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Journal of Bacteriology, December 1998, p. 6419-6423, Vol. 180, No. 23
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
secG and Temperature Modulate Expression
of Azide-Resistant and Signal Sequence Suppressor Phenotypes of
Escherichia coli secA Mutants
Visvanathan
Ramamurthy,
Vesna
Dapíc, and
Donald
Oliver*
Department of Molecular Biology and
Biochemistry, Wesleyan University, Middletown, Connecticut 06459
Received 11 May 1998/Accepted 5 September 1998
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ABSTRACT |
SecA is a dynamic protein that undergoes ATP-dependent membrane
cycling to drive protein translocation across the Escherichia coli inner membrane. To understand more about this process,
azide-resistant (azi) and signal sequence suppressor
(prlD) alleles of secA were studied. We found
that azide resistance is cold sensitive because of a direct effect on
protein export, suggesting that SecA-membrane interaction is regulated
by an endothermic step that is azide inhibitable. secG
function is required for expression of azide-resistant and signal
sequence suppressor activities of azi and prlD
alleles, and in turn, these alleles suppress cold-sensitive and
export-defective phenotypes of a secG null mutant. These
remarkable genetic observations support biochemical data indicating
that SecG promotes SecA membrane cycling and that this process is
dependent on an endothermic change in SecA conformation.
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TEXT |
Export of preproteins across the
inner membrane of Escherichia coli has been studied
extensively during the past decade. Both biochemical and genetic
approaches have resulted in the identification of many if not all of
the proteinaceous components of the translocation machinery (30,
36). Precursor proteins, synthesized with an amino-terminal
signal peptide, associate with chaperones, such as SecB protein, which
maintains them in an export-competent conformation (29).
They are then targeted to the plasma membrane, where they associate
with the translocase complex, which is composed of the membrane-dissociable SecA protein and the integral membrane protein, SecYEG, which are thought to form a translocation channel (5, 9,
14, 19). Central to preprotein assembly at the translocase complex is SecA protein, a 204-kDa homodimeric protein, which has been
shown to bind the signal peptide and mature region of the preprotein,
SecB protein, anionic phospholipids, and the amino-terminal portion of
SecY protein (1, 4, 8, 15, 17, 20, 32). SecA regulates these
diverse interactions and drives protein translocation by its ATPase
activity (23, 34). ATP binding to SecA catalyzes the initial
insertion of preprotein into the membrane, while hydrolysis promotes
translocation across the membrane (31). Protein
translocation appears to depend on the ability of SecA to undergo
multiple cycles of membrane insertion and retraction, and such
SecA-membrane cycling has been shown to depend on the function of the
high-affinity ATP-binding domain of SecA (11, 28). This
biochemical behavior of SecA has led to a model in which SecA has been
hypothesized to act like a molecular ratchet, utilizing its membrane
cycling activity to translocate proteins (12). Recently,
however, it has been argued that protein translocation can occur under
conditions in which SecA is permanently imbedded in the plasma membrane
(6). In addition to SecA-membrane cycling, it has been found
that SecG protein undergoes a topology inversion during protein
translocation, and this event appears to be coupled to the insertion
and retraction cycle of SecA protein, suggestive of a mechanistic
linkage of these two processes (25).
Sodium azide is a known inhibitor of many ATPases, and azide-resistant
mutants of Escherichia coli, denoted azi, have
been found to be alleles of secA (13, 16, 26).
Previous studies indicate that azide inhibits the translocation ATPase
activity of SecA (26). Azide has been shown to trap SecA in
the membrane-inserted state, as judged by the formation of a
protease-resistant and membrane-protected 30-kDa fragment of SecA
(35). In addition, previous genetic studies of signal
sequence suppressor alleles of secA, denoted
prlD, found that most such strains are altered in their
azide sensitivity or resistance, thus indicating a strong interconnection between these two properties of SecA protein
(16). Most prlD alleles are located in or
adjacent to the ATP-binding domains of SecA, which govern SecA membrane
cycling (16, 17, 23). In order to understand more about SecA
function and its regulation, we have performed further genetic
characterization of azi and prlD mutants.
The strains used in this study are described in Table
1, and where necessary they were
constructed by P1 transduction (22). The growth medium
employed in this study has been described previously (22).
