From the Department of Molecular and Cellular
Biology, Faculty of Biotechnology, University of Gdansk, 80-822 Gdansk,
Kladki 24, Poland, the § Department of Biochemistry, School
of Hygiene and Public Health, Johns Hopkins University, Baltimore,
Maryland 21205, and the ¶ Department de Biochimie Medicale, Centre
Medical Universitaire, 1, rue Michel-Servet, 1211 Geneva 4, Switzerland
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ABSTRACT |
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It has previously been established that sequences
at the C termini of polypeptide substrates are critical for efficient
hydrolysis by the ClpP/ClpX ATP-dependent protease. We
report for the bacteriophage The Hsp100 family of protein ATPases was originally identified in
Escherichia coli through the roles of individual ATPases as
regulatory components of certain ATP-dependent serine
proteases, such as ClpP (for reviews see Refs. 1-3). For example, the
ClpA and ClpX family members were isolated as factors that enabled the
ClpP catalytic component to hydrolyze certain polypeptide substrates in
an ATP-dependent manner (4-7). On its own, the ClpP
protease degrades only very small peptides, and such hydrolysis does
not depend on ATP (8, 9). In the presence of
ATP Hsp104, a Saccharomyces cerevisiae homologue of E. coli ClpB, has been shown to be essential for cellular
thermotolerance (10). Interestingly, overexpression of the molecular
chaperone Hsp70 largely reverses the temperature sensitivity exhibited
by the hsp104 mutant (11). These and other findings support
the idea that members of the Clp family of ATPases can perform certain chaperone functions in the absence of their partner protease subunit (12). This hypothesis has been confirmed by recent experimental findings, namely: (i) that Hsp104 participates in the disaggregation of
partially damaged or aggregated protein structures (13, 14) as well as
modulates the conformational transition between normal and altered
forms of prion-like factors (15), (ii) that bacterial ClpA protein
substitutes in vitro for the DnaK/DnaJ/GrpE chaperone machine in activation of the phage P1 RepA replication protein for
binding to its recognition sites in the P1 replication origin (16, 17),
and (iii) that ClpX protein enhances the binding of the Both the protease and chaperone activities of the Clp ATPases
depend on ATP hydrolysis. In the presence of ATP, the ClpA or Hsp104
proteins individually oligomerize to give rise to a hexameric, ring-like structure (25-27). Electron microscopic studies have shown
that ClpA or ClpX rings bind to the 7-fold symmetric ClpP component,
which itself is arranged in a barrel-like double-ring structure. These
interactions produce an overall structure that highly resembles the
eukaryotic 26 S proteosome (28-30).
It has been suggested that ClpA recognizes relatively unfolded
proteins, whereas ClpX is apparently much more specific. For example,
ClpX is known to recognize the We decided to test the universality of this proposed mechanism using
wild type and deletion mutant forms of the Plasmids and Mutagenesis--
Construction of plasmids that
overexpress wild type
For mutagenesis of a region encompassing the putative ClpX consensus
recognition site in the C-terminal region of Proteins--
Highly purified proteins (95% or greater purity)
were used in all experiments described in this paper. The
ClpX-overproducing strain was the kind gift of Drs. Satish Raina and
Dominique Missiakas (University of Geneva). The ClpX and ClpP proteins
were purified as described by Wojtkowiak et al. (6).
The wild type
The
Protease Assays--
The standard protease assay (150 µl) was carried out in 20 mM HEPES, pH 7.6, 10 mM MgAc2, 0.5% Brij, and 10 mM
ATP. Each assay included 5 µg of ClpP, 5 µg of ClpX, and 5 µg of
the appropriate substrate polypeptide. The reaction mixture was
incubated at 30 °C. At desired times, 25-µl portions of the
reaction mixture were withdrawn and processed by 12.5%
SDS-polyacrylamide gel electrophoresis.
Protein-Protein Interaction Assays--
The sensitive ELISA
assay used for monitoring protein-protein interactions has been
previously described in detail by Wawrzynow et al. (18). The
buffer B used in these experiments contained 25 mM
HEPES/KOH, pH 7.6, 150 mM KCl, 25 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol,
2.5% (v/v) glycerol, 0.05% Triton X-100, and 1 mM ATP.
