From the Department of Genetics, Groningen
Biomolecular Sciences and Biotechnology Institute, Kerklaan 30, 9751 NN
Haren, The Netherlands and the ¶ School of Microbiological,
Immunological and Virological Sciences, The Medical School, Newcastle
University, Framlington Place, Newcastle upon Tyne, NE4 4HH, United
Kingdom
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ABSTRACT |
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The type I signal peptidase SipS of
Bacillus subtilis is of major importance for the processing
of secretory precursor proteins. In the present studies, we have
investigated possible mechanisms of thermal inactivation of five
temperature-sensitive SipS mutants. The results demonstrate that two of
these mutants, L74A and Y81A, are structurally stable but strongly
impaired in catalytic activity at 48 °C, showing the (unprecedented)
involvement of the conserved leucine 74 and tyrosine 81 residues in the
catalytic reaction of type I signal peptidases. This conclusion is
supported by the crystal structure of the homologous signal peptidase
of Escherichia coli (Paetzel, M., Dalbey, R. E., and
Strynadka, N. C. J. (1998) Nature 396, 186-190).
In contrast, the SipS mutant proteins R84A, R84H, and D146A were
inactivated by proteolytic degradation, indicating that the conserved
arginine 84 and aspartic acid 146 residues are required to obtain a
protease-resistant conformation. The cell wall-bound protease WprA was
shown to be involved in the degradation of SipS D146A, which is in
accord with the fact that SipS has a large extracytoplasmic domain. As
WprA was not involved in the degradation of the SipS mutant proteins
R84A and R84H, we conclude that multiple proteases are responsible for
the thermal inactivation of temperature-sensitive SipS mutants.
Bacterial proteins that are exported from the cytoplasm via the
general secretion pathway are synthesized with an amino-terminal signal
peptide. The signal peptide is required for the targeting of
pre-proteins to the membrane and the initiation of protein translocation across this membrane (for review, see Ref. 1). During, or
shortly after, the translocation of secretory pre-proteins, signal
peptides are removed by type I signal peptidases
(SPases),1 which is a
prerequisite for the release of the mature protein from the
trans side of the membrane (for review, see Ref. 2).
Type I SPases belong to a special class of serine peptidases (peptidase
classification: clan SF, family S26; Ref. 3) with conserved serine and
lysine residues, which are essential for catalysis, most likely by
forming a serine-lysine catalytic dyad (see Refs. 2, 4, and 5). As
demonstrated by the crystallographic analysis of the type I SPase of
Escherichia coli (also known as leader peptidase), the
active site serine residue acts as a nucleophile attacking the carbonyl
carbon of the scissile peptide bond at the SPase recognition site,
whereas the unprotonated form of the lysine In the Gram-positive bacterium Bacillus subtilis five
chromosomally encoded type I SPases have been identified, which are denoted SipS, SipT, SipU, SipV, and SipW (11-13). SipS and SipT are of
major importance for protein secretion, whereas SipU, SipV, and SipW
contribute only to a minor extent to the processing of secretory
pre-proteins (12-14). Cells depleted of functional SipS and SipT stop
growing and lyse, showing that the presence of either SipS or SipT is
essential for growth and viability. This was demonstrated with a
B. subtilis strain in which the chromosomal copies of the sipS and sipT genes were disrupted and
functionally replaced by one of five different plasmid-borne genes for
temperature-sensitive SipS mutant proteins (i.e. L74A, Y81A,
R84A, R84H, and D146A; Ref. 13). In the present study, we have
investigated the mechanism of thermal inactivation of these five SipS
mutant proteins. The results show that SipS L74A and Y81A are
structurally stable at high temperature, indicating that the
substituted residues are, in addition to the putative active site
serine (Ser-43) and lysine residues (Lys-83), involved in catalysis. By
contrast, SipS R84A, R84H, and D146A are prone to proteolytic
degradation, in particular at high temperature, showing that the
residues at these positions are required to maintain resistance to
proteases. Interestingly, a cell wall-bound protease, WprA, was shown
to be involved in the degradation of SipS D146A, but not in the
degradation of SipS R84A and R84H.
Plasmids, Bacterial Strains, and Media--
Table I lists the
plasmids and bacterial strains used. TY medium was prepared as
described in Ref. 15. GCHE medium was prepared as described in Ref. 16.
Antibiotics were used in the following concentrations: chloramphenicol,
5 µg/ml; erythromycin, 1 µg/ml; kanamycin, 10 µg/ml.