The concentrations of ampicillin, kanamycin, and tetracycline used were 100, 15, and 10 µg ml
1, respectively. Sodium
azide was purchased from Mallinckrodt.
Azide resistance is cold sensitive.
Since previous studies
showed that the insertion of SecA into the membrane involves a
temperature-dependent unfolding of the protein (33) and
azide prevents retraction of SecA from the membrane (35), we
were interested in determining the effect of temperature on azide
resistance. azi and prlD mutants that are
normally azide resistant at 37°C were tested for their ability to
form colonies on Luria-Bertani (LB) plates containing azide at various
temperatures. Remarkably, none of the azi or prlD
mutants that were azide resistant at 37 or 42°C were able to grow on
LB plates containing 1 mM azide at 20°C, although growth was normal on LB plates (Table 2). The
azi and prlD mutants showed reduced azide
resistance at 30°C, where single-colony formation was inhibited at 1 mM azide and growth was blocked completely at 2 mM azide. These
findings indicate that azide resistance is cold sensitive.
To determine whether the cold sensitivity of azide resistance
correlates with reduction in the rate of protein secretion under
these
conditions, we determined the effect of temperature and
azide on the
rate of processing of maltose-binding protein (MBP)
and OmpA in
azi and
prlD mutants. The rate of protein
processing
is a valid measure of the rate of protein secretion, given
the
topology of signal peptidase I (
7). Even in the absence
of
azide, lowering the growth temperature of the
prlD2
mutant to
20°C reduced the rate of protein secretion, particularly
for OmpA
(Fig.
1), indicating that
protein export was somewhat cold sensitive
in this case. Furthermore,
while protein secretion in the
prlD and
azi
mutants was substantially resistant to the effects of
azide at 37°C,
it was not resistant at 20°C. These data demonstrate
that protein
export in
azi and
prlD strains is phenotypically
azide sensitive at low growth temperatures.
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FIG. 1.
Analysis of protein secretion of azi and
prlD mutants at low temperature in the presence or absence
of azide. Strains (left to right: DO168, KB313, DO309, and DO315) were
grown in M63 minimal medium (22) containing 0.4% glycerol,
0.4% maltose, and 20 µg (each) of 18 amino acids (lacking cysteine
and methionine) ml1 at 37°C until the mid-logarithmic
phase, when portions of each culture were shifted to the indicated
temperature. After 20 min, sodium azide was added to a final
concentration of 2 mM to the indicated cultures. Five minutes later, a
0.5-ml aliquot of each culture was pulse-labeled with 10 µCi of Tran
35S-label (>1,000 Ci mmol1; ICN) for 1 min,
followed by the addition of an equal volume of ice-cold 10%
trichloroacetic acid. MBP and OmpA were immunoprecipitated and analyzed
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
autoradiography as described previously (26). Three times
more sample was loaded on the gel for the samples labeled at 20°C.
The positions of the precursor and mature forms of MBP (preMBP and MBP,
respectively) and OmpA (proOmpA and OmpA, respectively) are given.
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Azide resistance is SecG dependent.
Since both SecG and azide
have been suggested to affect SecA membrane cycling (25,
35), we were interested in studying the effect of secG
function on azide resistance. Using P1 transduction, azi and
prlD alleles were introduced into KN370 containing a
secG deletion. Remarkably, all azi or prlD
secG double mutants were unable to form colonies on LB plates
containing 1 mM azide at 37°C (Table
3). To show that this result was directly
due to the lack of secG function and was not an effect of
the general strain background, these strains were transformed with
plasmids containing secG, secE and
secY, or secE, secY, and
secG expressed from the trc promoter
(10). An azide-resistant phenotype was recovered only in
strains containing plasmids with secG, demonstrating the
importance of this gene in azide resistance. Since overproduction of
SecYE protein alone was insufficient to promote an azide-resistant phenotype, it seems unlikely that the involvement of secG in
this case was indirect, for example, by promoting a higher level of activity of SecYE protein.