Single-stranded DNA Replication Assay--
The N-terminal Portion of
To exclude the possibility that the central portion of the
The smallest O protein fragment tested here that was efficiently
hydrolyzed by the ClpP/ClpX protease is the
Even though our protein truncation experiments suggest that the very
N-terminal portion of the Binding of
O fragments comprising the N-terminal 110 amino acids showed the
highest affinity for ClpX in the ELISA assay. Removal of the first 18 amino acids at the N terminus led to a significant drop in the
affinities of several O derivatives (O(19-110), O(19-139), and
O(19-299)) for ClpX (Fig. 4). This result suggests the possibility that there may be two or more interaction sites for ClpX in the N-terminal region of O, one of which must be comprised in part by
residues 1-18. We cannot, however, rule out an alternative possibility
that the N-terminal region of O contains a single high affinity site
for ClpX binding that is formed by amino acid residues on both
sides of residue 19.
Role of ClpX in the "Presentation" of In a recent publication, Levchenko et al. (36)
suggested that the ClpX protein recognizes specific sequences located
at or near the C terminus of its substrate proteins. Their results indicated that such sequences may be displayed in a relatively disordered conformation. Experiments performed on MuA transposase, selected mutants of Mu repressor, and SsrA-tagged protein have implicated C-terminal residues of the substrate, mainly nonpolar amino
acids, as being absolutely essential for ClpX recognition. As pointed
out by Levchenko et al. (36), a second group of ClpX substrates is composed of proteins that contain predominantly polar
amino acids near the C terminus, with arginine at the terminal position. In this respect, the In this report, we present evidence that certain of the predictions for
the first class of ClpX substrates) made by Levchenko et al.
(36) are not applicable to This study also revealed that for most O deletion mutant proteins there
is a close correlation between their relative affinity for ClpX and
their rate of hydrolysis by the ClpP/ClpX protease. However, neither
O(19-139) nor O(19-110) obeys this correlation. Both bind ClpX with
moderate affinity, yet are nearly completely resistant to
ClpP/ClpX-mediated proteolysis. We infer, therefore, that binding of
ClpX to a potential substrate polypeptide is not sufficient to ensure
hydrolysis of the polypeptide by the ATP-dependent ClpP/ClpX protease.
Although the N-terminal region of It is likely that relatively weak ClpX-binding sites are
exposed on
the surface of certain protein substrates of the ClpP/ClpX protease. In
this regard, it is important to stress that such ClpX substrates as MuA
transposase and Phd are hydrolyzed by the ClpP/ClpX protease much less
efficiently than Our results show that it is possible to identify proteins that bind to
ClpX but are not hydrolyzed by the ClpP/ClpX protease. This implies
that certain protein substrates that are hydrolyzed by the ClpP/ClpX
protease, such as It was proposed previously that ClpX could participate in regulatory
mechanisms that "decide" whether a particular protein substrate is
either repaired or destroyed (1, 2, 12). This decision could be based
on the affinity of ClpX for the various sites present on a given
protein substrate as well as on the accessibility of appropriate C- or
N-terminal amino acid sequences. The results presented here suggest at
least three possible outcomes for proteins that have ClpX recognition
sites. First, it is possible to find polypeptides (such as O(19-139))
that bind to ClpX but are resistant to hydrolysis by the ClpP/ClpX
protease. In such cases, ClpX is restricted to act only as a molecular
chaperone. Second, if the protein instead also contains an exposed C-
or N-terminal sequence that aids ClpX binding and/or function (such as
The extreme lability of the O protein in vivo was first
reported 25 years ago (47). Later reports demonstrated that the O
protein has a chemical half-life of just 1.5 min in O replication protein, however, that
N-terminal sequences play the most critical role in facilitating
proteolysis by ClpP/ClpX. The N-terminal portion of
O is degraded
at a rate comparable with that of wild type O protein, whereas the
C-terminal domain of O is hydrolyzed at least 10-fold more slowly.
Consistent with these results, deletion of the first 18 amino acids of
O blocks degradation of the N-terminal domain, whereas proteolysis
of the O C-terminal domain is only slightly diminished as a result of deletion of the C-terminal 15 amino acids. We demonstrate that ClpX
retains its capacity to bind to the N-terminal domain following removal
of the first 18 amino acids of O. However, ClpX cannot efficiently
promote the ATP-dependent binding of this truncated O
polypeptide to ClpP, the catalytic subunit of the ClpP/ClpX protease.
Based on our results with
O protein, we suggest that two distinct
structural elements may be required in substrate polypeptides to enable
efficient hydrolysis by the ClpP/ClpX protease: (i) a ClpX-binding
site, which may be located remotely from substrate termini, and (ii) a
proper N- or C-terminal sequence, whose exposure on the substrate
surface may be induced by the binding of ClpX.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S,1 a poorly
hydrolyzable ATP analogue, ClpP/ClpA-mediated hydrolysis of peptides of
moderate length is stimulated. This suggests that interactions between
ClpA and ClpP, promoted by nucleotide binding to ClpA, bring about an
activation of proteolysis by ClpP. Nevertheless, for both the ClpP/ClpA
(9) and ClpP/ClpX (6) proteases, ATP hydrolysis per se is required to
achieve processive degradation of larger polypeptides.
