THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 44, Issue of November 1, pp. 42325–42333, 2002
Printed in U.S.A.

Dual Recognition of the Bacterial Chemoreceptor by
Chemotaxis-specific Domains of the CheR Methyltransferase*
Received for publication, February 28, 2002, and in revised form, July 2, 2002
Published, JBC Papers in Press, July 5, 2002, DOI 10.1074/jbc.M202001200

Daisuke Shiomi‡, Igor B. Zhulin§, Michio Homma‡, and Ikuro Kawagishi‡¶
From the ‡Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602,
Japan, and the §School of Biology, Georgia Institute of Technology, Biology Department, Atlanta, Georgia 30332-0230

Adaptation to persisting stimulation is required forhighly sensitive detection of temporal changes of stimuli, and often involves covalent modification of receptors. Therefore, it is of vital importance to understandhow a receptor and its cognate modifying enzyme(s)modulate each other through specific protein-protein
interactions. In the chemotaxis of Escherichia coli,
adaptation requires methylation of chemoreceptors (e.g.
Tar) catalyzed by the CheR methyltransferase. CheR
binds to the C-terminal NWETF sequence of a chemoreceptor that is distinct from the methylation sites. However, little is known about how CheR recognizes its
methylation sites or how it is distributed in a cell. In thisstudy, we used comparative genomics to demonstratethat the CheR chemotaxis methyltransferase contains
three structurally and functionally distinct modules: (i)the catalytic domain common to a methyltransferase
superfamily; (ii) the N-terminal domain; and (iii) the␤-subdomain of the catalytic domain, both of which arefound exclusively in chemotaxis methyltransferases.
The only evolutionary conserved motif specific to CheR
is the positively charged face of helix ␣2 in the N-terminal domain. The disulfide cross-linking analysis suggested that this face interacts with the methylation helix of Tar. We also demonstrated that CheR localizes toreceptor clusters at cell poles via interaction of the␤-subdomain with the NWETF sequence. Thus, the two
chemotaxis-specific modules of CheR interact with distinct regions of the chemoreceptor for targeting to thereceptor cluster and for recognition of the substratesites, respectively.

In many sensory systems, transmembrane receptors recognize extracellular stimuli and transduce them into cytoplasmic
signals to trigger defined physiological responses. These initial
responses often diminish during persisting stimulation. The
latter process, termed adaptation or desensitization, is essential for the detection of temporal changes of stimuli and/or thehighly sensitive detection of stimuli over a comprehensive
range. Covalent modifications of a receptor are often required
for adaptation. In such cases, it is of vital importance to understand a regulated interplay between a receptor and modifying enzyme(s), including their mutual recognition and their* This work was supported in part by grants-in-aid for scientificresearch from the Japan Society for the Promotion of Science (to D. S.
and I. K.) and by start-up funds from Georgia Institute of Technology (toI. B. Z). The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 81-52-7892993; Fax: 81-52-789-3001; E-mail: i45406a@cc.nagoya-u.ac.jp.
This paper is available on line at http://www.jbc.org

subcellular localization, that assures spatially and temporallyorganized information processing.
Molecular mechanisms of adaptation have been well characterized in the chemotaxis of Escherichia coli and Salmonella
typhimurium (1– 6). The transmembrane chemoreceptors, also
known as the methyl-accepting chemotaxis proteins (MCPs),1
are methylated by the S-adenosylmethionine (AdoMet)dependent methyltransferase (MTase) CheR and demethylatedby the methylesterase CheB. In the resting state, an MCP is inequilibrium between methylation and demethylation. An attractant shifts the equilibrium toward methylation and a repellent toward demethylation. Each MCP has 4 –5 glutamate
residues located in two separate ␣ helices (the first and second
methylation helices (MH1 and MH2)) in the cytoplasmic domain (7, 8). CheR has to recognize these residues (i.e. thesubstrate sites), but this interaction between CheR and an
MCP has not been detected to date probably because it is weak
and/or transient.
In contrast, binding of CheR to the C-terminal pentapeptide
sequence (NWETF) of the two high abundance receptors, theserine chemoreceptor Tsr and the aspartate chemoreceptor
Tar, has been well characterized (9 –12). However, the low
abundance chemoreceptors (the ribose-galactose transducer
Trg and the dipeptide transducer Tap) do not have the sequence, indicating that the binding of CheR to the NWETF
sequence might not be essential for its catalytic activity itself.
The three-dimensional structure of CheR of S. typhimurium
revealed that the monomeric protein consists of two domains(the N-terminal domain with no assigned function and theMTase domain) and one subdomain (the ␤-subdomain) (13).
The co-crystal of CheR and the pentapeptide revealed that the␤-subdomain binds to the NWETF sequence (11). Mutagenesisof the NWETF sequence of Tar demonstrated that it binds to
CheR mainly through hydrophobic interaction (12). However, it
is not clear how the CheR molecule is oriented when it binds toa chemoreceptor, nor how the NWETF sequence is oriented
relative to the other part of the chemoreceptor molecule.
Recently, subcellular localization of some proteins involvedin chemotactic signal transduction (MCPs and Che proteins)
has been studied using immunoelectron and immunofluorescence microscopy and YFP fusion proteins (14 –18). These studies demonstrated that MCPs cluster with the histidine kinase
CheA and the adaptor protein CheW at cell poles. The localization and the clustering depend, at least to some extent, on
CheA and CheW, but not on CheR or CheB. The localizationand clustering of the chemotactic machinery at cell poles areproposed to be essential for amplification of input signals and
1
The abbreviations used are: MCP, methyl-accepting chemotaxis
protein; AdoMet, S-adenosylmethionine; GFP, green fluorescent protein; MBP, maltose-binding protein; MH, methylation helix; MTase,methyltransferase.