Transformation of B. subtilis--
B. subtilis was
transformed by growth in GCHE medium until an optical density at 600 nm
(OD600) of approximately 1.0, subsequent addition of DNA to
the culture, and continued growth for 4 h.
Western Blot Analysis--
Western blotting was performed using
a semi-dry system as described by Kyhse-Andersen (17). After separation
by SDS-polyacrylamide gel electrophoresis, proteins were transferred to
polyvinylidene difluoride membranes (Roche Molecular Biochemicals,
Mannheim, Germany). Proteins were visualized with specific antibodies
and horseradish peroxidase anti-rabbit IgG conjugates, using the ECL detection system of Amersham (Little Chalfont, United Kingdom).
Stability of SipS Mutant Proteins at 480C--
We
have shown previously that the SipS mutant proteins R84A, R84H, and
D146A are prone to proteolytic degradation at 37 °C, showing very
low levels of SPase activity (6). Nevertheless, at this temperature
sufficient active molecules of SipS R84A, R84H, or D146A are produced
in B. subtilis to replace the wild-type forms of SipS and
SipT (13). To investigate whether the inactivity of these three mutant
proteins at 48 °C (13) was due to increased proteolytic degradation,
Western blotting experiments were carried out with B. subtilis 8G5 sipS (
Similarly, the stability of the SipS mutant proteins L74A and Y81A,
which compared with SipS R84A, R84H, and D146A, showed a relatively
high activity at 37 °C (6) and which were unable to replace SipS and
SipT at 48 °C (13), was investigated by Western blotting, using
B. subtilis SipS D146A Is a Substrate for the Cell Wall-bound Protease
WprA--
As SipS is a type II membrane protein, the largest
(carboxyl-terminal) part of which is exposed to the extracytoplasmic
side of the membrane (11), it seems likely that proteases residing in
the membrane or cell wall are responsible for the degradation of the
SipS R84A, R84H, and D146A mutant proteins. As a first approach to
identify the proteases responsible for their degradation, we tested the
stability of SipS D146A, the most unstable mutant, in B. subtilis strains lacking (putative) membrane-bound proteases such
as FtsH, HtrA, protease IV or Tsp (for a review on the E. coli homologues of these proteases, see Ref. 18), or the cell wall-bound protease WprA (19, 20). Strikingly, none of the membrane-bound proteases appeared to be involved in the degradation of
SipS D146A (data not shown). In contrast, at 37 °C, B. subtilis W Suppression of the Temperature Sensitivity of B. subtilis In the present studies, we show that temperature-sensitive mutants
of SipS are inactivated via different mechanisms. Two major classes of temperature-sensitive SipS mutants were identified. The
first class of mutant proteins, consisting of SipS L74A and Y81A, is
structurally stable but has impaired catalytic activity at 48 °C,
while the second class of mutant proteins, consisting of SipS R84A,
R84H, and D146A, is sensitive to proteolysis, in particular at
48 °C.
The observation that SipS L74A and Y81A are structurally stable,
showing a particularly reduced enzymatic activity at 48 °C, indicates that leucine 74 and tyrosine 81 are involved in catalysis. This is a novel conclusion, which was not evident from previous site-directed mutagenesis experiments with SipS or other type I SPases
(for review, see Ref. 2), but which is supported by the recently
published crystal structure of the E. coli SPase I (5). As
evidenced by the latter structure, the side chains of phenylalanine
133, tyrosine 143, and methionine 270, and the main chain atoms of
methionine 270, methionine 271, glycine 272, and alanine 279 make van
der Waals contacts with the side chain of the active site lysine
residue 145. Thus, the In contrast to SipS L74A and Y81A, the thermal inactivation of the SipS
mutants R84A, R84H, and D146A seems to be based on proteolysis,
suggesting that these mutations strongly retard the folding of SipS to
a protease-resistant conformation or that they make the folded SipS
protein protease-sensitive. Based on the crystal structure of E. coli SPase I (5), we favor the latter hypothesis, because the
equivalent residues of arginine 84 and aspartic acid 146 of SipS in the
E. coli SPase I (i.e. arginine 146 and aspartic
acid 273) form a salt
bridge,2 which may be
required to rigidify the structure of type I SPases, such as SipS. Our
present results show that the wall-bound protease WprA (19), which was
shown recently to be involved in the degradation of folding
intermediates of secreted proteins (20), is involved in the degradation
of SipS D146A, but not of SipS R84A and R84H. Furthermore, depletion of
WprA did not result in the accumulation of SipS D146A to wild-type
levels. Thus, it seems that other, as yet unidentified, proteases at
the membrane-cell wall interface are also involved in the degradation
of structurally unstable SipS mutant proteins. These could even include
other type I SPases, such as SipU, SipV, or SipW, as suggested by the
observation that the homologous Sec11p subunit of the SPase complex in
the yeast endoplasmatic reticular membrane is involved in protein
degradation (21). Finally, we are currently unable to exclude the
possibility that cytosolic proteases are involved the degradation of
SipS mutant proteins, either before, during, or after membrane
insertion. The latter possibility is particularly intriguing since it
would require retrograde transport to the cytoplasm, as documented
recently for the degradation of ER lumenal proteins (22-25).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-amino group probably
serves to activate the hydroxyl group of the serine residue. A similar
catalytic mechanism has been proposed for the structurally related
LexA-like proteases (4-8), which employ a serine-lysine catalytic dyad
for self-cleavage (7, 9, 10).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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S; lacking the chromosomal
sipS gene) transformed with plasmid pS-R84A, pS-R84H, or
pS-D146A (Table I). As shown in Fig.
