Attractant binding alters arrangement ofchemoreceptor dimers within its cluster at a cell pole
Motohiro Homma, Daisuke Shiomi, Michio Homma, and Ikuro Kawagishi
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
Edited by David J. DeRosier, Brandeis University, Waltham, MA, and approved December 9, 2003 (received for review October 14, 2003)

Many sensory systems involve multiple steps of signal amplification to produce a significant response. One such mechanism maybe the clustering of transmembrane receptors. In bacterial chemotaxis, where a stoichiometric His-Asp phosphorelay from the kinase
CheA to the response regulator CheY plays a central role, thechemoreceptors (methyl-accepting chemotaxis proteins) cluster
together with CheA and the adaptor CheW, at a pole of a rodshaped cell. This clustering led to a proposal that signal amplification occurs through an interaction between chemoreceptor homodimers. Here, by using in vivo disulfide crosslinking assays, we
examined an interdimer interaction of the aspartate chemoreceptor (Tar). Two cysteine residues were introduced into Tar: one at
the subunit interface and the other at the external surface of the
dimer. Crosslinked dimers and higher oligomers (especially a deduced hexamer) were detected and their abundance depended on
CheA and CheW. The ligand aspartate significantly reduced theamounts of higher oligomers but did not affect the polar localization of Tar-GFP. Thus, the binding of aspartate alters the rate ofcollisions between Tar dimers in assembled signaling complexes,
most likely due to a change in the relative positions or trajectoriesof the dimers. These collisions could occur within a trimer-ofdimers predicted by crystallography, or between such trimers.
These results are consistent with the proposal that the interactionof chemoreceptor dimers is involved in signal transduction.

S

ensing and responding to extracellular signals are essential
for any living cell. One of the most extensively studied
sensing systems is the chemotaxis of Escherichia coli (1–4); i.e.,
migration toward or away from chemicals. The chemotactic
signal transduction involves a His-Asp phosphate relay from thehistidine kinase CheA to the response regulator CheY that isregulated by chemoreceptors or methyl-accepting chemotaxis
proteins (MCPs). The aspartate chemoreceptor Tar has a very
low threshold concentration (⬇3 ⫻ 10⫺8 M) of L-aspartate for
an attractant response (5–7). Moreover, E. coli responds to avery small change (⬍1%) in the receptor occupancy withaspartate (8). Therefore, an input signal has to be amplified to
produce a significant response. The flagellar motor switching by
phospho-CheY is a highly cooperative event that can account for
at least some degree of signal amplification (9, 10). In addition,
recent analyses by using fluorescence resonance energy transfer(11) suggested that much of the gain occurs at the receptor endof the signaling pathway. However, its mechanism remains to beelucidated: the gain could be achieved through the MCP–MCP
interaction (12, 13) or the involvement of CheB (11, 14).
MCP forms a homodimer, regardless of its ligand occupancy
state (15). Each subunit (⬇60 kDa) consists of two transmembrane helices (TM1 and TM2), the ligand-binding domain in the
periplasm, the signaling兾adaptation domain in the cytoplasm,
and the HAMP domain, which connects TM2 with the signaling兾
adaptation domain. The cytoplasmic domain forms a stablecomplex with the histidine kinase CheA and the adaptor protein
CheW (16, 17) that forms a cluster at a cell pole (18–20), leading
to a proposal that the lateral communication between MCP
dimers in the cluster plays a critical role in signal amplification(12, 13). In fact, chemically synthesized multivalent ligands
induce attractant responses of E. coli with lower thresholds than3462–3467 兩 PNAS 兩 March 9, 2004 兩 vol. 101 兩 no. 10