42325

42326

Receptor Recognition by Methyltransferase

for efficient methylation. The latter hypothesis assumes a highlocal concentration of CheR around the receptor/kinase cluster
to provide a molecular basis of efficient methylation of both
high abundance and low abundance receptors. Previous studies(10, 12, 19 –21) suggest that the NWETF sequence may serve toconcentrate CheR around MCPs, but no direct evidence has
been obtained.
Moreover, the information about the structure-function relation of CheR was limited although the three-dimensional structure of S. typhimurium CheR has been determined in theabsence and presence of the NWETF peptide (11, 13) and themutagenesis of the cysteine residues of S. typhimurium CheR
(22) was carried out. In this study, we took advantage of comparative genomic analysis to identify evolutionary conserved
and therefore structurally and functionally important residues
in the CheR protein family. Mutagenesis of some conserved
residues in E. coli CheR demonstrated that some of them arefunctionally important. Characterization of GFP-CheR revealed that CheR localizes to cell poles through the interaction
between its ␤-subdomain and the NWETF sequence of thechemoreceptor. Disulfide cross-linking assay was employed to
examine the interaction between CheR and Tar and demonstrated that the positively charged residues in helix ␣2 of CheR
are involved in the recognition of MH1. Thus, CheR interacts
with the chemoreceptor through two distinct chemotaxis-specific modules to achieve efficient adaptation.
EXPERIMENTAL PROCEDURES

Data Base Searches and Protein Sequence Analysis—BLAST
searches (23) of nonredundant and individual microbial genomes databases at the National Center for Biotechnology Information (Bethesda,
MD) were performed with default parameters. Position-specific iterative BLAST searches (23) were performed with defined parameters(BLOSUM62 matrix, an inclusion threshold of E ⫽ 0.01 and composition based statistics). Searches were iterated to convergence and repeated with all newly found homologues as queries. CheR and relatedsequences from unfinished microbial genomes were identified intBLASTn searches at www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi followed by gene finding and translation using the FramePlot
program at www.nih.go.jp/⬃jun/cgi-bin/frameplot.pl. Profile hiddenMarkov-model searches against SMART (24) and Pfam (25) data bases
were performed with default parameters. Multiple alignments wereconstructed using the ClustalX program (26). Visualization of the threedimensional structure of CheR was achieved using a Swiss-PDB viewer(27).
Bacterial Strains and Plasmids—All strains used in this work arederivatives of E. coli K12. Strain RP437 is wild-type for chemotaxis (28).
Strain RP4944 (⌬cheR his pyrC46 thyA araD139 ⌬lac-U169 nalA rpsLthi) lacks CheR,2 and RP8691 (⌬tsr-7021 zec::Tn10 ⌬cheR leuB6 his-4
metF159(Am) eda-50 rpsL163 thi-1 ara-14 lacY1 mtl-1 xyl-5 tonA31
tsx-78) lacks CheR and Tsr (29). Strain HCB436 (⌬tsr-7021 ⌬(tarcheB)2234 ⌬trg-100 zbd::Tn5 thr leu his met rpsL136) (30) lacks CheB
and CheR as well as MCPs, and strain HCB1262 (⌬(cheA-cheZ)::zeoR
thr leu his met rpsL136)3 lacks all of the Che proteins involved ingeneral chemotaxis as well as MCPs.
Plasmids used in this study are listed in Table I. Plasmid pTrcHisB(Invitrogen) carries the trc promoter, the lacIq and bla genes. PlasmidpBAD24 (31), which was provided by J. Beckwith, carries the araBAD
promoter, the araC gene encoding the positive and negative regulator,
and the bla gene. Plasmid pMAL-c2 (New England Biolabs) carries themutant malE gene, which encodes the mature maltose-binding protein(MBP) without its leader sequence, under the control of the tac promoter, the lacIq gene, and the bla gene. Plasmid pACYC184 and its
derivative pSU21 (32) carry the cat gene. The pSU21-based plasmid,
pDS2 (12), carries the wild-type cheR gene. The pACYC184-based plasmid, pLC113 (33), which was provided by J. S. Parkinson, carries thewild-type tar gene under control of the nahG promoter. Plasmid pEGFP(CLONTECH) encodes the enhanced green fluorescent protein.
Site-directed mutagenesis of the cheR and tar genes was performed
according to the two-step PCR method (34) using primers synthesized
2
3

J. S. Parkinson, personal communication.
H. C. Berg, personal communication.

by Life Tech Oriental (Tokyo) and Pyrobest DNA polymerase (Takara
Shuzo, Kyoto). For the tight regulation of CheR proteins (i.e. CheR,
His6-CheR, and GFP-CheR proteins), the coding regions were placeddownstream of the araBAD promoter on vector pBAD24. For the expression of MBP-Tar, the 3⬘ region of tar (encoding the cytoplasmic
domain, i.e. residues 215–553) was introduced into the vector pMAL-c2.
Swarm Assay of Chemotaxis—Swarm assays were performed essentially as described previously (10) using tryptone semisolid agar supplemented with appropriate antibiotics and, when necessary, with various concentrations of arabinose and fucose.
Analysis of Receptor Methylation by Immunoblotting—Receptor
methylation was monitored by immunoblotting as described previously(10) with anti-Tsr serum (38), which cross-reacts with Tar, and alkaline
phosphatase-conjugated anti-rabbit IgG (Vector Laboratories) as thefirst and the second antibodies (at the dilution of 1:3000).
Observation of Subcellular Localization of GFP-CheR—HCB436 cells
carrying pLC113 encoding wild-type Tar, pLC113-W550Op encoding
Tar-N*, or the vector pACYC184 were further transformed with
pBR322-based plasmids encoding the GFP-CheR fusion proteins. Cells
were grown in TG medium containing appropriate antibiotics and inducers with vigorous shaking at 30 °C, harvested at late exponential
phase, washed twice with MLM (10 mM potassium phosphate buffer(pH 7.0), 0.1 mM EDTA, 10 mM DL-lactate, 0.1 mM methionine), andresuspended in MLM. A small aliquot of the cell suspension was spotted
onto slide glasses coated with 0.5% agarose and was observed under afluorescence microscope (Olympus, BX50). The images were recorded
and processed by using a digital camera (Hamamatsu Photonics,
C4742-95) and imaging software (Scanalytics, IP Lab version 3.2).
Disulfide Cross-linking Assay—HCB1262 cells expressing any of theHis6-tagged CheR proteins were suspended in 2 ml of lysis buffer (50mM NaH2PO4 (pH 8.0), 300 mM NaCl, and 10 mM imidazole) containing
10 mM dithiothreitol, and sonicated in short pulses (10 s each) on ice.
After low speed (17,000 ⫻ g at 4 °C for 10 min) and high speed(100,000 ⫻ g at 4 °C for 30 min) centrifugation, the resulting supernatant was applied to a nickel-nitrilotriacetic acid-agarose column (Qiagen). Each His6-tagged CheR protein was eluted with 250 mM imidazole. HCB1262 cells expressing MBP-Tar-E308C were suspended in 2 mlof column buffer (20 mM Tris-HCl (pH 7.4), 200 mM NaCl, and 1 mM
EDTA) containing 10 mM dithiothreitol, and sonicated in short pulses
on ice. After low speed and high speed centrifugation, the supernatant
was applied to the amylose column (New England BioLabs). MBP-TarE308C was eluted with 10 mM maltose. Each His6-tagged CheR protein
and MBP-Tar-E308C were mixed and incubated at 37 °C for 20 min in
the presence or absence of 0.1 mM I2. The mixture was divided into two
aliquots, which were boiled at 100 °C for 3 min with or without 2-mercaptoethanol and were subjected to SDS-PAGE followed by immunoblotting with anti-CheR serum (12), at a dilution of 1:2000, or anti-MBP
serum (New England BioLabs) at a dilution of 1:10000. The first antibodies were detected with horseradish peroxidase-conjugated anti-rabbit IgG (New England BioLabs) at the dilution of 1:3000 and with an
ECL detection kit (Amersham Biosciences).
RESULTS