1 (A and B), SipS
R84A, R84H, or D146A were detectable at 37 °C, but not at 48 °C.
Furthermore, at 37 °C all three mutant proteins were present in
reduced amounts compared with wild-type SipS, the D146A mutant protein
being most unstable. Examination of the accumulation of the hybrid
precursor pre(A13i)-
-lactamase (11) in B. subtilis
ST
(lacking the chromosomal sipS and sipT genes)
transformed with pS-R84A, pS-R84H, or pS-D146A, showed that SPase
activity was strongly reduced at 48 °C, compared with B. subtilis
ST transformed with pGDL41, specifying wild-type SipS
(Fig. 1C). Thus, impaired SPase activity at 48 °C was
paralleled by the disappearance of the SipS mutant proteins, indicating
that SipS R84A, R84H, and D146A were inactivated by proteolysis.
Plasmids and bacterial strains
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Fig. 1.
Thermal inactivation of SipS mutant
proteins. Cells of B. subtilis S (lacking the
chromosomal sipS gene) containing pGDL41 (wild-type
(wt)), pS-L74A, pS-Y81A, pS-R84A, pS-R84H, or pS-D146A were
grown in TY medium until the mid-exponential growth phase
(OD600
0.7). Next, the cultures were divided into two
aliquots, which were incubated for 3 h at either 37 (A)
or 48 °C (B). Cells were separated from the growth medium
by centrifugation, and the cellular levels of the different SipS
proteins were analyzed by SDS-polyacrylamide gel electrophoresis and
Western blotting. The position of SipS is indicated. C, the
accumulation of pre(A13i)-
-lactamase in B. subtilis
ST
containing pGDL41 (wild-type (wt)), pS-L74A, pS-Y81A,
pS-R84A, pS-R84H, or pS-D146A was determined by Western blotting as in
B. p, precursor; m, mature.
S transformed with plasmids pS-L74A or
pS-Y81A. Unexpectedly, both at 37 and 48 °C, the levels of SipS L74A
and Y81A were comparable with those of wild-type SipS (Fig. 1,
A and B). The impaired SPase activity of SipS
L74A and Y81A at 48 °C was reflected by the increased accumulation of pre(A13i)-
-lactamase in B. subtilis
ST transformed
with plasmids pS-L74A or pS-Y81A, as compared with B. subtilis
ST producing wild-type SipS (Fig. 1C).
These results show that leucine 74 and tyrosine 81 are very important
for catalysis at 48 °C, but not for protease resistance of SipS.
S (depleted of WprA, lacking wild-type SipS)
accumulated approximately 10-fold higher levels of SipS D146A than
B. subtilis
S (Fig. 2),
showing that WprA is involved in the degradation of SipS D146A. Interestingly, the levels of SipS R84A and R84H were not increased in
the absence of WprA, indicating that these SipS mutants are not
substrates for WprA. Despite the improved stability at 37 °C, SipS
D146A was not detected immunologically in cells of B. subtilis W
S (pS-D146A) grown at 48 °C (data not shown).