O
replication initiator to the
replication origin (18) and promotes
the binding of the RK2 plasmid-encoded TrfA protein to the
oriRK2 sequence (19). Yet perhaps the most convincing evidence for the notion that Clp ATPases have intrinsic chaperone activity comes from investigations of the bacteriophage Mu DNA replication system. Initially it was discovered that deletion of the
E. coli clpX gene (but not the clpP gene)
resulted in a total inactivation of Mu DNA replication in
vivo (20). Later, studies of the mechanism of Mu DNA replication
in a reconstituted multiprotein system showed that ClpX acts at a step
prior to initiation of DNA replication to promote the disassembly of an
extremely stable MuA transposase nucleoprotein complex (21-23). It is
suggested that ClpX-mediated release of MuA transposase from a
transposition intermediate triggers initiation of Mu DNA replication by
providing access of host replication proteins to Mu DNA ends (24).
O (6, 7), P1 Phd (31), Mu
Repvir (32), MuA transposase (21),
s (33),
and UmuD' (34) proteins, each of which seemingly exists in a native,
properly folded state. These findings support the hypothesis that the
Clp ATPases are specificity factors that are capable of recruiting
different protein substrates and, subsequently, transferring them to
the ClpP subunit for proteolysis (6, 7). It is still not certain which
sequences or protein structures in substrate proteins are recognized by
the Clp/Hsp100 family (2, 35). For several protein substrates,
e.g. MuA transposase (36), mutants of bacteriophage Mu
repressor (32), or SsrA-tagged polypeptides (37), it has been shown
that C-terminal sequences are primarily responsible for their targeting
to the ClpP/ClpX protease. Recently, the presence of PDZ-like domains
has been detected in ClpX and other Clp/Hsp100 family members. These
structural motifs have been suggested to mediate the binding
specificity of these proteins to different protein substrates (36).
Furthermore, it was postulated that the initiating event during
substrate selection by ClpX is the formation of a complex between ClpX
and exposed C-terminal residues of a substrate protein (36).
O replication initiator
protein. Wild type O protein, a highly effective substrate of the
ClpP/ClpX protease, is composed of N- and C-terminal structural domains, which are connected by a relatively unstructured linker region
(38). Contrary to published results with MuA transposase, mutants of Mu
repressor, and SsrA-tagged polypeptide substrates, which demonstrated
the importance of C-terminal sequences for hydrolysis by the ClpP/ClpX
protease, we provide evidence that it is the N-terminal portion of the
O replication protein that is primarily required for its efficient
hydrolysis by this ATP-dependent protease. In addition, we
also demonstrate that stable binding of substrate polypeptides to ClpX
is not sufficient to ensure proteolysis by ClpP/ClpX. We suggest that
in addition to a ClpX-binding site, special N-terminal or C-terminal
sequences must be present in the ClpP/ClpX substrate polypeptide.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
O protein (plasmid pRLM77) or certain
truncated versions of O (O(1-126/181-299) from plasmid pRLM186;
O(1-162) from plasmid pRLM84; O(1-139) from plasmid pRLM146;
O(19-139) from plasmid pRLM147; O(1-110) from plasmid pRLM150; and
O(19-110) from plasmid pRLM151) have been described (38). Plasmids
that overexpress O(150-299) (plasmid pRLM216) and O(19-299) (plasmid
pRLM312) will be described in detail elsewhere. In brief, plasmid
pRLM77 contains a 1.5-kilobase pair AluI fragment harboring
the
O gene inserted into the multiple cloning site
present on expression plasmid pRLM76 (39). Plasmid pRLM84 was
constructed from plasmid pRLM77 by digesting the plasmid at the unique
EcoRI site, located within the
O gene,
filling in the cohesive end with the Klenow fragment of DNA polymerase I, and religating. This creates a 4-base pair duplication that results
in an in-frame chain termination codon. Plasmid pRLM186 was constructed
by digesting plasmid pRLM77 with EcoRI, treating the
linearized DNA with Bal31 nuclease, and religating. Plasmids that
overproduce truncated
O proteins were identified and sequenced so
that the end points of each internal deletion in the O gene could be defined. A plasmid producing an O protein, deleted for amino
acid residues 127-180, was named pRLM186. Plasmids pRLM146, pRLM147,
pRLM150, pRLM151, pRLM216, and pRLM311 each contain a segment of the
O gene generated by PCR-mediated amplification of DNA
sequences carried on the
dv-like plasmid pRLM4 (40). The primers
used at one end for PCR amplification carried in order (5' to 3') were
a BamHI restriction site, a strong ribosome-binding site
from gene 10 of phage T7, a properly spaced ATG initiation codon,
followed by the desired O coding sequence. The primers used
for synthesis of the complementary strand contained in order (5' to 3')
were XbaI, PstI, and SalI restriction
sites, two tandem chain termination anti-codons, and a sequence
complementary to the codons for the desired new C terminus. The
PCR-amplified DNA preparations were digested with both BamHI
and PstI or with both BamHI and SalI.