Comparative Protein Sequence Analysis of the CheR Protein—Initial analysis of the domain architecture of the CheR
protein identified not only homologous CheR proteins from avariety of species, but also several types of multidomain proteins with a core domain corresponding to that of CheR. (Fig 1).
Position-specific iterative BLAST searches with the N-terminal
domain (residues 1–90) of S. typhimurium CheR (13) retrieved
only corresponding domains from homologous CheR proteinsfrom bacterial and archaeal species, indicating that this domain is present exclusively in chemotaxis-related MTases(Figs. 1 and 2A). Searches with the catalytic domain (residues
91–286) retrieved sequences of nonchemotactic AdoMetdependent MTases, including DNA MTases and rRNA dimethyltransferases, from both prokaryotes and eukaryotes (Figs. 1
and 2B): hundreds of functionally divergent MTases were identified in the third and forth iterations with a high degree ofstatistical significance (expectation value E ranged from 10⫺4
to 10⫺8).
Domain organization of all types of MTases retrieved during
the data base searches is shown in Fig. 1. FrzF of Myxococcus
xanthus, a known functional CheR homolog (39), contains sev-

Receptor Recognition by Methyltransferase

42327

TABLE I
Plasmids used in this study
PlasmidpBAD24
pSU21
pDS2
pDS80
pDS81A
pDS81E
pDS82
pDS83A
pDS83E
pDS84
pDS85A
pDS85E
pDS86
pDS87
pDS88A
pDS88E
pDS89
pDS90
pDS91
pDS92
pDS93
pDS94
pDS200pDS201
pDS202pDS203pDS204pDS205pDS206pDS207pDS208pDS209pDS210pDS220pDS221
pDS222
pDS223
pDS224
pLC113pDS1015
pDS850pDS851
pMAL-c2
pEGFP
pTrcHisB

Relevant phenotype

CheR
CheR
CheR-K46A
CheR-K46E
CheR-V50A
CheR-R53A
CheR-R53E
CheR-L54A
CheR-R57A
CheR-R57E
CheR-R98A
CheR-D154A
CheR-R187A
CheR-R187E
CheR-H192A
CheR-R197A
CheR-H192A/R197A
CheR-C7S
CheR-C229S
CheR-C7S/C229S (CheR-CS)
His6-CheR-CS
His6-CheR-CS-K46C
His6-CheR-CS-R53C
His6-CheR-CS-L54C
His6-CheR-CS-L55C
His6-CheR-CS-R56C
His6-CheR-CS-R57C
His6-CheR-CS-L58C
His6-CheR-CS-R59C
His6-CheR-CS-S60C
His6-CheR-CS-K46C/R53A
GFP
GFP-CheR
GFP-CheR-R53A
GFP-CheR-D154A
GFP-CheR-H192A/R197A
Tar
Tar-W550Op (Tar-N*)
MBP-Tar-EEEE-E308C
MBP-Tar-QQQQ-E308C

FIG. 1. Domain architecture of CheR and related MTases. Domain architecture of the MTase superfamily was determined by position-specific iterative BLAST and profile hidden-Markov-model based
searches. Abbreviations: NCheR, the N-terminal domain found exclusively in chemotaxis MTases; CheW, a domain homologous to the CheW
docking protein (43); T, the tetratrico peptide repeat (40); PAS, the PAS
domain (42). Green oval depicts the MTase (catalytic) domain and the
purple oval depicts the methylesterase (MEase (catalytic) domain. Red
rectangle within the MTase domain corresponds to the ␤-subdomain of
the CheR protein. Species abbreviations are the same as in a complete
list of abbreviations shown in Fig. 2.

eral tetratrico peptide repeats in its C terminus. Tetratrico
peptide repeats is thought to facilitate protein-protein interactions in various signal transduction proteins (40, 41). An unusual fusion protein containing catalytic domains of methyles-

Vector

pSU21
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24pACYC184
pACYC184
pMAL-c2
pMAL-c2

Source

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New England Biolabs
Clontech
Invitrogenterase and MTase as well as several PAS domains that may
also facilitate protein-protein interactions (42) have been identified in several proteobacterial species, e.g. in Sinorhizobium
meliloti (Fig. 1). Finally, a CheR-like protein from spirochetes
lacks the N-terminal domain of a classical CheR, but instead
has a CheW domain in its N terminus. The CheW protein is
known to directly interact with MCPs (43).
Multiple sequence alignments of the N-terminal CheR-specific domains (Fig. 2A) revealed a conservation pattern centered on hydrophobic and turn-like residues comprising ␣-helices. Residue Gly-39, which determines a crucial turn following
helix ␣1, is strictly conserved in all CheR homologs. The only
two other positions where strict conservation occurs are positively charged residues Lys/Arg-46 and Arg-53 of helix ␣2. Suchstrict conservation of a positive charge across long evolutionary
distances clearly indicates functional significance, especiallyfor Arg-53.
Catalytic domains from most AdoMet-dependent MTases,
including CheR, share a signature sequence (G/A)X(G/A/S)XG(Fig. 2B) involved in the binding of AdoMet (44). Interestingly,
the ␤-subdomain is present in all CheR homologs and CheRrelated proteins, such as PilK, FrzF, CheW3, and Smb20515,
but is missing from all nonchemotactic single-domain MTases,
such as COQ3 (Figs. 1 and 2B). This finding is consistent withthe functional role of the ␤-subdomain, i.e. interaction with

