Journal of Bacteriology, June 2000, p. 3111-3116, Vol. 182, No. 11
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
CII by
Escherichia coli FtsH (HflB)

Department of Molecular Genetics and Biotechnology, The Hebrew University-Hadassah Medical School, Jerusalem,1 and The Protein Research Center, Department of Biology, The Technion, Haifa,2 Israel
Received 1 June 1999/Accepted 8 March 2000
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ABSTRACT |
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FtsH (HflB) is a conserved, highly specific, ATP-dependent protease
for which a number of substrates are known. The enzyme participates in
the phage
lysis-lysogeny decision by degrading the lambda CII
transcriptional activator and by its response to inhibition by the
CIII gene product. In order to gain further insight into the mechanism
of the enzymatic activity of FtsH (HflB), we identified the peptides
generated following proteolysis of the phage
CII protein. It was
found that FtsH (HflB) acts as an endopeptidase degrading CII into
small peptides with limited amino acid specificity at the cleavage
site.
-Casein, an unstructured substrate, is also degraded by FtsH
(HflB), suggesting that protein structure may play a minor role in
determining the products of proteolysis. The majority of the peptides
produced were 13 to 20 residues long.
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INTRODUCTION |
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Degradation of regulatory proteins
by ATP-dependent proteases is an important mechanism for the rapid
control of gene activity in all organisms. In bacteria, proteolysis
acts on key regulatory transcription factors which regulate the heat
shock response, stationary-phase and SOS stress responses, capsular
polysaccharide biosynthesis, and the control of the lysis-lysogeny
decision of phage
(12, 14, 27).
FtsH, a membrane-bound Zn2+ metalloprotease, which was
originally identified as an hflB mutation (high-frequency
lysogenization by phage
), is highly conserved in all organisms, is
the only known essential protease in Escherichia coli, and
does not participate in cell division (28, 35). The name
FtsH was coined before the discovery of a second mutation in the
ftsI gene, in the strain carrying the temperature-sensitive
ftsH mutation, that is responsible for cell filamentation
(7). Following infection by bacteriophage
, the
CII
regulatory protein, which activates transcription of the CI repressor
from the pE promoter, is rapidly degraded by FtsH (18, 20, 30,
32). The
CIII peptide extends the half-life of CII, thereby
prolonging CI repressor synthesis, which in turn promotes the lysogenic
pathway (4, 21).
In higher organisms, FtsH orthologs are found in mitochondria and chloroplasts. It was suggested that in yeast mitochondria FtsH plays a key role in maintaining membrane integrity, possessing an ATP-dependent chaperone-like activity and a protease activity that degrades unassembled membrane components (5, 25). Similar roles have been suggested for FtsH in E. coli (2, 3). Recently, mutations in the gene coding for paraplegin, a human ortholog of FtsH, were demonstrated to be responsible for hereditary spastic paraplegia (9). The mutations lead to mitochondrial defects, suggesting that paraplegin is a nucleus-encoded mitochondrial protein (9, 11).
FtsH belongs to a large class of ATPase-containing proteins belonging to the AAA protein superfamily (ATPases associated with different cellular activities), which is characterized by a highly conserved domain of 230 to 250 amino acid residues that contains an ATP binding consensus and ATPase activity (8, 10). These proteins are found in all organisms and play essential roles in the cell cycle, vesicular transport in the Golgi complex, mitochondrial assembly, proteasomes, and cellular metalloproteases. The crystal structure of the N-ethylmaleimide-sensitive fusion protein (NSF) D2 hexameric AAA motif has been determined recently (24, 37).
FtsH is a highly discriminatory protease; the number of known
substrates is rather small and includes the heat shock sigma factor
32, phage
CII, CIII, and Xis proteins, SecY, YccA,
subunit a of the membrane-embedded F0 part of the H+-ATPase
proteins, and SsrA tagged proteins (1, 15, 16, 20, 23, 32,
34). However, little is known about the mechanism by which these
substrates are selected. It was proposed that a different process is
used by FtsH to select soluble and membrane-bound substrates
(19). Nothing is known about the proteolytic products generated by FtsH and the amino acid specificity of FtsH surrounding the cleavage sites.
In this study we monitored the degradation of purified CII in vitro by glutathione S-transferase (GST)-FtsH. We utilized reverse-phase high-performance liquid chromatography (HPLC) and electrospray mass spectrometry (MS) to identify peptide products larger than 3 amino acid residues that were produced by FtsH proteolysis. It was found that CII degradation by FtsH is processive and that only small peptides, 4 to 26 residues long, could be identified. Our results suggest that FtsH can hydrolyze peptide bonds with limited specificity at the cleavage sites.