These DNA fragments were subsequently inserted by ligation between the
BamHI and PstI polylinker sites present on
plasmid pRLM76 (pRLM146, pRLM147, pRLM150, and pRLM151) or between the
BamHI and SalI polylinker sites present on
plasmid pRLM156 (41) (pRLM216 and pRLM311). The expression vector for O(19-299), pRLM312, was constructed by ligating the appropriate BamHI/SalI fragment of pRLM311 between the
BamHI and SalI sites of pET21(+) (Novagen). All
plasmid sequences derived from PCR amplification were verified by DNA
sequence analysis using the dideoxynucleoside triphosphate chain
termination method.
O, the 488 base pairs
BamHI-PstI fragment of pRLM216 was cloned into BamHI-PstI pGEM3Zf(
) vector (Promega) to give
plasmid pMZ2. This plasmid was subsequently transformed into the
bacterial strain CJ236 dut
ung
, and single-stranded, uracil-containing DNA was
produced by mobilization with helper phage VCSM13 (Stratagene),
according to the method of Kunkel et al. (42). Primer
extension reactions were performed in the presence of 5%
Me2SO in the reaction mixture. The mutations resulting in
O(150-299)-D287A and the C-terminal truncation
O(150-284),
missing the final 15 amino acids of O, were constructed by
oligonucleotide-directed mutagenesis using the primers: D287A 5'-GTGTTTGTCAGGGCGAGTTTTGGTTTGC-3' and PstI-stop284
5'-CTGTGTTTGTCAGCT GCAGTTATGGTTTGCT-3', respectively. Candidate mutant
plasmids were initially screened for loss of a TaqI
restriction site (O(150-299)-D287A) or for a specific
BamHI-PstI digestion pattern (O(150-284)). The BamHI-PstI inserts of positive clones were
verified by the dideoxy sequencing technique using
SequenaseTM version 2.0. Finally, the
BamHI-PstI inserts were excised and recloned into
pHE6 (43) to yield the expression plasmids pMZ5 and pMZ6, encoding
O(150-299)-D287A and
O(150-284), respectively.
O protein, O(1-299), deletion mutant proteins
harboring the N-terminal domain of O (
O(1-162),
O(1-139), and
O(19-139)), the central deletion mutant protein,
O(1-126/181-299), and
O(19-299) were each purified as described
by Roberts and McMacken (44). Polypeptides containing the
C-terminal domain of
O, namely
O(150-299),
O(150-284),
and
O(150-299)-D287A, were purified as follows. After cell lysis
(24 g of cells) in the presence of lysozyme and spermidine and
centrifugation (44), the supernatant (120 ml) was mixed with ammonium
sulfate (0.3 g/ml supernatant) and stirred for 30 min at 4 °C.
Following centrifugation at 70,000 × g for 30 min at
4 °C, the pellet was resuspended and dialyzed against buffer A (50 mM Tris/HCl, pH 7.4, 0.1 M KCl, 0.1 mM EDTA, 10% (v/v) glycerol, 5 mM
dithiothreitol, 0.02% Triton X-100) and applied to a Pharmacia
Q-Sepharose (2.5 × 14 cm) column that had been equilibrated with
buffer A. The void volume fractions were pooled (60 ml) and applied to
a Whatman P11 phosphocellulose column (1.5 × 8 cm) that was
equilibrated with buffer A. Bound proteins were eluted with a linear
gradient (120 ml) of 0.1-0.7 M KCl in buffer A. Fractions
containing significant quantities of O protein were pooled (35 ml) and
applied to a hydroxylapatite column (1.5 × 5 cm) that was
equilibrated with buffer A. Bound protein was eluted with a linear
gradient (120 ml) of 0.1-0.7 M KCl in buffer A. The
fractions containing pure protein were pooled, frozen, and stored at
80 °C prior to use.