42328

Receptor Recognition by Methyltransferase

FIG. 2. Multiple sequence alignments of the CheR-specific N-terminal domain (A) and a portion of the MTase (catalytic) domain
including the ␤-subdomain (B). Each sequence is identified by the NCBI gene identification number or by the sequencing center preliminary
identification number (shown in the last column). The secondary structure (E for ␤-sheets and H for ␣-helices) shown above the alignments is basedon the known three-dimensional structure of S. typhimurium CheR. The signature sequence involved in binding to AdoMet (44) is indicated bythree stars, and a critical histidine residue (His-192) in the ␤-subdomain is highlighted in black (panel B). Conserved amino acid residues areReceptor Recognition by Methyltransferase

FIG. 3. A, swarming abilities of RP4944 cells expressing mutant
CheR proteins. Aliquots (2 ␮l each) from fresh overnight cultures werespotted onto tryptone semi-solid agar (0.3%) supplemented with 50
␮g/ml ampicillin, 0.1 mM arabinose (the inducer), and 0.1 mM fucose(the anti-inducer) and then the plate was incubated at 30 °C for 8 h. The
swarm sizes of the three different colonies were measured for eachmutant. The swarm diameter of cells carrying the vector pBAD24(none) was subtracted from those of cells expressing each mutant CheR
to obtain net swarm sizes. The relative swarm diameter was defined as
the net swarm size normalized to that of cells expressing wild-type
CheR. B, methylation levels of Tar co-expressed with each mutant
CheR. Methylation of a receptor protein increases its mobility in SDSPAGE and therefore can be detected by immunoblotting with anti-Tsr
serum. The lower band is of an unidentified protein unrelated to Tar.

MCPs (11). CheR proteins from all chemotactic species retainthe ␤-subdomain, although in many species all MCPs lack the
NWETF-type sequence (data not shown). On the other hand,residues His-192 and Arg-197 of S. typhimurium CheR, whichinteract with the Trp residue of the NWETF pentapeptide (11),and residue Arg-187, which forms a salt bridge with the Glu
residue of the pentapeptide (11), are absent from many CheR
homologs (Fig. 2B).
Mutagenesis of Some Conserved Residues in E. coli
CheR—We replaced some conserved residues of E. coli CheR by
Ala or Glu. The resulting mutant proteins were expressed instrain RP4944 (⌬CheR) at levels similar to that of wild-type
CheR (data not shown). Swarming abilities of these transformants were examined (Fig. 3A). RP4944 cells expressing either
MTase domain mutants (R98A or D154A) failed to swarm, as
expected. Among the ␤-subdomain mutations, H192A, R197A,
and H192A/R197A significantly slowed the swarming rate. The
defects in swarming were suppressed by the overproduction ofthese three mutant CheR proteins (data not shown). Thus,
these three mutations may decrease the affinity of CheR for an

42329

MCP, consistent with the crystallographic study (11), whichsuggested that His-192 and Arg-197 interact with the Trp
residue of the pentapeptide sequence. In contrast, R187A andR187E did not affect the ability of CheR to support swarming.
Residue Arg-187 is suggested to form a salt bridge with the Glu
residue of the NWETF sequence (11), but the E551A mutationof E. coli Tar had little effect on its methylation (12). Taken
together, the salt bridge, if it is formed, may not play a criticalrole in the CheR function in vivo. Among the N-terminal domain mutations, V50A and L54A had no effect, but R53A and⌬1– 89, in which residues 1– 89 were deleted, impaired swarming, fully supporting predictions that resulted from our in silico
analysis.
Next, we examined methylation levels of Tar in RP8691(⌬Tsr ⌬CheR) cells expressing the mutant CheR proteins byimmunoblotting with anti-Tsr, which cross-reacts with Tar(Fig. 3B). In RP8691 cells carrying the vector, methylation of
Tar was hardly detected in the absence of aspartate but wasslightly enhanced by the addition of aspartate, suggesting thatthe host strain has a residual activity of CheR. In the absenceof aspartate, the methylation level of Tar in RP8691 cells
expressing wild-type CheR was not much different from that ofcells carrying the vector, but was greatly increased by theaddition of aspartate, as expected. The MTase domain mutant(CheR-D154A) did not show any methylating activity, nor did
the N-terminal domain mutant (CheR-R53A). Among the␤-subdomain mutants, CheR-R197A and CheR-H192A/R197A
were slightly impaired in the methylating activity. These results are consistent with those of the swarming assay (Fig. 3A).
Subcellular Localization of GFP-CheR—Previous studies
demonstrated that MCPs together with CheA and CheW form
clusters and localize to cell poles (14, 16, 17), and that CheY
and CheZ also localize to cell poles in the presence of MCPs
(18). CheR may also target to the receptor/kinase cluster. To
visualize subcellular localization of CheR, we constructed aplasmid encoding GFP-CheR. Swarming ability of RP4944 cells
expressing GFP-CheR was similar to those of cells expressing
CheR (data not shown). We then examined subcellular localization of GFP-CheR in the presence of 1 mM arabinose (Fig. 4).
With the lower concentrations of arabinose, the fluorescencebleached quickly, but arabinose concentration did not seem toaffect localization of GFP-CheR (data not shown). In HCB436(⌬MCPs ⌬CheR ⌬CheB) cells, GFP-CheR localized to cell polesin the presence of wild-type Tar, but not in the presence of themutant Tar protein lacking the C-terminal NWETF sequence(Fig. 4, left panels). These results demonstrate that CheR istargeted to the MCP cluster through its binding to the NWETF
sequence.
We next examined whether mutations in the CheR part of
GFP-CheR affect its localization. The three severe mutations
characterized above (R53A, D154A, and H192A/R197A), each
representing one of the three domains, were tested. GFP-CheR
with the D154A mutation (in the catalytic domain) localized tocell poles (Fig. 4, right middle panel) although the D154A
mutation impairs the MTase activity (see Fig. 3, A and B).

