may restore this packing by creating a salt bridge between TM1 and
TM2'.
It is striking that A198D and A198E had such different effects. An Asp
residue at position 198 might not extend far enough to form a salt
bridge with Lys-19' or, if a salt bridge does form, it might affect the
receptor structure and function. Hydrophobic interaction between TM1
and TM2' might be important for the receptor architecture, and a
charge-neutralizing salt bridge might restore such interactions. A
similar salt bridge between TM helices has been implicated in the
structural stabilization of wild-type lactose permease (38, 39).
The interhelical salt bridges have another, perhaps more important,
implication for signal transduction. They would probably be maintained
upon binding and release of a small molecule-like aspartate. The
enthalpies ( Recent studies have provided good evidence that TM1 and TM1' do not
have to move relative to one another for effective TM signaling to take
place after ligand binding (10-15). Rather, vertical displacement,
tilting, or rotation of TM2 relative to TM1 seems to be the critical
element in signaling. These findings predict that ligand binding may
cause both intra- and intersubunit displacement of TM2. The multiple
interhelical salt bridges that are compatible with receptor function
argue that such a displacement cannot be too large. These small
movements of TM2, however, may trigger structural changes in the
cytoplasmic domains that are essential for signal production. Such
changes have been suggested to occur between subunits (41-45) as well
as within a subunit (or between dimers) (30, 46, 47).
The mode(s) of suppression by W192R and V202L are still unknown. These
mutations might exert their effects within a subunit or they might be
effective only when they are placed in both subunits of a dimer.
Moreover, A19K can also be suppressed by substitutions in the
cytoplasmic "linker" region, which is contiguous from TM2 (16).
Further investigation of these suppressors should help elucidate the
mechanism of receptor signaling.
We thank Dr. K. Oosawa for strains, plasmids,
and helpful discussion and Dr. M. D. Manson (Texas A & M
University) for critically reading the manuscript.
*
This work was supported in part by a grant-in-aid for
scientific research from the Ministry of Education, Science and Culture of Japan (to I. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Kowa Research Institute, Kowa Co., Ltd., 1-25-5 Kannon-dai, Tsukuba, Ibaraki 305-0856, Japan.
¶
To whom correspondence should be addressed. Tel.:
81-52-789-2993; Fax: 81-52-789-3001; E-mail:
i45406a{at}nucc.cc.nagoya-u.ac.jp.
The abbreviations used are:
TM, transmembrane; MSA, minimal semisolid agar; TM1, transmembrane region 1; TM2, transmembrane region 2; TSA, tryptone semisolid agar.
Intersubunit Interaction between Transmembrane Helices of the
Bacterial Aspartate Chemoreceptor Homodimer*
,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-helices. In addition to supporting the
molecular architecture of the proteins, these TM domains must also play
a critical role in signal transduction across the cytoplasmic membrane.
-helices (1, 2, 3, and 4), which form a
four-helix bundle. The longer helices, 1 and 4, are contiguous with TM1 and TM2, respectively, and they form a quasi four-helix bundle
with the 1' and 4' helices of their partner subunit. Recent
studies suggest that the -helical pair TM1/1-TM1'/1', which
constitutes an interface between the two subunits of the receptor
dimer, is rather static (10-15). In contrast, binding of ligand to the
receptor dimer is thought to cause a displacement (a tilt, rotation or
vertical slide) of 4/TM2 relative to the TM1/1-TM1'/1' pair.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
thi thr leu met eda
rpsL 
(tar-tap)5201 tsr-1 recA) (20) and KO607 (tsr-7021 (tar-tap)5201
trg-100 recA) (21), both of which were provided by K. Oosawa of Nagoya University, were used as the plasmid hosts in
chemotaxis assays. Strain DH5 (F
- recA1 hsdR17 endA1 gyrA96 supE44 relA1
thi-1 (argF-lacZYA)U169 
80dlacZM15) (22) was used for plasmid
construction. A pBR322-based plasmid, pAK101, carries the wild-type
tar gene (20). Its derivatives carrying tar genes
encoding Tar-A19K or Tar-A19K with suppressors (W192R, A198E, V201E,
V202L) were provided by K. Oosawa. Plasmid pIT6 was constructed by
subcloning a tar-containing DNA fragment into the plasmid
vector pSU18 (23), which contains the P15A replicon and the
chloramphenicol acetyltransferase gene.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Properties of the Tar-A19K · Sup homodimers
)
adaptation was observed.
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Fig. 1.
The strategy for intersubunit
suppression. A, a functional (+) homodimer of Tar with
A19K (star) and its suppressor (Sup) (circle);
B, co-expression of Tar-A19K and Tar-T154P·Sup. Tar-Sup is
functional, and so the mutation T154P (triangle), which
causes a severe defect in aspartate sensing, was introduced into
Tar-Sup. The function of the heterodimer of Tar-A19K and
Tar-T154P·Sup (right) can be monitored, because neither of
the homodimers (left and center) can mediate an
aspartate response ( ).
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Fig. 2.
Swarming abilities of RP4372recA
cells co-expressing Tar-A19K and Tar-T154P·Sup from compatible
plasmids. Overnight cultures were spotted onto MSA containing
ampicillin, chloramphenicol, and 0.1 mM aspartate. The
plate was incubated at 30 °C. The Tar proteins present are:
a, wild-type Tar; b, no Tar; c,
Tar-A19K; d, Tar-T154P; e, Tar-A19K + Tar-T154P;
f, Tar-A19K + Tar-T154P·W192R; g, Tar-A19K + Tar-T154P·A198E; h, Tar-A19K + Tar-T154P·V201E;
i, Tar-A19K + Tar-T154P·V202L; j,
Tar-T154P·W192R; k, Tar-T154P·A198E; l,
Tar-T154P·V201E; m, Tar-T154P·V202L.