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TABLE 3.
secG is required for azide resistance, while
azi and prlD alleles suppress the cold
sensitivity of secG strainsa
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|
To determine whether the observed azide-sensitive phenotype of
azi or
prlD secG double mutants correlates
with a reduced
rate of protein export under these conditions, the rate
of MBP
and OmpA secretion was investigated. While azide addition caused
a significant inhibition of MBP and OmpA secretion in the
azi and
prlD single mutants, more severe
inhibition of protein export
was noted for the
azi or
prlD secG double mutants (Fig.
2). These
effects were not due to a
decrease in SecA protein levels in any
of these strains as monitored by
pulse-labeling and immunoprecipitation
or by Western blotting (up to
2 h in the presence of 2 mM azide
[data not shown]). These data
are consistent with the observed
azide sensitivity of growth of the
azi or
prlD secG strains.
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FIG. 2.
Analysis of the azide sensitivity of protein secretion
of azi or prlD secG double mutants. Strains
(from left to right: DG313, DG101, DG309, DG313.2, DG101.2, and
DG309.2) were grown at 37°C, treated with sodium azide (where
indicated), and radiolabeled, and MBP and OmpA were analyzed as
described in the legend to Fig. 1.
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|
Deletion of
secG results in a cold-sensitive phenotype in
certain strain backgrounds, such as C600 and W3110 (
24). In
some
cases, the cold-sensitive phenotype is manifested only when the
unc genes encoding F
1F
0-ATPase are
deleted also (
10). MC4100
secG mutants were
not cold sensitive, whether they contained
unc+
or
uncB-C alleles (results not shown), indicating that the
status
of the
unc locus need not determine the
cold-sensitive phenotype
of
secG mutants. The reason for
such variation among different
strain backgrounds is unclear. Because
MC4100
secG derivatives
are not cold sensitive, we tested
azi and
prlD derivatives of
this strain for the
dependence of azide resistance on
secG function.
Even in
this strain background, we found that
secG function affected
the level of azide resistance, although not as severely as KN370
derivatives.
azi and
prlD MC4100 derivatives were
able to form
colonies on LB plates containing 3.5 mM azide, while their
isogenic
secG counterparts were only able to form
colonies on LB plates
containing 1 mM azide, with the exception of the
prlD22 secG mutant, which formed colonies on LB plates
containing up to 2
mM azide (results not
shown).
Cold sensitivity caused by secG deletion can be
suppressed by azi and prlD alleles.
Loss
of secG function leads to cold sensitivity of growth and an
accumulation of preproteins at low temperature (24).
Overexpression of acidic phospholipids has been shown to suppress this
phenotype (18), suggesting that the loss of secG
function may relate to a defect in SecA-membrane binding or insertion
(which requires anionic phospholipids [3, 33]). Since
purified SecA proteins containing the azi and
prlD mutations displayed increased membrane ATPase activity,
even at 28°C (28a), indicating enhanced SecA-membrane interaction, we speculated that azi and prlD
alleles may be able to suppress the cold sensitivity of secG
mutants. To this end, the growth property of the azi or
prlD secG derivatives was investigated. Nearly all of
these double mutants were able to form colonies on LB plates at 20°C
(Table 3), indicating that azi and prlD alleles
suppressed the cold sensitivity caused by the secG mutation. One exception to this pattern of suppression was the prlD20
secG mutant, which was unable to form colonies at 20°C. The
lack of suppression in this case seems to relate to the cold
sensitivity of prlD20 strains (data not shown). Our findings
are consistent with those of an earlier study showing that
secA36, which leads to azide resistance, can suppress a
secG defect in protein export at 20°C (21).
In order to determine whether the growth observed for
azi or
prlD secG double mutants at low temperatures correlates
with
an increase in the rate of protein export, secretion of MBP and
OmpA was investigated. The
azi or
prlD secG
mutants displayed
an increased rate of protein secretion at 20°C
compared to the
isogenic
secG parent (Fig.
3). These results suggest that a direct
mechanism of suppression of the
secG growth defect by
these
secA alleles is most probable.
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FIG. 3.
Analysis of protein secretion of azi or
prlD secG double mutants at low temperature. Strains
(from left to right: DG100, DG313, DG101, and DG309) were grown at
37°C, shifted to the temperature indicated for 20 min, and subjected
to radiolabeling, and MBP and OmpA were analyzed as described in the
legend to Fig. 1, except that an equal amount of each sample was loaded
on the gel.