Similarly, at 48 °C, SipS R84A and R84H were not detected in cells
of B. subtilis W
S transformed with pS-R84A or pS-R84H
(data not shown).
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Fig. 2.
Stabilization of SipS D146A by WprA
depletion. To investigate the role of WprA in the degradation of
SipS mutant proteins, B. subtilis S (lacking the
chromosomal sipS gene) was transformed with chromosomal DNA
of B. subtilis KS408 wprA::pMutin2. In
the resulting strain W
S, the transcription of the wprA
gene is IPTG-dependent (20). Cells of B. subtilis
S or W
S, which were transformed with pGDL41
(wild-type (wt)), pS-R84A, pS-R84H, or pS-D146A, were grown
at 37 °C in TY medium without IPTG until the beginning of the
post-exponential growth phase. Next, the cellular levels of SipS
wild-type and mutant proteins were analyzed by SDS-polyacrylamide gel
electrophoresis and Western blotting as in Fig. 1, A and
B. To visualize the lack of effect of WprA depletion on
wild-type SipS, which is produced in much higher amounts than SipS
R84A, R84H, or D146A (see Fig. 1), the film was exposed for a shorter
period of time. The position of SipS is indicated.
ST
(pS-D146A) by WprA Depletion--
We have shown previously that the
production of SipS D146A, to levels that are below the detection limit
for Western blotting, can be sufficient for growth of strains lacking
SipS and SipT, provided that the temperature is not raised above
42 °C (13). To determine whether the depletion of WprA in B. subtilis
ST (pS-D146A) could suppress the temperature
sensitivity of this strain, even though the depletion of WprA did not
result in the accumulation of detectable amounts of SipS D146A at
48 °C (see above), a wprA mutation was introduced into
this strain. As shown in Fig. 3, the
depletion of WprA resulted in the suppression of the temperature
sensitivity of B. subtilis
ST (pS-D146A); while B. subtilis
ST (pS-D146A) stopped growing and started to lyse upon
a temperature shift from 37 to 48 °C, the corresponding
WprA-depleted strain continued to grow normally at 48 °C. These
observations show that depletion of WprA resulted in increased levels
of active SipS D146A even at 48 °C.
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Fig. 3.
Suppression of the temperature sensitivity of
B. subtilis ST (pS-D146A) by depletion
of WprA. B. subtilis
ST (pS-D146A) (lacking the
chromosomal sipS and sipT genes; indicated with
the
) or B. subtilis W
ST (pS-D146A) (indicated with
the
) were grown in TY medium without IPTG at 37 °C. When the
cells reached the mid-exponential growth phase (OD600
0.7; indicated with an arrow), the temperature was shifted
to 48 °C and incubation continued. Samples were taken for optical
density readings at half-hourly intervals.
DISCUSSION
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES
-amino group of lysine 145 is located in a
hydrophobic environment, which is probably essential to lower its
pKa to such an extent that it can function as a
general base (5). As leucine 74 and tyrosine 81 of SipS are the
equivalents of phenylalanine 133 and tyrosine 143 of E. coli
SPase I, it seems likely that their side chains are required to lower
the pKa of the active site lysine residue 83 of
SipS. Even though the latter hypothesis provides an explanation for the
reduced activities of the SipS L74A and Y81A mutant proteins, it does
not explain why these mutant proteins display some residual activity at
37 °C (6, 13), but not at 48 °C. It is, however, conceivable that
the pKa of lysine 83 is further increased at
elevated temperature due to local unfolding events. Alternatively,
local unfolding in the L74A and Y81A mutant proteins at 48 °C may
affect the interaction between the active site serine 43 and lysine 83 residues.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. E. Dalbey for communicating the fact that R146 and D273 of E. coli SPase I form a salt bridge and J. D. H. Jongbloed, M. L. van Roosmalen, and other members of the European Bacillus Secretion Group for stimulating discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by Biotechnology Grant Bio2-CT93-0254 from the European Union.
Supported in part by Biotechnology Grants Bio2-CT93-0254 and
Bio4-CT96-0097 from the European Union.
** To whom correspondence should be addressed. Present address: Dept. of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. Tel.: 31-50-3633079; Fax: 31-50-3632348; E-mail: j.m.van.dijl{at}farm.rug.nl.
2 R. E. Dalbey, personal communication.
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ABBREVIATIONS |
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The abbreviations used are:
SPase, signal
peptidase;
IPTG, isopropyl--D-thiogalactopyranoside.
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