colored according to 90 (A) or 100% (B) consensus (shown below alignments): polar (p, KRHEDQNST) in red; hydrophobic (h, LIVMYFW) and thealiphatic subset (l, ILV) with yellow background; turn-like (t, ACDEGHKNQRST) in green; small (s, ACDGNPSTV) in blue; tiny (u, AGS) in whitewith blue background; charged (c, DEHKR) and the positively charged subset of these (⫹, HKR) in white with red background. The species
abbreviations are: Aful, Archaeoglobus fulgidis; Atha, Arabidopsis thaliana; Atum, Agrobacterium tumefaciens; Bbro, Bordetella bronchiseptica;
Bbur, Borrelia burgdorferi; Bcep, Burkholderia cepacia; Bhal, Bacillus halodurans; Bste, Bacillus stearothermophilus; Bsub, B. subtilis; Cace,
Clostridium acetobutylicum; Cbot, Clostridium botulinum; Ccre, Caulobacter crescentus; Cele, Caenorhabditis elegans; Cjej, C. jejuni; Drad,
Deinococcus radiodurans; Ecol, E. coli; Eclo, Enterobacter cloaceae; Lino, Listeria innocua; Lmon, Listeria monocytogenes; Magn, Magnetococcus
sp.; Mxan, M. xanthus; Neur, Nitrosomonas europeae; Paby, Pyrococcus abyssi; Paer, Pseudomonas aeruginosa; Phor, Pyrococcus horikoshii; Reut,
Ralstonia eutropha; Rcen, Rhodospirillum centenum; Rsph, Rhodobacter sphaeroides; Save, Streptomyces avermitilis; Smel, S. meliloti; Spom,
Schizosaccharomyces pombe; Sput, Shewanella putrefaciens; Styp, Salmonella enterica serovar Typhimurium; Tmar, Thermotoga maritima; Tpal,
Treponema pallidum; Vang, Vibrio anguillarum; Vcho, V. cholerae; Vpar, Vibrio parahemeolyticus; Ypes, Yersinia pestis.

42330Receptor Recognition by Methyltransferase
TABLE II
Relative swarm diameter of RP4944 (⌬CheR) cells expressing eachmutant CheR protein with an Ala or Glu substitution in helix ␣2
Swarming was examined with tryptone semi-solid agar (0.3%) supplemented with 50 ␮g/ml Ap and the indicated concentrations of arabinose (ara) and fucose (fuc). The relative swarm diameter was normalized to that of cells expressing wild-type CheR in the presence of 0.1 mMarabinose and 0.1 mM fucose.
[Ara], [Fuc] (mM)
Mutant

None
WT
K46A
K46E
R53A
R53E
R57A
R57E

FIG. 4. Subcellular localization of the wild-type and mutant
versions of GFP-CheR. The fusion proteins were expressed in
HCB436 cells carrying the vector plasmid (none) or the plasmid encoding wild-type (NWETF) or the truncated (N*) Tar protein. Sodium
salicylate (0.1 ␮M) and arabinose (1 mM) were added to induce theexpression of Tar and GFP-CheR, respectively. The images are shownfor cells expressing wild-type GFP-CheR in the presence of wild-type
Tar (Tar) or Tar-W550Op (Tar-N*) and in the absence of Tar (none) ora mutant version of GFP-CheR with the R53A mutation (the N-terminal
domain), the D154A mutation (the catalytic domain), or the H192A/
R197A mutations (the ␤-subdomain) in the presence of wild-type Tar.

GFP-CheR with the H192A/R197A mutation (in the ␤-subdomain) did not localize to cell poles (Fig. 4, right lower panel),
supporting the previous conclusion that the mutation may decrease the affinity of CheR for the NWETF sequence. Interestingly, GFP-CheR with the R53A mutation (in the N-terminal
domain) localized to cell poles (Fig. 4, right upper panel), although the mutation severely impaired the CheR activity (Fig.
3, A and B). These results suggest that the targeting of CheR tocell poles does not depend on the MTase activity and the function of the N-terminal domain of CheR, but depends primarilyon the binding of the ␤-subdomain of CheR to the C-terminal
NWETF sequence of high abundance MCPs.
Mutagenesis of Positively Charged Residues in Helix ␣2 of
CheR—The R53A mutation severely impaired the CheR function without affecting its subcellular localization. However,cells expressing CheR-R53A produced larger swarm with increasing levels of expression (Table II), raising a possibility
that residue Arg-53 is not directly involved in the catalytic
activity. Helix ␣2 of CheR that contains Arg-53 has a face withmany positively charged residues (Fig. 5, left) that constitutes
a part of the “receptor interaction opening” together with theactive center and the ␤-subdomain (13). On the other hand, thefirst methylation helix (MH1) of Tar that contains three ofthe four methylation sites has many residues with negatively
charged or polar side chains (Fig. 5, right). Therefore, it isreasonable to speculate that the positively charged face of helix
␣2 may interact with the negatively charged face of MH1. To
examine this possibility, we substituted Ala or Glu for some of0, 00.1, 0.1

1, 0

5, 0

10, 0

0
0
0
0
0
0
0
0

0
1.00
0.16
0
0
0
1.18
1.18

0
0.94
0.73
0
0.24
0
1.33
1.33

0
0.22
0.64
0
0.59
0
1.50
1.27

0
0.18
0.52
0
0.54
0
1.35
1.20

FIG. 5. Helical wheel presentations of helix ␣2 of CheR (left)
and the first methylation helix (MH1) of Tar (right). Positively
charged residues of CheR and the methylation sites of Tar are highlighted by asterisks and boxes, respectively. Residues with negatively
charged and polar side chains of Tar are indicated with closed and open
circles, respectively. Residue Glu-308, which was replaced by Cys, isunderlined. The relative efficiency of cross-linking of the Cys-replaced
CheR proteins with Tar is indicated with ⫹. The cross-linking at L58C
was only barely detectable (⫾).

the positively charged residues in helix ␣2 of CheR (K46A/E,
R53A/E, and R57A/E).
Swarming abilities of RP4944 cells (⌬CheR) expressing the
mutant CheR proteins were examined (Table II). Expression of
wild-type CheR with 0.1 mM arabinose and 0.1 mM fucose
resulted in the largest swarm ring. Overproduction of CheR
impaired swarming presumably because overmethylation of
MCPs caused tumbling-biased swimming and/or the titrationof AdoMet by excess CheR impaired cell growth. Cells expressing CheR-K46A or R53A required higher concentrations ofarabinose for swarming than cells expressing wild-type CheR.
As the concentration of arabinose increased, the former cells
swarmed faster. However, cells expressing CheR-K46E or
R53E failed to swarm even in the presence of higher concentrations of arabinose. Thus, a Glu substitution seems to be
more severe than an Ala substitution. Overproduction of CheRR57A or R57E allowed the cells’ swarming presumably because
these proteins have weak activities or are unstable. These
results suggest that the positive charges of helix ␣2 are important for the CheR function. Especially, Arg-53 seems to be
critical, which is consistent with the results described above(Fig. 3).
Disulfide Cross-linking between CheR and MH1 of Tar—To
detect directly the interaction between CheR and MH1 of Tar

