(Received for publication, April 28, 1995; and in revised form, August 2, 1995)
From the
Earlier studies have suggested that CheZ, the phosphatase of the
signaling protein CheY in bacterial chemotaxis, may be in an oligomeric
state both when bound to phosphorylated CheY (CheYP) (Blat, Y.,
and Eisenbach, M.(1994) Biochemistry 33, 902-906) or
free (Stock, A., and Stock, J. B.(1987) J. Bacteriol. 169,
3301-3311). The purpose of the current study was to determine the
oligomeric state of free CheZ and to investigate whether it changes
upon binding to CheY
P. By using either one of two different sets
of cross-linking agents, free CheZ was found to be a dimer. The
formation of the dimer was specific, as it was prevented by SDS which
does not interfere with cross-linking mediated by random collisions.
The dimeric form of CheZ was confirmed by sedimentation analysis, a
cross-linking-free technique. In the presence of CheY
P (but not in
the presence of non-phosphorylated CheY), a high molecular size
cross-linked complex (90-200 kDa) was formed, in which the
CheZ:CheY ratio was 2:1. The size of the oligomeric complex was
estimated by fluorescence depolarization to be 4-5-fold larger
than the dimer, suggesting that its size is in the order of 200 kDa.
These results indicate that CheZ oligomerizes upon interaction with
CheY
P. This phosphorylation-dependent oligomerization may be a
mechanism for regulating CheZ activity.
Bacteria such as Escherichia coli or Salmonella typhimurium use chemotaxis to navigate toward favorable environments and retreat from non-favorable ones(1) . The sensory information from the receptors is integrated by a cytoplasmic signal transduction network of chemotaxis proteins (see for review, (2, 3, 4, 5) ) and transmitted to the flagella by the signaling molecule CheY (6, 7) . CheY interacts with the switch-motor complex at the base of the flagellum (6, 7, 8, 9, 10) and changes the direction of rotation from the default direction, counterclockwise(9, 10, 11, 12, 13, 14, 15, 16, 17) to clockwise(12, 15, 17) , and thereby causes the cell to reorient(18) . The clockwise causing activity of CheY is regulated by phosphorylation(19) . The phosphorylation level is determined by the kinase CheA and the phosphatase CheZ(20, 21, 22) . The activity of the kinase, CheA, is modulated by chemotactic stimuli via the membrane chemotaxis receptors and the chemotaxis protein CheW(23, 24, 25) . On the other hand, regulation of CheZ activity by chemotactic stimuli has not been demonstrated.
In a previous study we found that the binding of CheZ
to phosphorylated CheY (CheYP) is 2 orders of magnitude higher
than to non-phosphorylated CheY, and that several molecules of CheZ can
bind to a single CheY
P molecule(26) . Earlier observations
indicated that CheZ can be in two oligomeric forms, 115 and >500
kDa, as estimated by size-exclusion chromatography (27) (the
molecular size of the monomer is 23.9 kDa(27) ). The
observations of both studies taken together suggest that the oligomeric
state of CheZ is modulatable. Here we examine this possibility and
demonstrate that CheZ is a dimer which oligomerizes upon interaction
with CheY
P. The possibility that this phosphorylation-dependent
oligomerization is a regulation mechanism for CheZ activity is
investigated in a subsequent work (28) .
Figure 1:
Autoradiogram of DMS-mediated
cross-linking of CheZ. The cross-linking was carried out as described
under ``Experimental Procedures.''
[C]CheZ (3.2 nCi) was used for each reaction.
The top band in each of the lanes belongs to nonspecific
aggregates unable to enter the separating
gel.
A mixture of EDC and
NHS mediates the cross-linking of amines to carboxyl
groups(38) . CheZ exposed to this mixture formed a single
defined band at 46 kDa (Fig. 2A, lane 2). In the
presence of non-phosphorylated CheY, most of CheZ remained as a dimer (lane 3). A sharp change in the oligomeric state of CheZ was
observed when CheY was added together with AcP. Under these conditions
all the CheZ formed a >94 kDa cross-linking product (lane
4), which appeared on a 10% SDS-PAGE as a 90-200-kDa smear
and a distinct band at 145 kDa (Fig. 2B). As in the
case of DMS, AcP in the absence of CheY did not form the >94-kDa
complex (Fig. 3A, lane 3). These results confirm that
an oligomeric complex of CheZ is formed in the presence of CheYP.