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|
Signal sequence suppressor activity of prlD alleles is
secG dependent.
Since secG function is
needed for azide resistance, we were interested whether it was also
required for the signal sequence-suppressor activity of prlD
alleles, since both of these properties would result in an increased
demand on the protein translocation system. To this end,
prlD or azi alleles were introduced into
secG+ or secG derivatives of MM2
that contain the malE14-1 allele, which is a defect in the
signal sequence of MBP (2). As expected, prlD
secG+ strains were able to suppress the signal
sequence defect, resulting in a Mal+ phenotype, while the
azi-4 secG+ strain was Mal (Fig.
4). Remarkably, all prlD
secG double mutants were Mal, indicating that
secG function is required for prlD-mediated signal sequence suppression. In a second assay system that measures lamB14D signal sequence suppressor activity by the strain's
sensitivity to lambda phage adsorption and killing by cross-streaking,
all prlD secG double mutants had a lambda-resistant
phenotype, except for the prlD22 secG double mutant
(prlD22 is the strongest signal sequence suppressor
[16]), which was weakly lambda sensitive (data not
shown). We conclude that secG function is needed for the
expression of the signal sequence suppressor activity of
prlD alleles.
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FIG. 4.
secG function is required for signal sequence
suppression by prlD alleles. prlD or
azi MM2 derivatives with (secG+) or
without (secG) secG function were tested for
their ability to suppress the malE14-1 signal sequence
mutation by overnight growth on maltose-tetrazolium plates at 37°C.
Red colonies (Mal) indicate little or no suppressor
activity, while white colonies (Mal+) indicate good
suppressor activity.
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|
In this work, we have shown a remarkable genetic interaction between
the SecA and SecG proteins. This conclusion is based
on the fact that
the azide-resistant and signal sequence-suppressor
properties of
azi and
prlD mutants are SecG dependent, and yet
azi and
prlD alleles suppress the cold
sensitivity of
secG mutants.
The requirement of SecG for
expression of the
azi and
prlD phenotypes
presumably relates to the ability of SecG to increase the pool
of
biochemically activated, SecYEG-bound SecA protein. Thus, in
the
azi or
prlD secG double mutants, there is
insufficient activated
SecA protein to promote the azide-resistant and
signal sequence
suppression activities of SecA, which place an unusual
demand
on the translocation process. However, there is sufficient
activated
SecA protein under this circumstance to promote normal
protein
translocation at low temperature in the absence of
secG function.
The ability of these mutations to activate
SecA protein in the
absence of
secG function as well as to
promote suppression of
signal sequence defects may relate to their
predicted destabilization
of the compact quaternary structure of SecA
protein that is likely
to require a conformational change to promote
biochemical activation
(based on the atomic structure of SecA)
(
16a).
A second important conclusion from our study is that azide resistance
is cold sensitive. It is tempting to speculate that
this cold
sensitivity is another manifestation of the inherent
cold sensitivity
of the protein export process that has been observed
previously
(
27) and that its biochemical basis rests on an endothermic
transition of SecA conformation that has been noted previously
(
3,
33), and which is required to promote SecA membrane interaction
and cycling. Presumably SecG, as well as perhaps other Sec components,
normally helps to overcome this cold-sensitive step, which is
azide
inhibitable and which can be partially compensated for by
azi and
prlD mutations, thereby bypassing the
strict requirement
for SecG function. This step is likely to correspond
to one in
the membrane insertion-retraction cycle of SecA, consistent
with
the ability of azide to block SecA membrane retraction and SecG
to
promote SecA membrane cycling (
25,
35). Further elucidation
of this complex system will require biochemical studies that are
currently under
way.
| |
ACKNOWLEDGMENTS |
We thank John Hunt for productive discussions on SecA structure and
mechanism and Bill Wickner, Hajime Tokuda, and Tom Silhavy for
provision of strains.
This work was supported by grant GM42033 from the National Institutes
of Health to D.O.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT
06459. Phone: (860) 685-3556. Fax: (860) 685-2141. E-mail: doliver{at}wesleyan.edu.
Present address: Department of Biochemistry/HHMI, University of
Washington, Seattle, WA 98195.
| |
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Journal of Bacteriology, December 1998, p. 6419-6423, Vol. 180, No. 23
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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