O(1-110) N-terminal fragment of
O was purified from the
membrane fraction of the crude lysate. After cell lysis (40 g of cells)
with lysozyme and centrifugation of the lysate at 70,000 × g for 60 min at 4 °C, the membrane-containing pellet was
resuspended in 50 ml of buffer B (50 mM Tris/HCl, pH 7.4, 0.2 M KCl, 1 mM EDTA, 5 mM
dithiothreitol, 10% (v/v) glycerol, 0.04% Triton X-100, 4 M urea) and sonicated. Following gentle overnight shaking,
the suspension was centrifuged at 70,000 × g for 30 min at 4 °C. The supernatant (70 ml) was dialyzed against 2 liters
of buffer C (50 mM Tris, pH 7.4, 0.2 M KCl, 1 mM EDTA, 5 mM dithiothreitol, 10% (v/v)
glycerol, 0.04% Triton X-100) and applied to a P11 phosphocellulose
column (2.5 × 14 cm) equilibrated with buffer C. The column was
washed with several column volumes of buffer C, and the bound proteins
were subsequently eluted with a linear gradient (400 ml) of KCl
(0.2-0.8 M in buffer C). Fractions containing significant
amounts of the N-terminal domain of
O protein were pooled, dialyzed
against buffer C, and applied to a 6-ml Resource MonoS column
(Pharmacia) that was equilibrated with buffer C. The bound protein was
eluted with a 50-ml KCl gradient (0.2-0.5 M in buffer C)
and was stored at
80 °C.
O(19-110) protein was purified as described above for
O(1-110), except that a hydroxylapatite column (1.2 × 4.8 cm)
was substituted for the Resource MonoS column during the final
purification step. A linear gradient (50 ml) from 0.1 to 0.7 M KCl in buffer C was used to elute bound protein from the
column. Fractions containing homogeneous O(19-110) were frozen in
liquid nitrogen and stored at
80 °C. The N-terminal sequences of O
and all purified O deletion mutant proteins were determined using an
ABI 473A Applied Biosystems Microsequencer system.
single-stranded
DNA replication assays were performed as described (45, 46) with
modifications as follows. M13mp9 viral DNA (165 ng) was used as the
single-stranded DNA template, and reaction mixtures also contained 90 ng of E. coli GrpE protein. DNA synthesis was measured after
a 20-min incubation at 30 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
O Is Important for the
ClpP/ClpX-dependent Hydrolysis of
O--
It has
previously been shown that the ClpX and ClpA ATPases recognize specific
protein substrates, leading to selective ClpP-dependent proteolysis (6, 18). Subsequent studies led to the hypothesis that
sequences at the C termini of substrates of the ClpP/ClpX and ClpP/ClpA
proteases are required for targeting the substrate polypeptide to the
ClpX or ClpA ATPases (36, 37). We decided to investigate whether this
C-terminal substrate recognition mechanism applies uniformly to all
physiological substrates of ClpX. We tested wild type
O replication
protein, which is known to be a particularly good substrate of the
ClpP/ClpX protease both in vivo and in vitro, and
various truncated derivatives of O (Fig. 1) for their sensitivity to hydrolysis by
the ClpP/ClpX protease. We were surprised to find that the C-terminal
domain of
O protein, represented by
O(150-299), is hydrolyzed
much more slowly by the ClpP/ClpX protease than either wild type
O(1-299) or a N-terminal domain fragment,
O(1-162) (Figs.
2 and 3).
Control experiments indicate that both the N- and C-terminal domains of
O are apparently folded correctly, even in the absence of the other
domain, because each individual domain retains its relevant biological
activity. Thus, the
O(1-162) polypeptide, which contains the
origin recognition elements of O, has been shown to bind specifically
to the O binding sites present in ori
DNA (38). Likewise,
the C-terminal O(150-299) fragment remains capable of functionally
interacting with the
P-DnaB complex to support the transfer of DnaB
helicase onto DNA (45), as indicated by the significant levels of DNA
replication obtained (Table I) when the
O(150-299) protein replaces wild type O protein in the
single-stranded replication reaction (45, 46). The N-terminal origin
recognition domain alone is incapable of supporting this
single-stranded DNA replication reaction (Table I).
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Fig. 1.
Representations of the structures of the
various O deletion mutant proteins used in
this work.
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Fig. 2.
Kinetics of ClpP/ClpX-mediated hydrolysis of
wild type O and of O derivatives composed of
either the N- or C-terminal domain. Proteolysis was carried out as
described under "Materials and Methods."
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Fig. 3.
Kinetics of hydrolysis of various
O deletion mutant proteins by the ClpP/ClpX
protease. Proteolysis assays were performed as described under
"Materials and Methods." The times listed under
t1/2 indicate the measured half-lives of the
individual polypeptides in the proteolysis reaction. The amount of wild
type or mutant
O polypeptide present in each sample was calculated
after densitometric analysis, using a Bio-Rad densitometer, of the
relevant Coomassie Blue-stained band.