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Fig. 3.
Aspartate-sensing abilities of heterodimers
containing Tar-A19K and Tar-T154P·Sup. Various concentrations of
aspartate were added to a suspension of cells expressing wild-type Tar
( 
) or co-expressing Tar-A19K with Tar-T154P (
), Tar-T154P·W192R
(
), Tar-T154P·A198E (
), Tar-T154P·V201E (
), or
Tar-T154P·V202L (
). After 20 s, the smooth-swimming fractions
of the cells were measured.
-amino group of Lys-19 and the 
-carboxyl group of the
suppressing Glu residue (Glu-198 or Glu-201).
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Fig. 4.
Schematic representation of the TM domains of
the Tar homodimer. The relative orientation of the putative
-helices (TM1, TM1', TM2, and TM2') is based on
disulfide-crosslinking studies (10, 11, 31, 32). The view is from the
periplasmic surface (after Ref. 10). Closed circles,
positions at which substitutions created class 1 suppressors;
gray circles, positions at which substitutions generated
class 2 suppressors; hatched circles, positions at which Asp
or Glu substitutions did not fully suppress A19K. The circle
with a black center at position 198 indicates that A198E
suppressed, whereas A198D did not.
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Fig. 5.
Suppression of A19K by Asp or Glu
substitutions in TM2. RP4372recA cells expressing each
mutant Tar were examined for their swarming ability in MSA containing
ampicillin and 0.1 mM aspartate as described in the legend
to Fig. 2. The Tar proteins present are: a, wild-type Tar;
b, no Tar; c, Tar-A19K; d,
Tar-A19K·A198D; e, Tar-A19K·V201D; f,
Tar-A19K·I204D; g, Tar-A19K·L205D; h,
Tar-A19K·A208D; i, Tar-A19K·A198E; j,
Tar-A19K·V201E; k, Tar-A19K·I204E; l,
Tar-A19K·L205E; m, Tar-A19K·A208E.
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Fig. 6.
Aspartate-sensing ability of homodimers of
Asp- or Glu-substituted Tar-A19K. Responses to aspartate mediated
by wild-type Tar ( ), Tar-A19K (), or Tar-A19K with A198D (),
A198E (
), V201D (), V201E (
), I204D (), I204E (
), L205D
(), L205E (
), A208D (
), or A208D (
) were determined as
described in the legend to Fig. 3. A, cells that showed
aspartate responses comparable with that mediated by wild-type Tar;
B, cells that showed marginal responses to aspartate;
C, cells that did not respond to aspartate.
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Fig. 7.
Expression levels and methylation patterns of
Asp- or Glu-substituted Tar-A19K proteins. RP4372recA
cells expressing wild-type or mutant Tar receptors were incubated with
(+) or without ( ) 10 mM aspartate (Asp), and
whole cell lysates were subjected to SDS-polyacrylamide gel
electrophoresis and immunoblotted with anti-Tsr serum. None,
no Tar; WT, wild-type Tar; A19K, Tar-A19K;
A198D etc., Tar-A19K·A198D etc.;
Nonsp., a band cross-reacting nonspecifically with the
serum.
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Fig. 8.
Trans suppression of A19K by Asp or Glu
substitutions in TM2. RP4372recA cells co-expressing
Tar-A19K and Tar-T154P with an Asp or Glu substitution from compatible
plasmids were examined for their swarming ability as described in the
legend to Fig. 2. The Tar proteins present are: a, wild-type
Tar; b, no Tar; c, Tar-A19K; d,
Tar-T154P; e, Tar-A19K + Tar-T154P; f, Tar-A19K + Tar-T154P·A198D; g, Tar-A19K + Tar-T154P·V201D;
h, Tar-A19K + Tar-T154P·I204D; i, Tar-A19K + Tar-T154P·L205D; j, Tar-A19K + Tar-T154P·A208D;
k, Tar-A19K + Tar-T154P·A198E; l, Tar-A19K + Tar-T154P·V201E; m, Tar-A19K + Tar-T154P·I204E;
n, Tar-A19K + Tar-T154P·L205E; o, Tar-A19K + Tar-T154P·A208E; p, Tar-T154P·A198D; q,
Tar-T154P·V201D; r, Tar-T154P·I204D; s,
Tar-T154P·L205D; t, Tar-T154P·A208D; u,
Tar-T154P·A198E; v, Tar-T154P·V201E; w,
Tar-T154P·I204E; x, Tar-T154P·L205E; y,
Tar-T154P·A208E.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-carboxyl groups of the glutamate residues of the class 1 suppressors are likely to form an intersubunit salt bridge with the
-amino group of Lys-19'. Consistent with this, all of the same-site
pseudorevertants isolated from A19K are to introduce uncharged residues
(Ile, Thr, and Gln) (16).
H) of formation of the various salt bridges
cannot be calculated precisely, but they should be larger than the
enthalpy (18 kcal/mol) reported for serine binding to Tsr (40).
Therefore, the salt bridges presumably restrict the potential ability
of TM1 and TM2' to move relative to one another during the
signal-transduction cycle.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Dept. of Bacteriology, Institute of Medical
Science, University of Tokyo, Minato-ku, Tokyo 108-0071, Japan.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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