corresponding monovalent ligands presumably by promoting
MCP clustering to increase sensitivity (21, 22).
A highly ordered structure of an MCP cluster has been
proposed. The cytoplasmic fragment of the serine chemoreceptor Tsr crystallizes with a unit of a trimer of dimers (23). Genetic
analyses support the putative contacts among three dimers (24).
It has been proposed that trimer units of MCP dimers assemble
into a lattice-like matrix, in which the excitation of an MCP
dimer can be propagated to other dimers through their physicalcontacts (25, 26). Nevertheless, a direct physical interaction
between MCP dimers has not been established, although Tar and
Tsr can be crosslinked with a chemical crosslinker (24).
In this study, we performed in vivo disulfide crosslinking assays
and showed that the interdimer crosslinking of Tar is sensitive tothe attractant binding. We also showed that the attractant does
not significantly alter the localization of Tar-GFP. Taken together, we suspect that the attractant may not induce a global
redistribution of Tar in a cell and that the ligand may alter thearrangement of Tar dimers within a polar cluster.
Materials and Methods
Bacterial Strains and Plasmids. RP2859 (J. S. Parkinson, personalcommunication) lacks Tap, CheB, and CheR. HCB339 (27),
HCB436 (28), and RP3098 (29) lack all four MCPs. In addition,
HCB436 lacks CheB and CheR and RP3098 lacks all Che
proteins.
The entire tar gene and its coding region were cloned into the
low-copy-number vector pWSK29 (30) and the expression vector
pBAD24 (31), yielding pOH251 and pHS5 (H. Sakamoto andI.K., unpublished work), respectively. The pBAD24 derivativewith the P15A replicon (pBAD33) (31) was used as a vector
compatible with the pBR322-based CheA兾CheW-expressing
plasmid pDV4 (32). Mutagenesis was performed essentially as
described (33).
The tar-egfp fusion was constructed by replacing the tar stopcodon by GCC (Ala; boxed) using the reverse primer:
5⬘-CGTTAGTAAATACTCG GGC AAATGTTTCC-3⬘ andigating the AvaI site (underlined) to that of the GFP-encoding
vector pEGFP (Clontech). The tar-egfp coding region was placeddownstream of the trc promoter of the vector pTrcHisC(Invitrogen).
Swarm Assay of Chemotaxis. Aliquots of fresh overnight cultures

were spotted onto tryptone semisolid agar (1% tryptone, 0.5%
NaCl, and 0.3% agar) supplemented with appropriate antibiotics, which was then incubated at 30°C.

In Vivo Disulfide Crosslinking Assay. Fresh overnight cultures werediluted with TG broth [1% tryptone, 0.5% NaCl, and 0.5%
(wt兾vol) glycerol] supplemented with appropriate antibiotics

This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: Cu-phenanthroline, Cu(II)(o-phenanthroline)3; MCP, methyl-accepting chemotaxis protein; 2ME, 2-mercaptoethanol; NEM, N-ethylmaleimide.
*To whom correspondence should be addressed. E-mail: i45406a@cc.nagoya-u.ac.jp.
© 2004 by The National Academy of Sciences of the USA

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0306660101

and兾or inducers. After vigorous shaking at 30°C for 3.5 h, cells
were harvested and suspended in SDS-loading buffer [35 mM
Tris䡠HCl (pH 6.8)兾6.7% glycerol兾1% SDS兾0.0007% bromophenol blue] supplemented with 2.5 mM N-ethylmaleimide (NEM)
and 2.5 mM EDTA. Samples were boiled and subjected tononreducing SDS兾PAGE. Immunoblotting was carried out essentially as described (34), with the anti-Tar antibody raised
against the C-terminal peptide [NH2-(C)PRLRIAEQDPNWETF-COOH] (Sawady Technology, Tokyo).
To examine the effect of an attractant, cells were washed twicewith MLM [10 mM potassium phosphate buffer (pH 7.0)兾0.1
mM EDTA兾10 mM DL-lactate兾0.1 mM methionine], resuspended in MLM, and divided into two aliquots. Aspartate or␣-methylaspartate (1 mM) was added to one of the aliquots. The
samples were incubated for 10 min at room temperature, andtreated with 200 ␮M Cu(II)(o-phenanthroline)3 (Cu-phenanthroline) supplemented with or without 1 mM aspartate or methylaspartate for 10 min. The reactions were terminated with 2.5
mM NEM and 2.5 mM EDTA. The pH values of the samples
were kept between 6.6 and 6.7 during preparation.
Fluorescence Microscopy. Microscopy was performed essentially as
described (35). Cells were grown in TG broth containing appropriate antibiotics and inducers with vigorous shaking at 30°C,
were harvested at late exponential phase, were washed twice,
and were suspended in MLM supplemented with 10 mM aspartate, 10 mM serine, or none. A small aliquot of the cellsuspension was spotted onto a slide glass coated with 0.5%
agarose and was observed under an Olympus BX50 fluorescencemicroscope. The images were recorded and processed by usinga C4742–95 digital camera (Hamamatsu Photonics, Hamamatsu
City, Japan) and IP LAB VER. 3.2 imaging software (Scanalytics,
Billerica, MA).