Activity of O protein and O deletion mutant proteins in the
single-stranded replication reaction
single-stranded replication assays were performed as
described under "Materials and Methods." The concentrations of O or
O deletion mutant proteins in the reaction mixture were:
O(1-299),
180 nM;
O(19-139), 200 nM;
O(150-299), 280 nM.
O
protein provides the structural motif that permits binding to ClpX, we
tested a
O mutant with a large central deletion,
O(1-126/181-299). This O deletion mutant protein retains significant functional activity in an in vitro
DNA replication
system (38).
O(1-126/181-299) is almost as sensitive to
ClpP/ClpX-mediated proteolysis as wild type
O (Fig. 3). In
addition, the deletion of the C-terminal 15 amino acids from the
O(150-299) polypeptide, resulting in an O(150-284) truncated
fragment, only slightly alters its sensitivity to proteolysis (Fig. 3).
This result indicates that the
O protein behaves differently than
the MuA transposase or a related fusion protein, Arc-MuA, where
deletion of the last 8 amino acids from their C termini almost
completely stabilized MuA against ClpP/ClpX-dependent
proteolysis (36). It was noted previously that the aspartic acid at
position 287 of
O is a part of the LDL/A consensus sequence present
in all known ClpX protein substrates (2). To test this putative
recognition sequence, we introduced a point mutation, D287A, in the
O(150-299) polypeptide. However, contrary to expectation, this
sequence alteration did not substantially affect the rate of
degradation of the O C-terminal domain (Fig. 3).
O(1-110) N-terminal polypeptide (Fig. 3). We discovered, however, that further deletion of
the first 18 amino acids (MTNTAKILNFGRGNFAGQ) from this truncated substrate, resulting in
O(19-110), renders it almost completely resistant to protease action (Fig. 3). Yet O(19-110) retains the capacity to form a homodimer that binds specifically to the O recognition sites in ori
(38). We presume, therefore,
that a significant proportion of O(1-110) is folded correctly. We
performed the same experiments with
O(1-139) and
O(19-139)
and again found that deletion of the first 18 amino acids at the N
terminus causes a severe inhibition of ClpP/ClpX-dependent
proteolysis (Fig. 3). Interestingly, the
O(19-299) protein lacking
the first 18 amino acids is hydrolyzed but at a much slower rate than
the wild type
O, with kinetics comparable with those for
degradation of the C-terminal domain of O (i.e. O(150-299);
Fig. 3).
O protein is largely responsible for
targeting this phage replication initiator to the ClpP/ClpX protease,
we observed that O fragments comprised solely of the C-terminal domain
are moderately sensitive to this ATP-dependent protease
(Fig. 3). This result indicates the presence of one or more
"weaker" protease recognition sites within the C-terminal half of
the
O polypeptide.
O Deletion Mutant Proteins to the ClpX
ATPase--
We used a modified ELISA technique to determine the
relative binding affinity of ClpX for the various
O protein
constructs used in this work (Fig. 4).
For most substrates that are sensitive to the ClpP/ClpX protease, there
is a direct correlation between the apparent affinity of the substrate
for ClpX and the rate of substrate hydrolysis. The single exception,
O(19-299), has a slightly higher affinity for ClpX than
O(150-299) (Fig. 4) yet yields similar rates of degradation (Fig. 3).
Nevertheless, it is also apparent from these results that ClpX is
capable of binding with moderate affinity to polypeptides that are
inert as substrates for ClpP/ClpX. For example, O(19-139) and
O(19-110) each form stable complexes with ClpX yet are totally
resistant to proteolysis. We conclude that stable binding of ClpX to a
polypeptide is not sufficient to ensure hydrolysis of the polypeptide
by ClpP/ClpX.
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Fig. 4.
Binding of ClpX to various
O protein derivatives. The appropriate
O
protein derivative (50 µl, at a protein concentration of 10 µg/ml
in phosphate-buffered saline buffer) or bovine serum albumin was fixed
onto ELISA plate wells as described previously (18) and incubated for
1 h at room temperature. Increasing amounts of ClpX in buffer B
were then added. Following a 30-min incubation at room temperature, the
ELISA plates were washed once with buffer B and three times with
phosphate-buffered saline buffer supplemented with bovine serum
albumin. Following these steps, 100 µl of a 1:10,000 dilution of
anti-ClpX serum was added, and the plates were incubated for an
additional 2 h at room temperature. The amount of ClpX protein
stably retained in each well was determined by use of a TMB peroxidase
EIA substrate kit (Bio-Rad). Absorbance at 490 nm was measured using a
microplate reader. Each indicated absorbance value represents the
average value obtained in four independent experiments.