Receptor Recognition by Methyltransferase

42331

FIG. 6. Disulfide cross-linking between CheR and Tar. A, disulfide cross-linking between His6-CheR-CS-R53C (lanes labeled with R53C)
and MBP-Tar-EEEE-E308C with (lanes labeled with ⫹) or without (lanes labeled with ⫺) an oxidant (0.1 mM I2). The Cys-less parental protein
His6-CheR-CS (lanes labeled with CS) was also tested as a control. B, disulfide cross-linking between the Cys-replaced derivatives of His6-CheRCS-CheR and MBP-Tar-EEEE-E308C. Lane 1, His6-CheR-CS; lane 2, -K46C; lane 3, -R53C; lane 4, -V55C; lane 5, -R56C; lane 6, -L58C; lane 7,-R59C; lane 8, -S60C; lane 9, -K46C/R53A. C, effect of amidation of Tar on its interaction with CheR. The K46C, R53C, or S60C derivatives of
His6-CheR-CS were mixed with the EEEE (lanes labeled with E) or QQQQ (lanes labeled with Q) version of MBP-Tar-E308C. The samples were
subjected to SDS-PAGE followed by immunoblotting with anti-CheR or anti-MBP serum as indicated.

that had been eluded from conventional biochemical assays, weemployed a disulfide cross-linking assay. Wild-type CheR has
two Cys residues (Cys-7 and Cys-229), but consistent withprevious reports (22, 45), the substitution of Ser (C7S/C229S)
had little effect on CheR function (data not shown). Cys-scanning mutagenesis was carried out for residues (Lys-46 andArg-53 through Ser-60) in helix ␣2 of His6-tagged Cys-less
CheR (named His6-CheR-CS). All of the resulting proteins werefunctional, but the L54C and R57C versions were not used in
the following assay because of their low yields (data notshown).
Residue Glu-308 (underlined) of E. coli Tar lies in the consensus sequence around methylation sites (bold) ((E/Q)(E/
Q)XXA(S/T)X) (46). This position was used as a target forcross-linking with CheR. A cytoplasmic fragment (residues

215–553) of the deamidated (EEEE) or the amidated (QQQQ)
derivative of Tar-E308C was fused to the cytoplasmic version of
the maltose-binding protein (named MBP-Tar-EEEE(orQQQQ)-E308C). Gln residues are known to mimic methylated
Glu residues. RP437 (wild type for chemotaxis) cells expressing
each MBP-Tar protein failed to swarm (data not shown), a
dominant negative effect that suggests that Tar fragments arecorrectly folded to interact with some Che proteins.
We first identified the Tar-CheR cross-linked product. MBPTar-EEEE-E308C (about 79 kDa) was mixed with His6-CheRCS-R53C (about 34 kDa) in the presence of 0.1 mM I2 (anoxidant) and detected by immunoblotting with anti-CheR andanti-MBP sera (Fig. 6A). The R53C protein, but not its Cys-lessparental protein, gave a Tar-CheR cross-linked product with anapparent molecular mass a little higher than expected (about42332

Receptor Recognition by Methyltransferase

113 kDa) but lower than that of the cross-linked dimer of
MBP-Tar-EEEE-E308C (about 158 kDa). These cross-linked
products were not detected in the presence of
2-mercaptoethanol.
MBP-Tar-EEEE-E308C was then mixed with each His6CheR derivative, and incubated at 37 °C for 20 min without
addition of any oxidant (Fig. 6B). The K46C protein was mostefficiently cross-linked to Tar (lane 2). The cross-linking of the
L58C protein to Tar was only barely detectable and the V55C
protein was much less effectively cross-linked to Tar than the
K46C, R56C, R59C, and S60C proteins (lanes 2 and 4 – 8). The
R53C protein was not effectively cross-linked to Tar (lane 3)
presumably because Arg-53 is particularly important to recognize MH1. Indeed, the introduction of the R53A mutation into
the K46C protein reduced the efficiency of cross-linking (compare lanes 2 and 9). These results suggest that the Cys residues
in the positively charged face of ␣2 are more efficient for crosslinking to Tar than those in the opposite face and that thecross-linking reflects the ability of CheR to recognize MH1.
We also examined the effect of amidation of Tar on crosslinking with CheR. The His6-CheR-CS proteins with K46C,
R53C, or S60C were mixed with the EEEE or QQQQ version of
MBP-Tar-E308C (Fig. 6C). The K46C protein was effectively
cross-linked to MBP-Tar-E308C regardless of its amidation
state. However, the R53C and S60C proteins were less effectively cross-linked to the amidated (QQQQ) fragment than tothe deamidated (EEEE) one. This result reinforces the electrostatic nature of the interaction between MHs of MCPs andhelix ␣2 of CheR.
DISCUSSION

In this study, we examined how the MTase CheR interacts
with its substrate, i.e. the chemoreceptor (MCP). More specifically, we asked how CheR is targeted to the receptor/kinase
clusters at cell poles and how CheR recognizes the methylationsites of the chemoreceptor. We found that these two processes
result from the distinct functions of CheR that reside in its two
distinct domains.
Comparative protein sequence analysis of CheR homologs
and related proteins revealed that all of the CheR proteins
have two domains and one subdomain: the CheR-specific Nterminal domain, the catalytic domain, and the ␤-subdomain.
This organization is consistent with the crystallography of S.
typhimurium CheR, suggesting that all of the CheR proteinsshare a common three-dimensional structure. It is also possiblethat they share common basic mechanisms for catalysis andsubstrate recognition. However, MCPs of many bacteria lackthe C-terminal NWETF-type sequence that serves as a primarybinding site of CheR in E. coli and S. typhimurium although allof the CheR proteins have the ␤-subdomain, which binds to the
NWETF sequence in the case of S. typhimurium and E. coli
CheR (Ref. 11 and this study). The ␤-subdomain can be divided
into two groups (11) (Fig. 2B): longer ␤-loops (e.g. E. coli, S.
typhimurium, and S. meliloti) and shorter ␤-loops (e.g. Vibrio
cholerae and Bacillus subtilis). The CheR proteins of the bacteria whose MCPs contain the C-terminal NWETF-like motif
belong to the former group. This difference in the length of the␤-loop might reflect the differences in the mode of receptor
recognition by CheR.
In contrast to the recognition of the C-terminal tail of MCPs,
little was known about the recognition of the methylation sitesof MCPs by CheR. X-ray crystallography raised a possibility
that the positively charged face of helix ␣2 of CheR might beinvolved in the interaction with MCPs (13). However, such aninteraction had not been detected biochemically. Here, the mutagenesis and the disulfide cross-linking analyses indicatedthat the positively charged face in helix ␣2 of CheR is involved