Results
Construction and Expression of Cys-Replaced Tar Receptors. Hazel-

Homma et al.

Fig. 1. Introduction of Cys residues into Tar. (A) Strategy for detecting aninterdimer interaction by using in vivo disulfide crosslinking. Wild-type Tar isdevoid of Cys residue. Two subunits within a Tar homodimer can becrosslinked at position 36 (Left). If another Cys is introduced at an appropriate
position on the external surface of the dimer, the double-mutant Tar protein(Right) would form a trimer, a tetramer, or higher oligomers by means ofintersubunit disulfide bonds. (B) The three-dimensional structure of theperiplasmic fragment of Tar of Salmonella typhimurium (41). (Upper) Side
view. (Lower) Top view. Residues replaced by Cys are marked. Position 143 is
Asn in S. typhimurium and Tyr in E. coli.

to that of chromosome-encoded wild-type Tar (data notshown). The Cys mutation(s), S36C, D142C, Y143C, and兾or
G144C, were introduced into these plasmids. The resulting
plasmids were introduced into strain HCB339 (⌬MCP). Transformant cells were tested for their swarming abilities intryptone semisolid agar. HCB339 cells carrying any pOH251derivative plasmid swarmed (Fig. 2B). In the presence of 200
␮M arabinose, cells carrying any pHS5-derivative plasmid also
swarmed (data not shown). These results indicate that the Cys
substitutions per se did not affect receptor function. In thefollowing experiments, we used the pOH251-derivative plasmids unless otherwise noted, but essentially similar results
were obtained with the pHS5 derivatives.
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CELL BIOLOGY

bauer and coworkers (36, 37) and Maruyama et al. (38) showedthat introduction of Cys residues into appropriate positions of achemoreceptor yields disulfides within or between its subunits invivo. The rationale of detection of intra- and interdimer disulfides of the chemoreceptors was described by Bass and Falke(39). If a Cys residue is introduced at an appropriate position of
the subunit interface within a Tar dimer, the two partner
subunits will be crosslinked. For the S36C mutant, this result has
been demonstrated (15, 40). If multiple Tar dimers are in close
contact with each other, introduction of a Cys at the externalsurface of a Tar dimer would yield a disulfide between two
subunits from neighboring dimers. Therefore, a mutant Tar
protein with both inward and outward Cys residues would forma trimer, a tetramer, or higher oligomers by means of intersubunit disulfide bonds (Fig. 1A). Based on the crystal structure of
the periplasmic fragment of Tar (41), we chose positions 142–
144, which are far from position 36 (Fig. 1B), and do not directly
involved in aspartate binding.
To prevent inappropriate crosslinking, overproduction ofthese mutant proteins should be carefully avoided. We constructed a low-copy-number plasmid carrying the tar gene with
its native promoter (pOH251) and a plasmid carrying thetar-coding region downstream of the tightly regulatablearaBAD promoter (pHS5), and introduced them into strain
HCB436, which lacks any chemoreceptor, the methyltransferase CheR, and the methylesterase CheB (⌬MCP ⌬CheRB).
Immunoblotting with anti-Tar antibody revealed that theexpression level of Tar from plasmid pOH251 was about twice
as much as that of chromosome-encoded wild-type Tar and not
more than the total amounts of chromosome-encoded Tar and
Tsr (Fig. 2A). In the presence of 200 ␮M arabinose, theexpression level of mutant Tar from plasmid pHS5 was similarFig. 3. In vivo intra- and interdimer crosslinking of the Cys-replaced Tarproteins. Whole-cell lysates of HCB436 cells carrying pWSK29 (vector), pOH251(wild-type), or its derivative (mutated residues are indicated) were subjected
to nonreducing (⫺2ME) and reducing (⫹2ME) SDS兾PAGE and immunoblottedwith anti-Tar.