O to ClpP--
To
investigate the role of ClpX in delivering the
O substrate to the
ClpP subunit of the protease, we made use of the nucleotide analogue
ATP
S, which was previously shown to block the
ClpP/ClpX-dependent hydrolysis of
O (18). Using an
ELISA assay, we determined the minimal requirements for ClpP to enter
into a protein complex with O or O deletion mutant proteins (Fig.
5). Our results indicate that
O does
not enter into a complex with the ClpP protein unless both ClpX and
ATP
S are present. The fact that the association of
O with ClpP
depends on ClpX suggests that at least in the presence of ATP
S, a
O-ClpX-ClpP intermediate complex is formed. Interestingly the
O(19-139) truncated protein, which binds moderately well to ClpX
(Fig. 4) but is not hydrolyzed by ClpP/ClpX protease (Fig. 3),
forms only a relatively weak complex with ClpP, even when both ClpX and
ATP
S are present (Fig. 5). This result may indicate that the first
18 amino acids of
O protein are required for efficient
ClpX-dependent presentation of protein substrate to ClpP
protease.
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Fig. 5.
The binding of O to
ClpP depends on the simultaneous presence of ClpX and ATP.
A,
O(1-299). B,
O(1-139) or
O(19-139). Wild type or mutant O protein (50 µl, at a protein
concentration of 10 µg/ml in phosphate-buffered saline buffer) was
fixed onto the wells of an ELISA plate as described previously (18).
Subsequently, increasing amounts of ClpP (in the presence of 100 ng of
ClpX in buffer B) were added in the presence (open squares)
or absence (closed squares) of 1 mM ATP
S. In
a control experiment, increasing amounts of ClpP in the presence of
ATP
S (but in the absence of ClpX) were added (open
circles, A). After a 30-min incubation at room
temperature, the ELISA plates were washed once with buffer B and three
times with phosphate-buffered saline supplemented with bovine serum
albumin. Next, anti-ClpP serum (100 µl of a 1:8,000 dilution) was
added, and the plates were incubated for an additional 2 h at room
temperature. The amount of ClpP protein retained in individual wells
was determined using a TMB peroxidase EIA substrate kit (Bio-Rad). The
absorbance at 490 nm was measured using a microplate reader. In a
control experiment, when bovine serum albumin instead of
O was
used, complexes containing ClpP were not detected (data not
shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
O replication protein, whose 10 C-terminal residues (residues 290-299) are -NTDWIYGVDL, apparently does not fit neatly into either of the known categories of ClpX substrates.
O protein. Specifically, we showed that
the N-terminal portion of
O is more efficiently hydrolyzed by the
ClpP/ClpX protease than is the O C-terminal region. The following
experimental results support this conclusion: (i) the wild type
O
protein, O(1-299), is a much better substrate for ClpP/ClpX than
either the O(19-299) or O(150-299) deletion mutant proteins, even
though all three polypeptides share the same C-terminal sequence; (ii)
three O deletion mutant proteins, O(1-162), O(1-139), and O(1-110),
that each harbor a seemingly folded N-terminal domain, are rapidly
hydrolyzed by ClpP/ClpX with kinetics similar to those for wild type
O protein; (iii) the further removal of the first N-terminal 18 amino acids from
O(1-110) or
O(1-139) converts each to a form
that is largely resistant to ClpP/ClpX-mediated proteolysis; (iv) the
deletion of the first 18 amino acids at the N terminus of wild type
O(1-299) results in a partial stabilization of this protein against
hydrolysis by the ClpP/ClpX protease; and (v) deletion of the 15 C-terminal residues of O(150-299) causes only a slight reduction in
the rate of proteolysis by ClpP/ClpX.
O plays the primary role in
substrate recognition by ClpX, our investigations indicate that the
C-terminal domain of O retains some sensitivity to the ClpP/ClpX
protease. This finding suggests that a weaker ClpX recognition site(s)
is located within the C-terminal region of the
O polypeptide. Interestingly, O(19-299) and O(150-299), which possess different N-terminal sequences but identical C-terminal portions, are hydrolyzed by ClpP/ClpX protease with similar kinetics. This may imply, when the
18 amino acids at the N terminus are absent, that the C-terminal domain
of
O determines the rate of ClpP/ClpX-dependent proteolysis.
O.2,3
These ClpX substrates are hydrolyzed at rates roughly comparable with
or even lower than that of the
O(150-299). Thus, it is certainly
possible that some proteins recognized by ClpX may possess multiple
sequences or structural elements with varying degrees of affinity for ClpX.