Figure 2:
Autoradiogram of EDC-NHS-mediated
cross-linking of CheZ. The cross-linking was carried out for 1 h as
described under ``Experimental Procedures.''
[C]CheZ (3.2 nCi) was used for each reaction. A, 15% polyacrylamide gel. B, as lane 4, but
on 10% polyacrylamide gel.
Figure 3:
The dependence of CheZ oligomerization on
CheYP. The cross-linking was carried out as in Fig. 2A, except for the modifications indicated below.
Unless mentioned otherwise, the CheY and CheY57DE concentrations were
20 µM. A, cross-linking of
[
C]CheZ in the presence of varying
concentrations of CheY. B, quantification of the dependence of
the amount of oligomer formed on the concentration of CheY. The
relative intensity of each oligomer band was measured by a
PhosphoImager and calculated relatively to the intensity of the band
formed in the presence of 20 µM CheY. The results are the
mean of two experiments, one of which is the experiment shown in panel
A. C, cross-linking of [
C]CheZ under
conditions which prevent the phosphorylation of
CheY.
To confirm that the cross-linking products observed in this study resulted from specific interactions, we repeated all the above mentioned experiments in the presence of SDS (0.5%). (SDS does not interfere with cross-linking mediated by random collisions(39) .) Only the monomeric form of CheZ was observed; the formation of all the cross-linking products was prevented by SDS (data not shown), indicating that the interactions were specific.
The EDC-NHS-mediated cross-linking of CheZ (Fig. 2) was much more efficient than the DMS-mediated cross-linking (Fig. 1). This may be the consequence of the small number of lysine residues and the high number of aspartate and glutamate residues in CheZ (6 residues versus 37, respectively(40) ). For this reason the subsequent cross-linking studies were carried out with EDC-NHS.
If
the phosphorylation-dependent oligomerization of CheZ is
physiologically significant, it should depend on the concentration of
CheYP. As shown in Fig. 3, A and B, the
amount of the oligomer was indeed dependent on the concentration of
CheY added in the presence of access AcP. Furthermore, CheY57DE which
cannot be phosphorylated due to the substitution of Glu for the
phosphorylation site, Asp
(26, 41) , did
not promote the oligomer formation even in the presence of AcP (Fig. 3C, lane 7). Similarly, depletion of
Mg
(necessary for CheY phosphorylation(42) )
also prevented the formation of the oligomer (lane 5).
Figure 4:
The
amount of CheY associated with the oligomer.
[C]CheZ (3.2 nCi, specific activity 126 Ci/mol)
or [
C]CheY (20 nCi, specific activity 50 Ci/mol)
(the radiolabeled protein in each lane is indicated by an asterisk) were cross-linked by EDC-NHS as in Fig. 2A, only that the cross-linking was carried out
for 40 min.
Figure 5: Size estimation of CheZ by zonal centrifugation on a sucrose gradient. The positions of the molecular size marker proteins are indicated by arrows.