Fig. 2.
Expression levels and swarming abilities of cells expressing the
mutant Tar proteins. (A) Expression levels of the wild-type Tar protein fromvarious plasmids. Whole-cell lysates were subjected to SDS兾PAGE and immunoblotting with anti-Tar. HCB436 (⌬MCP ⌬CheRB) was used as the plasmid
host unless otherwise noted. Vector, pWSK29; Tsr, the Tsr-encoding plasmid
pHS301; pOH341, the pBAD33-based plasmid (200 ␮M arabinose); pHS5, thepBAD24-based plasmid (200 ␮M arabinose); pOH251, the pWSK29-based
plasmid; and chromosome, RP2859 (⌬Tap, ⌬CheRB) cells carrying pWSK29. (B)Swarming abilities of HCB339 cells (⌬MCP) expressing the mutant Tar proteinsfrom the pWSK29-based plasmids. Overnight cultures were spotted onto
tryptone semisolid agar supplemented with 50 ␮g兾ml ampicillin, and the plate
was then incubated at 30°C for 11 h. Mutated residues are indicated below thecolonies. Vector, pWSK29.

Detection of the Intra- and Interdimer Crosslinking Between Tar
Monomers in Vivo. We examined the crosslinking of the Cys-

substituted Tar proteins expressed in HCB436 cells by immunoblotting in the absence and presence of a reducing agent
2-mercaptoethanol (2ME) (Fig. 3). To avoid crosslinking during
denaturation, the SDS-loading buffer was supplemented with 2.5
mM NEM and 2.5 mM EDTA. All of the mutants gave bands
with apparent molecular masses of ⬇60 kDa, which correspondto their monomers. In addition, Tar-S36C gave a band with anapparent molecular mass of ⬇120 kDa, and the D142C, Y143C,
and G144C proteins gave bands with apparent molecular massesof ⬇150 kDa. Moreover, Tar-S36C&D142C gave two additional
bands with higher apparent molecular masses: ⬇240 and 360
kDa when roughly estimated by extrapolating the fitting curve ofmobility of standard proteins. These additional bands may
correspond to the intradimer crosslinked dimers, the interdimer
crosslinked dimers, and the crosslinked oligomers, respectively,
because they disappeared on treatment with 2ME. In thefollowing experiments, we focused on the D142C mutation,
which caused the most efficient interdimer crosslinking.
Effect of CheA and CheW on the Intra- and Interdimer Crosslinking.

Because CheA and CheW are required for the optimal polar
clustering of the MCPs (18, 19, 42), the lack of these proteinsshould affect the interdimer crosslinking. To avoid any complexity from the formation of oligomers, we used the single
3464 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0306660101