FIG. 7. Two modes of interaction between CheR (left) and MCP(right). The ␤-subdomain and the ␣2 helix of CheR interact with the
C-terminal pentapeptide (NWETF) sequence and the MHs, respectively. The CheR structure (green) determined in the presence of thepentapeptide (blue) (11) is shown with the crystal structure of thecytoplasmic fragment of Tsr (8) by placing the ␣2 helix of CheR to face
the first three methylation sites (violet) of one subunit (blue) of the Tsr
dimer. The other subunit of Tsr is shown in gray. The NWETF sequenceis tentatively connected to the main part of the cytoplasmic domain
with a broken line, although it is not clear whether CheR can bindsimultaneously to these two parts of the MCP. CheR residues involved
in receptor recognition and catalysis are shown in red and yellow,
respectively.

in the recognition of MH1 of Tar. This is the first direct demonstration of the interaction between CheR and a methylation
helix of any MCP. Among the residues tested, Arg-53 seems tobe the most important residue for the recognition of MH1. This
residue is strictly conserved among all of the CheR proteinsexcept for the Campylobacter jejuni homolog, in which thecorresponding residue is Lys.
Thus, effective methylation requires two types of interaction
between CheR and MCPs: one between the ␤-subdomain and
the NWETF sequence (for the targeting of CheR to cell poles)
and the other between the positively charged face in helix ␣2and the negatively charged face of an MH (for the recognition ofsubstrate sites) (Fig. 7). An E. coli cell expresses some 5,000
monomers of MCPs and only several hundred molecules of
CheR (47). Therefore, the targeting of CheR to the C-terminal
tail of MCPs may be required to concentrate CheR moleculesaround receptor/kinase clusters at cell poles. Increased probability of CheR to collide with MCP molecules may then allow it
to interact with the negatively charged face of an MH. This
interaction between helix ␣2 and an MH is predicted to be weak
and/or transient. CheR might slide on the negatively charged
face of MH1, which contains three methylation sites with intervals of two turns of the helix, to monitor the methylationsites.
It is still unknown how CheR is oriented when it binds to an
MCP. The binding of the NWETF sequence to the ␤-subdomain
was visualized by x-ray crystallography (11). However, it isunclear how the rest of the MCP molecule is oriented andwhether CheR can catalyze methylation of an MCP molecule
while it is anchored to the C-terminal tail of the same moleculeor the partner subunit of the same dimer. It was also suggested
that CheR can catalyze methylation of a neighboring MCP
molecule within an MCP cluster (19, 20). This interdimer methylation explains why a low abundance MCP can be methylated
efficiently in the presence of a high abundance MCP (48).
Again, it is not clear whether this can be achieved without dissociation of CheR from the NWETF sequence. In any case, theReceptor Recognition by Methyltransferase
simultaneous anchoring and catalysis would require large flexibility of the MCP molecule. Because the affinity of CheR for thepentapeptide is not very high, it is also possible that CheR is
dissociated from the NWETF sequence during catalysis.
We also examined subcellular localization of CheR. GFP-CheR
localized to cell poles only in the presence of an MCP with the
NWETF sequence. The mutagenesis of the CheR part suggested
that the targeting of CheR to cell poles depends primarily on theinteraction between CheR and the NWETF sequence. These results are consistent with the hypothesis that the NWETF sequence serves to concentrate CheR around MCPs at cell poles.
It should be noted that the abilities of the wild-type andmutant versions of GFP-CheR to localize to cell poles (Fig. 4,
right panels) appeared to vary: wild-type ⬎ R53A ⬎ D154A. In
the experimental conditions applied, the GFP-CheR proteins
were mildly overproduced relative to chromosome-encoded
CheR. Therefore, the methylation level of Tar would be different from one strain to another. This may suggest two possibilities: the methylation levels of MCPs might affect the subcellular localization of: (i) CheR and/or (ii) MCPs themselves. The
high abundance MCPs (Tar and Tsr) localize to cell poles andform clusters with CheA and CheW, whereas the low abundance MCPs (Tap and Trg) also localize to cell poles but do notform a cluster (16). The latter receptors are poor substrates for
CheR because they lack the C-terminal NWETF sequence.
Taken together, it is possible that the methylation levels of
MCPs would be critical for their clustering at cell poles. However, the polar localization of the high abundance MCPs does
not seem to require CheR and CheB (15). Further analyses are
required to clarify this issue.
The Cys-substituted CheR proteins were more effectively
cross-linked to Tar-EEEE than to Tar-QQQQ. This may result
from the electrostatic nature of the interaction between MHs of
MCPs and helix ␣2 of CheR. It is also possible that receptor
amidation (and hence methylation) alters the conformation of
the MHs to reduce its affinity to helix ␣2 of CheR. In any case,this finding is consistent with the notion that upon methylation, an MCP becomes a poorer substrate of CheR.
CheB also binds to the C-terminal NWETF sequence of
MCPs (49, 50) and has to recognize MHs of MCPs. However,
CheB may be different from CheR in these respects. The affinity of CheB for the pentapeptide is much lower than that of
CheR and the cellular concentration of CheB is much higher
than that of CheR. Therefore, it is hard to imagine that the
NWETF sequence serves to recruit CheB around MCPs. As forthe substrate recognition, CheB recognizes methylated Glu
residues to hydrolyze them, whereas CheR recognizes unmethylated Glu residues. Consistent with this consideration,
E. coli CheB does not have a positively charged cluster in the
primary sequence. Thus, it is intriguing to compare the mechanisms of receptor recognition of CheB with those of CheR.
Acknowledgments—We thank Drs. J. Beckwith and J. S. Parkinson
for providing plasmids and bacterial strains. We acknowledge the following genome sequencing centers (and their funding agencies) for
providing access to incomplete genome data: the Sanger Centre (Beowulf Genomics), the Joint Genome Institute (United States Department of Energy), the Institute for Genome Research (United States
Department of Energy), and the University of Oklahoma (National
Science Foundation).