To enable the measurement of the
rotational diffusion of CheZ by fluorescence depolarization, we had
first to fluorescently label CheZ. For this purpose we replaced
phenylalanine 214 of CheZ with cysteine by using a mismatch primer and
PCR, as described under ``Experimental Procedures.'' The
mutated protein, CheZ214FC, retained normal CheY phosphatase activity (Fig. 6) and therefore could serve as a model for wild-type
CheZ. CheZ214FC contains a single cysteine residue and therefore could
be labeled at a specific site by fluorescein-5-maleimide. The
anisotropy, which reflects the rotational diffusion and therefore also
the size of the fluorescein-labeled
CheZ214FC(43, 44) , was dependent on the concentration
of added CheY (Fig. 7A). In the absence of AcP, the
anisotropy increased only moderately with the concentration of CheY and
was not saturated within the concentration range of the experiment
(0-45 µM CheY). This moderate change in anisotropy
was probably the result of the low-level binding of non-phosphorylated
CheY to CheZ(26) . In the presence of AcP, the anisotropy was
increased over 2-fold, indicating a large decrease in the rotational
diffusion of CheZ upon CheY phosphorylation. The increase in anisotropy
was saturated already at 4 µM CheY. The anisotropy change
was rapid; it was completed prior to the first measurable time point
(30 s). In order to estimate the magnitude of the size change, the
anisotropy of CheZ was measured at varying viscosity values in the
presence or absence of CheY
P. The data, presented in the form of a
Perrin plot (43, 44) , are shown in Fig. 7B. In the Perrin plot the slopes of the curves
are inversely related to the molecular dimension and therefore to the
molecular size(43, 44) . The slope was 4.8-fold
smaller in the presence of CheY
P than in its absence, suggesting
that upon CheY phosphorylation CheZ formed a complex which is
approximately 4-5-fold larger in volume than the CheZ dimer. The
fact that we did not detect CheY
P-induced oligomerization of CheZ
in size-exclusion chromatography, but did observe it using fluorescence
depolarization, suggests that the oligomer is unstable and dissociates
in the absence of CheY
P.
Figure 6:
Phosphatase activity of CheZ214FC. The
fraction of CheYP out of total CheY (0.26 ± 0.06) in the
absence of CheZ was considered as 100%. The results are the mean
± S.D. of three independent experiments.
, wild type;
, CheZ214FC.
Figure 7:
Fluorescence depolarization studies of
fluorescein-labeled CheZ214FC. The experiment was carried out in the
absence () or presence (
) of 18 mM AcP. A,
the effect of CheY on the fluorescence anisotropy (A =
(I
- I
)/(I
+ 2I
)) (44) of CheZ. The measured
solution contained fluorescein-labeled CheZ214FC (0.2 µM),
Tris-HCl (50 mM, pH 7.9), MgCl
(5 mM) and
increasing concentrations of CheY
. B, Perrin plot
of the fluorescence anisotropy of fluorescein-labeled CheZ214FC (1.0
µM in the same buffer conditions as in A). When
present, the concentration of CheY
was 20 µM.
The medium viscosity was increased by sucrose up to 4.6 cPoise. In this
viscosity range the change in fluorescence anisotropy reflects mainly
the rotation of the whole CheZ-fluorescein conjugate, and to a much
lesser extent the free rotation of the probe (fluorescein). The latter
rotation is not expected to be affected by the presence of CheY and
AcP. Therefore, to a first approximation, the slopes of these lines,
can be taken as the inverse of the respective molecular volumes (43) .
In this study we have shown that CheZ is a dimer which
further oligomerizes upon interaction with CheYP. The significance
of these findings is discussed below.
The different approaches used in this study to probe the molecular size of CheZ have yielded conflicting results. When run on a size-exclusion column, CheZ appears as a tetramer ( (27) and this study). In the cross-linking experiments, CheZ appeared as a dimeric protein (Fig. 1Fig. 2Fig. 3). In zonal centrifugation, the estimated size of CheZ was between a monomer and a dimer (Fig. 5). Since it is well known, both experimentally (45, 46, 47) and theoretically(48) , that the molecular size of non-spherical proteins can be overestimated severalfolds by size-exclusion chromatography(45, 46, 47, 48) , and that the size can be underestimated in zonal centrifugation(47) , it is reasonable to assume that CheZ is a non-spherical dimer.