mutants and compared the intensities of the crosslinked dimers.
Strain RP3098 was used as the host strain lacking CheA and
CheW. Because no flagellar or chemotaxis promoter including
the tar promoter is active in this strain, only the plasmids with thearaBAD promoters can be used. Tar-D142C was less effectively
crosslinked when expressed in RP3098 than in HCB436 (40–60% decrease), whereas the efficiency of crosslinking at position
36 was not significantly affected (Fig. 4A).
We then examined whether the expression of CheA and CheW
restores the interdimer crosslinking between Tar monomers in
RP3098 (Fig. 4B). Strain RP3098 carrying each pBAD33-based
plasmid (S36C or D142C) was transformed with pDV4 (CheA
and CheW) or the vector pBR322. Crosslinking at position 142
was more effective when expressed in the presence of CheA and
CheW than in their absence, whereas the efficiency of crosslinking at position 36 was not significantly affected. It should be
noted that the total amount of either mutant Tar protein was not
significantly varied. We therefore conclude that the interdimer
crosslinking of Tar is not an artifact from sample preparation,
but that it reflects a CheA兾CheW-dependent interaction of Tar
dimers in their native setting.
Detection of Higher Oligomers of Tar. To promote the formation ofhigher oligomers, we then used a catalyst for oxidation, Cuphenanthroline (Fig. 5). HCB436 cells expressing each mutant
Tar protein were treated with various concentrations of Cuphenanthroline for 10 min, followed by the addition of the stop
solution containing NEM and EDTA. Cu-phenanthroline enhanced crosslinking at position 36 and the efficiency reachedalmost 100% at 200 ␮M oxidant. Crosslinking at position 142 wasalso enhanced, but its efficiency was substantially less than thatat position 36. For the S36C&D142C mutant, four major oligomers (named A, B, C, and D) were detected, with B and C
corresponding to the 360- and 240-kDa bands described above.
To estimate their sizes, the crosslinked dimer formed in theabsence of NEM was excised from the gel, treated with Cuphenanthroline, and subjected to SDS兾PAGE again (data notshown). Two bands corresponding to oligomers B and C weredetected, which led us to a tentative assignment of theseoligomers as a hexamer and a tetramer, although other possibilities are not excluded. The deduced hexamer became predominant over other possible oligomers. This result is reminiscent of the crystal unit of a cytoplasmic fragment of Tsr thatconsists of a trimer of dimers (23). The deduced crosslinked
Homma et al.

hexamer might reflect the trimerization of MCP dimers in vivo.
Moreover, Tar-S36C&D142C was less effectively crosslinked
when expressed in strain RP3098 (⌬MCP ⌬Che), although asmall amount of the oligomer was detected in the presence of 50
␮M or higher concentrations of Cu-phenanthroline (data notshown), suggesting that CheA and CheW enhance or stabilize,
but are not prerequisite for, the receptor oligomerization.
Effect of the Ligand Aspartate on the Interdimer Crosslinking and theFormation of the Deduced Hexamer. Is the interdimer interactionand兾or the formation of the deduced hexamer of Tar involved insignaling? To address this question, we next examined whether
a ligand has any effect on crosslinking. HCB436 cells expressing
each mutant Tar protein were washed and divided into two
aliquots (see Materials and Methods). The Tar-specific attractant
aspartate was added to one of the aliquots. The samples wereincubated for 10 min at 30°C, and for further treatment with orwithout Cu-phenanthroline. Tar-D142C was slightly lesscrosslinked when treated with aspartate in the absence and thepresence of 10 ␮M Cu-phenanthroline, whereas the efficiency of
crosslinking at position 36 was not significantly influenced byaspartate at any concentration of Cu-phenanthroline (Fig. 6A).
Tar-S36C&D142C was clearly less crosslinked when treated withaspartate (Fig. 6A). Similar results were obtained with a nonmetabolizable analog, ␣-methyl-DL-aspartate (data not shown),
but the effect of methylaspartate was lost when the T154P
mutation, which abolishes aspartate binding (43), was introduced
into Tar-S36C&D142C (Fig. 6B). These results indicate that the
Homma et al.