42333
REFERENCES

1. Manson, M. D. (1992) Adv. Microbial. Physiol. 33, 277–346
2. Parkinson, J. S. (1993) Cell 73, 857– 871
3. Blair, D. F. (1995) Annu. Rev. Microbiol. 49, 489 –522
4. Stock, J. B., and Surette, M. G. (1996) in Escherichia coli and Salmonella
typhimurium: Cellular and Molecular Biology (Neidhardt, F. C., Curtiss, R.,
III, Ingram, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S.,
Riley, M., Schaechter, M., and Umbarger, H. E., eds) pp. 1103–1129, 2ndEd., American Society for Microbiology, Washington, D. C.
5. Falke, J. J., Bass, R. B., Butler, S. L., Chervitz, S. A., and Danielson, M. A.
(1997) Annu. Rev. Cell Dev. Biol. 13, 457–512
6. Armitage, J. P. (1999) Adv. Microb. Physiol. 41, 229 –289
7. Bass, R. B., and Falke, J. J. (1999) Structure 7, 829 – 840
8. Kim, K. K., Yokota, H., and Kim, S.-H. (1999) Nature 400, 787–792
9. Wu, J., Li, J., Li, G., Long, D. G., and Weis, R. M. (1996) Biochemistry 35,4984 – 4993
10. Okumura, H., Nishiyama, S., Sasaki, A., Homma, M., and Kawagishi, I. (1998)
J. Bacteriol. 180, 1862–1868
11. Djordjevic, S., and Stock, A. M. (1998) Nat. Struct. Biol. 5, 446 – 450
12. Shiomi, D., Okumura, H., Homma, M., and Kawagishi, I. (2000) Mol. Microbiol. 36, 132–140
13. Djordjevic, S., and Stock, A. M. (1997) Structure 5, 545–558
14. Maddock, J. R., and Shapiro, L. (1993) Science 259, 1717–1723
15. Lybarger, S. R., and Maddock, J. R. (1999) J. Bacteriol. 181, 5527–5529
16. Lybarger, S. R., and Maddock, J. R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97,
8057– 8062
17. Skidmore, J. M., Ellefson, D. D., McNamara, B. P., Couto, M. M., Wolfe, A. J.,
and Maddock, J. R. (2000) J. Bacteriol. 182, 967–973
18. Sourjik, V., and Berg, H. C. (2000) Mol. Microbiol. 37, 740 –751
19. Li, J., Li, G., and Weis, R. M. (1997) Biochemistry 36, 11851–11857
20. Le Moual, H., and Koshland, D. E., Jr. (1997) Biochemistry 36, 13441–13448
21. Feng, X., Lilly, A. A., and Hazelbauer, G. L. (1999) J. Bacteriol. 181,3164 –3171
22. Subbaramaiah, K., Charles, H., and Simms, S. A. (1991) J. Biol. Chem. 266,
19023–19027
23. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W.,
and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389 –3402
24. Schultz, J., Milpetz, F., Bork, P., and Ponting, C. P. (1998) Proc. Natl. Acad.
Sci. U. S. A. 95, 5857–5864
25. Bateman, A., Birney, E., Durbin, R., Eddy, S. R., Howe, K. L., andSonnhammer, E. L. (2000) Nucleic Acids Res. 28, 263–266
26. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins,
D. G. (1997) Nucleic Acids Res. 25, 4876 – 4882
27. Guex, N., and Peitsch, M. C. (1997) Electrophoresis 18, 2714 –2723
28. Parkinson, J. S., and Houts, S. E. (1982) J. Bacteriol. 171, 6271– 6278
29. Ames, P., Yu, Y. A., and Parkinson, J. S. (1996) Mol. Microbiol. 19, 737–746
30. Wolfe, A. J., Conley, M. P., Kramer, T. J., and Berg, H. C. (1987) J. Bacteriol.
169, 1878 –1885
31. Ames, P., Studdert, C. A., Reiser, R. H., and Parkinson, J. S. (2002) Proc. Natl.
Acad. Sci. U. S. A. 99, 7060 –7065
32. Guzman, L. M., Belin, D., Carson, M. J., and Beckwith, J. (1995) J. Bacteriol.
177, 4121– 4130
33. Bartolomé, B., Jubete, Y., Martinez, E., and Cruz, F. D. (1991) Gene (Amst.)
102, 75–78
34. Landt, O., Grunert, H. P., and Hahn, U. (1990) Gene (Amst.) 96, 125–128
35. Deleted in proof
36. Deleted in proof
37. Deleted in proof
38. Iwama, T., Homma, M., and Kawagishi, I. (1997) J. Biol. Chem. 272,
13810 –13815
39. Sogaard-Andersen, L., and Kaiser, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93,
2675–2679
40. Lamb, J. R., Tuqendreich, S., and Hieter, P. (1995) Trends Biochem. Sci. 20,
257–259
41. Das, A. K., Cohen, P. W., and Barford, D. (1998) EMBO J. 17, 1192–1199
42. Taylor, B. L., and Zhulin, I. B. (1999) Microbiol. Mol. Biol. Rev. 63, 479 –506
43. Liu, J. D., and Parkinson, J. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,
8703– 8707
44. Malone, T., Blumenthal, R. M., and Cheng, X. (1995) J. Mol. Biol. 253,
618 – 632
45. Subbaramaiah, K., and Simms, S. A. (1992) J. Biol. Chem. 267, 8636 – 8642
46. Kehry, M. R., Engstrom, P., Dahlquist, F. W., and Hazelbauer, G. L. (1983)
J. Biol. Chem. 258, 5050 –5055
47. Simms, S. A., Stock, A. M., and Stock, J. B. (1987) J. Biol. Chem. 262,
8537– 8543
48. Yamamoto, K., Macnab, R. M., and Imae, Y. (1990) J. Bacteriol. 172, 383–388
49. Barnakov, A. N., Barnakov, L. A., and Hazelbauer, G. L. (1999) Proc. Natl.
Acad. Sci. U. S. A. 96, 10667–10672
50. Barnakov, A. N., Barnakova, L. A., and Hazelbauer, G. L. (2001) J. Biol. Chem.
276, 32984 –32989