The major finding of this study is that, in the
presence of CheYP, there is further oligomerization of the CheZ
dimer. However, it is not possible to determine, on the basis of the
results, whether CheY
P is an integral part of the oligomer at a
CheZ:CheY
P ratio of 2:1, or whether it is just bound to an
oligomer comprising CheZ alone. The results of the fluorescence
depolarization implied that the oligomer is about 4-5 times
larger than the CheZ dimer formed in the absence of CheY
P. This
suggests that the size of the oligomer is in the order of 200 kDa. This
size is about at the top of the size range observed in the
cross-linking experiments (Fig. 2B). The size
estimation of the oligomer from the fluorescence depolarization is only
a first degree approximation, because it was based on the assumption
that both the oligomer and the CheZ dimer are
spherical(43, 44) , an assumption which, according to
our own results, is probably incorrect. Determination of the exact size
or shape is beyond the resolution of the applied methods. A better
estimation of the size of the oligomer is rather difficult by currently
available methods because, at this stage, there is no obvious way to
separate the oligomer from CheY
P and maintain it in a stable form.
For example, techniques in which the shape contribution can be
estimated (e.g. light scattering) cannot distinguish between
the oligomer and CheY
P in the mixture. Nevertheless, these
difficulties in size and shape estimation do not affect the conclusions
reached in this study, as neither the exact size nor the shape of the
oligomer are necessary for the conclusions. It should be noted that the
CheZ oligomer, observed in this study, is different from the CheZ
homopolymer (27) and the CheZ-CheA
multimeric
complex(49) , observed earlier, in the sense that the oligomer
of this study is not stable and it readily dissociates in the absence
of CheY
P.
In the fluorescence depolarization experiments, the
anisotropy of fluorescein-labeled CheZ was increased to a large extent
in the presence of CheYP (Fig. 7A). This
observation could, in principle, be attributed either to a significant
increase in the molecular volume of CheZ, or to a large conformational
change at the vicinity of the fluorescein moiety that restricts its
free rotation. The following observations strongly suggest that a
significant increase in the molecular volume of CheZ, i.e. CheZ oligomerization, is the mechanism responsible for the
anisotropy change. (i) To a first approximation, the Perrin plot (Fig. 7B) is composed of two distinct rotations: a fast
rotation of the probe, and a slow rotation of the whole protein. To
determine the rotational freedom of the probe, we extrapolated the
straight line of the Perrin plot to 1/anisotropy = 0. Since the
straight line shown in the figure represents the rotation of the
protein, the extrapolated value represents the hypothetical case in
which the protein rotation is frozen but the probe rotates freely. As
shown in Fig. 7B, both the dimeric and the oligomeric
forms of CheZ fall in the same anisotropy range (1/anisotropy values of
4.0 and 4.8 for the dimer and oligomer, respectively). This indicates
that the rotation of the probe itself is not significantly affected by
the oligomerization, and therefore that the anisotropy change is not
the result of a change in the probe rotation. (ii) An increase in
anisotropy could, in principle, be due to a decrease in the lifetime of
the excited state of the probe, reflected in a reduced efficiency of
the fluorescence. However, the large oligomerization-dependent change
in anisotropy was accompanied by only a minor reduction (7%) in the
fluorescence efficiency. The lack of substantial changes in the
fluorescence efficiency and in the motional freedom of the probe, is
supported by the observation that both the 214F
C substitution (Fig. 6) and the conjugation of this cysteine with fluorescein
maleimide (
)did not affect the activity of CheZ.
It is
well known in a variety of systems, including bacterial signal
transduction systems(50, 51) , that oligomerization
regulates protein activity. Accordingly, it is conceivable that the
oligomerization may either activate or inhibit the phosphatase activity
of CheZ. In the first possibility, a burst of CheY phosphorylation will
activate CheZ and will thereby promote faster deactivation of CheY.
Fast deactivation of CheY will prevent non-beneficial too long periods
of tumbling. If the other possibility is correct and oligomerization
inhibits the phosphatase activity of CheZ, the oligomerization may
serve as an amplification step in which phosphorylation of CheY leads
to oligomerization of CheZ, inhibition of its phosphatase activity,
and, consequently, further increase in the level of CheYP. The
results described in the subsequent paper (28) suggest that the
first possibility is the correct one.
This paper is dedicated to Julius Adler on the occasion of his sixty-fifth birthday.