O, possess at least two structural elements that
define its vulnerability to the protease. One element is presumably a
ClpX recognition motif, whereas the other element apparently is
composed, at least in part, of a specific sequence at the N terminus or
C terminus of the polypeptide. The latter sequence may play a role in
efficient delivery of a ClpX-bound polypeptide to the active site of
the ClpP protease, perhaps by enabling efficient unfolding of the
substrate. As shown in this paper, one of the ClpX recognition sites in
O is located internally, somewhere within the 19-110-amino acid
fragment of O. The demonstration that ClpX recognition sites are not
necessarily located at polypeptide termini agrees with a previous
genetic analysis of the stationary phase sigma factor
(
s), a study concluding that ClpX recognizes an internal
amino acid sequence in this substrate protein (33). Regardless of which portion of a protein is primarily recognized by ClpX, it is likely that
the final outcome, in term of proteolysis, will depend not only on the
presence of an intrinsic high affinity ClpX recognition sequence but
also on the availability of a special C- or N-terminal sequence to make
contact with ClpX and/or ClpP during delivery of the substrate
polypeptide to the ClpP catalytic subunit. This precise situation was
encountered previously by Laachouch et al. (32). They fused
the C-terminal 7 amino acids of the ClpX-sensitive Mu repressor vir3061
protein to the C termini of CcdA and CcdB proteins encoded by plasmid
F. Fusion of this sequence to CcdB did not endow a sensitivity to
ClpP/ClpX, whereas fusion of the same sequence to CcdA did engender a
partial sensitivity to this ATP-dependent protease.
O and O(1-139)), then such polypeptides may be immediately
targeted to ClpP and hydrolyzed. Third, a native protein could contain
both a ClpX-binding site(s), located distantly from polypeptide
termini, as well as a "proper" C- or N-terminal sequence for ClpX
that is not exposed on the surface of the protein. In this situation,
ClpX action would be restricted to binding, but such binding events may
in turn result in partial unfolding of the substrate protein. In the
event that the special C- or N-terminal sequence of the substrate polypeptide is still not accessible once all ClpX-induced structural rearrangements have been completed, the substrate could dissociate from
ClpX and have an opportunity to refold. Repeated cycles of ClpX
binding, followed by substrate unfolding, dissociation, and refolding
could result in the repair of a partially denatured protein substrate.
Thus, ClpX would act as a molecular chaperone in this instance. On the
other hand, if the initial binding of ClpX and unfolding of the
substrate results in the formation of a stable complex between ClpX and
the special sequence at the N or C terminus, then the refolding
reaction may be largely precluded. In this case, ClpX would rapidly act
to "deliver" the partially unfolded protein substrate to the ClpP
protease for degradation.
-infected cells
(48-50) and that an ATP-dependent protease mediated this rapid turnover (50, 51). In light of the identification of the
responsible protease as the ClpP/ClpX protease (6, 7) and of recent
findings that O protein bound to the
replication origin is, both
in vivo (52) and in vitro (53), stabilized against proteolytic attack, it is likely that the studies reported here
will provide useful insights into the O turnover process in
vivo. For example, the rapid degradation of free intracellular O
protein suggests that the various ClpX recognition motifs in O
identified in the present study are freely accessible to ClpX in
vivo. In contrast, the resistance of O protein assembled at ori
(the O-some) to ClpX/ClpP attack makes it likely that
one or more of the ClpX recognition motifs in the O-some nucleoprotein structure are inaccessible to ClpX. It is also conceivable that protein-protein and protein-DNA interactions within the O-some stabilize O protein against a critical ClpX-mediated unfolding step
that is required for initiation of proteolysis.
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ACKNOWLEDGEMENTS |
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We thank Drs. Satish Raina and Dominique Missiakas for providing the ClpX protein overproducing plasmid and Dr. Alicja Wegrzyn for the ClpP overproducing plasmid. We thank the Foundation for Polish Science (BITECH program) for fermentor equipment.
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FOOTNOTES |
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* This work was supported by Grant 6P04A01712 from the Polish State Committee for Scientific Research, National Institutes of Health Grants GM36526 and GM32253 from the U. S. Public Health Service, and Project 7PLPJ048480 and Grant 31-47283-96 from the Swiss National Foundation.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.
To whom correspondence should be addressed. E-mail:
zylicz{at}biotech.univ.gda.pl.
2 H. Nakai, personal communication
3 M. Yarmolinsky, personal communication.
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ABBREVIATIONS |
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The abbreviations used are:
ATPS, adenosine
5'-O-(3-thiotripho-sphate);
ELISA, enzyme-linked immunosorbent
assay;
PCR, polymerase chain reaction.
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REFERENCES |
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