Fig. 5. Detection of higher oligomers. (A) Effect of an oxidant on crosslinking. Cells were treated with 0 (⫺), 20 (⫹), or 200 ␮M (⫹⫹) Cu-phenanthroline
for 10 min. The reaction was terminated by the addition of the stop solution
supplemented with 2.5 mM NEM and 2.5 mM EDTA. Samples were subjected
to nonreducing SDS兾PAGE and immunoblotting with anti-Tar. Major oligomers are labeled A, B, C, and D. (B) Deduced orientation of the periplasmic
domains of Tar in the putative hexamer unit. A view from outside the cell. EachTar dimer (ellipse) is believed to be related by threefold symmetry. Filled
circles, residue 142; arrows, interactions between dimers that are influenced
by attractant binding.

aspartate binding to Tar decreases the interdimer crosslinking
and especially the formation of the higher crosslinked oligomers,
and that it may decrease the interdimer interaction within
and兾or between trimer units.
This effect of aspartate might result from its interference withpolar localization of Tar. To address this issue, GFP was fusedto the C terminus of Tar. Without induction, the fusion protein
was expressed at a level similar to that of chromosome-encodedwild-type Tar and complemented the defect of a tar mutant (datanot shown). Whereas the fluorescence signal was accumulated atpoles of HCB436 cells (CheA⫹ CheW⫹), Tar-GFP was evenly
distributed throughout the surface of RP3098 cells (CheA⫺
CheW⫺; Fig. 7). This result is consistent with the report on theeffects of CheA and CheW on the localization and clustering of
MCPs (18, 19) and on the interdimer crosslinking of Tar (results
described above), suggesting that the localization of Tar-GFP
reflects that of wild-type Tar. By contrast, aspartate (up to 10mM) did not significantly alter the localization of fluorescencesignals at cell poles (Fig. 7). This finding suggests that theligand-induced decreases in crosslinking of Tar does not result
from global redistribution of the protein in a cell. The simplest
explanation is that aspartate alters the relative positions or
trajectories of Tar dimers in assembled signaling complexes,
yielding changes in collision rates between dimers within or
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CELL BIOLOGY

Fig. 4. Effect of CheA and CheW on the intra- and interdimer crosslinking of
the Cys-replaced Tar proteins. (A) Effect of cytoplasmic Che proteins on
crosslinking. HCB436 (⌬MCP ⌬CheRB) and RP3098 (⌬MCP ⌬Che) cells expressing Tar-S36C or D142C were subjected to nonreducing SDS兾PAGE, followed byimmunoblotting with anti-Tar. (B) Effect of CheA and CheW on crosslinking.
RP3098 cells expressing Tar-S36C or D142C were transformed with pDV4 (⫹) or
pBR322 (⫺). Samples were subjected to reducing (anti-Tar**) or nonreducing(others) SDS兾PAGE, followed by immunoblotting with anti-Tar (anti-Tar**)or anti-CheA antibodies as indicated. *, chemiluminescence; **, chemical
staining.

Fig. 6. Effects of the ligand aspartate on the intra- and interdimer crosslinking of the Cys-replaced Tar proteins. (A) Effect of aspartate on crosslinking. HCB436
cells expressing Tar-S36C, Tar-D142C, or Tar-S36C&D142C were washed with MLM, incubated in MLM supplemented with 1 mM aspartate (⫹) or none (⫺) at 30°C
for 10 min, and then treated with indicated concentrations (␮M) of Cu-phenanthroline in the presence or absence of aspartate or serine at 30°C for 10 min. The
reaction was terminated on ice by the addition of stop solution containing 2.5 mM NEM and 2.5 mM EDTA. Samples were subjected to nonreducing SDS兾PAGE
and immunoblotting with anti-Tar. (B) Effect of the T154P mutation, which abolishes aspartate-binding. HCB436 cells expressing Tar-S36C&D142C or
Tar-S36C&D142C&T154P were incubated with or without 1 mM ␣-methyl-DL-aspartate (MeAsp) in the presence of 200 ␮M Cu-phenanthroline. (Upper) Oligomer
B. (Lower) Monomer.

between trimers of dimers. The possibility that the aspartate
effects arise from a slowing of subunit exchange is eliminated byour preliminary observation that aspartate increased crosslinking of some other Cys mutants (e.g., M110C; data not shown).
Discussion
Disulfide crosslinking assays have been used to examine spatial
proximity of residues in Tar (15, 40, 44), because the formationof a disulfide bond is highly sensitive to collisions between two
thiol groups (during which the proximity should be 1.9 Å or less).
This technique has also been used to probe the structures (andtheir changes on signaling) of the periplasmic domain (38, 45,46), the transmembrane domain (36, 37, 47, 48), and of thecytoplasmic domain (39, 49–51) of MCPs. Some of these studies
were carried out in vivo (36–38). Here, we extend this techniqueto probe the in vivo interaction between Tar homodimers, andthe results are consistent with the model that interdimer interactions are involved in receptor signaling.
Considering that the structure of Tar should be fairly flexible(26), crosslinking at positions 142–144 may not necessarily imply
that these positions constitute an interdimer contact site. In fact,
Mehan et al. (52) recently showed that coupling of a bulky probe
to Cys-139 that may be in direct contact with position 142 had
no effect on kinase activation, providing strong evidence that thisregion does not constitute a contact surface between receptor
dimers. Moreover, the effect of aspartate on the interdimer
interaction in the periplasmic domain might not be direct, butrather, it might be exerted through the cytoplasmic contacts (23,24, 26). A structural model for the intact MCP (26) proposes thatthe receptors in a trimer of dimers make contacts to othertrimers in their periplasmic domains. Because this periplasmic
contact is also trimeric, the deduced crosslinked hexamer could
result from the interactions between, rather than within, trimersof dimers.
This study provides information about relative orientations or
trajectories of the periplasmic domains of MCP dimers in theirpolar cluster that may be modulated by attractant binding. The
effect of aspartate on disulfide bond formation between Cys-142
residues could result from (i) slowing of subunit exchange, (ii)slowing of collisions between dimers, (iii) altered trajectories ofcollisions between dimers, or (iv) altered relative positions of the
dimers. Our preliminary observation that aspartate increased
crosslinking of some other Cys mutants, including S36C&M110C(data not shown), argues against the first two possibilities.
Therefore, we suspect that the ligand alters either the positions
or trajectories of Tar dimers within a putative trimer of dimers,
or between such trimers. The results seems to contradict theprediction of Kim et al. (26) that ligand binding simply enhances
the dynamic motions of the dimers. However, if the trimer
stability is reduced by the attractant, intratrimer crosslinking
might be reduced. In this context, it is intriguing that effects ofaspartate on the crosslinking were much more profound with the
double Cys mutant protein than with the single Cys mutant
protein (Fig. 6). The 36–36⬘ crosslink might reduce flexibility ofFig. 7. Subcellular localization of Tar-GFP in vivo. HCB436 (⌬MCP ⌬CheRB) or RP3098 (⌬MCP ⌬Che) cells carrying the plasmid encoding Tar-GFP werepreincubated in MLM supplemented with 10 mM aspartate, 10 mM serine, or nothing. Cells were spotted onto a slide glass coated with 0.5% agarose and wereobserved under a fluorescent microscope.
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Homma et al.

the receptor dimer and might restrict the 142–142⬘ crosslink
within the trimer unit. It should be noted that the double mutant
produced the 36–36⬘ crosslinked dimers efficiently and thiscrosslinking was little affected by the addition of the attractant
under the condition applied (Fig. 6).
Recent in vitro studies revealed rather low cooperativity forthe kinase regulation by MCPs (53–55). Nevertheless, in vivo
studies with fluorescence resonance energy transfer between
CheY-YFP and CheZ-CFP revealed significant degrees of signal
amplification especially in the presence of CheB (11). It should

We thank Dr. John S. Parkinson (University of Utah, Salt Lake City) for
providing strains and for critically reading the manuscript, Dr. Tohru
Umemura for providing initial experimental setup assistance, and Dr.
Toshiharu Yakushi for helpful discussions and encouragement. This
work was supported in part by grants-in-aid for scientific research fromthe Japan Society for the Promotion of Science (to D.S. and I.K.).

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CELL BIOLOGY

be possible to use our crosslinking assay to test whether receptor–receptor interactions are, indeed, important for signal
amplification.

Homma et al.

PNAS 兩 March 9, 2004 兩 vol. 101 兩 no. 10 兩 3467