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MATERIALS AND METHODS |
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Strains.
Strain A9286 is a derivative of BL21(DE3)
(33) carrying plasmid pET-CII. Strain A8926 is a derivative
of W3110 carrying sfhC zad-220::Tn10
ftsH3::kan (28,
29). Strain A9241 is strain A2097 (22) carrying
plasmid pLCIIH6.
Plasmids.
Plasmid pET-CII, which carries the cII
gene fused to a His6 tag at the region corresponding to the
N terminus, was derived from pET-15b (Novagen). In this plasmid a
thrombin cleavage site is engineered between the His6 and
CII sequences. The PCR product of the cII gene was
inserted between the NdeI and BamHI restriction sites. The oligonucleotides used for the PCR were
5'-CGAATTCAACCACACCTA-3' and
5'-CGGGATCCTCAGAACTCCATCTGG-3'. Plasmid pLCII-H6 was
constructed by inserting a PCR product carrying the cII gene
fused to the His6 sequence at the region corresponding to
the C terminus between the NdeI and HindIII
restriction sites of plasmid pTG44 (a
pL expression plasmid from
our collection). The oligonucleotides used for the PCR were
5'-GGGCATCAAATTAAACCAC-3' and
5'-AACCAAGCTTAGTGGTGATGGTGATGGTG-3', and pHG326 (from our
collection) was used as the template. Plasmid pGST-FtsH was constructed
by fusion of the FtsH gene with the first 9 bp deleted to pGEX-2T by
insertion of a BglII-EcoRI PCR fragment of FtsH
into the BamHI-EcoRI sites of the vector. This plasmid was introduced into a strain in which FtsH had been deleted (A8926) to generate strain A9390. The sequences of all inserts were
confirmed by DNA sequencing.
Protein purification.
His6-CII, expressed in
strain A9286 following 3 h of
isopropyl-
-D-thiogalactopyranoside (IPTG) induction at
37°C, was bound to Ni-nitrilotriacetic acid (NTA) beads (Qiagen RA
97008). This protein was eluted by thrombin cleavage to generate CII.
To obtain CII protein carrying additional residues at the amino
terminus, His6-CII protein was eluted with 500 mM imidazole
in 20 mM Tris (pH 8.0)-150 mM NaCl. To obtain CII protein carrying
additional residues at the carboxy terminus, CII-His6,
expressed in strain A9241 following a 30-min heat induction at 42°C,
was bound to Ni-NTA beads and obtained by elution with 500 mM imidazole
in 20 mM Tris (pH 6.8)-300 mM NaCl.
Proteolysis experiments. Degradation reactions were performed as previously described (32). For the addition of "Complete" inhibitor mix (Boehringer Mannheim), a tablet was dissolved in 1 ml, and 0.5 µl was added to the reaction mixture. For the identification of peptides following proteolysis of CII, the proteolysis reactions were carried out at 42°C with 50 pmol of CII and 10 pmol of GST-FtsH.
Liquid chromatography and MS. The peptides produced by FtsH proteolysis were resolved by reverse-phase HPLC on a 1- by 150-mm Vydac C18 column with a linear gradient of 4 to 65% (1%/min) acetonitrile in 0.025% trifluoroacetic acid (TFA), at a flow rate of 40 ml/min. The MS analysis was done in the positive-ion mode using repetitively a full MS scan followed by an MS-MS experiment (collision-induced fragmentation) on the most abundant ion of the MS scan. The MS and MS-MS data from the run were compared to the simulated proteolysis and fragmentation of the substrates by using Sequest software (J. Eng and J. Yates, University of Washington).
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RESULTS |
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Purification and characterization of CII protein.
For rapid
and simple purification of CII, the cII gene was cloned
into pET15b (Novagen) expressing a His6-CII protein fusion in which 6 histidines were added to the N terminus (see Materials and
Methods). Soluble CII protein was purified by binding to agarose-Ni beads and released from the beads by thrombin cleavage (Fig.
1A). This highly purified CII protein was
rapidly degraded by FtsH (data not shown). Degradation of CII is
absolutely dependent on the presence of ATP and is partially inhibited
by the addition of o-phenanthroline (Fig. 1B). As expected,
no inhibition was observed in the presence of a protease inhibitor
cocktail that is known to inhibit a large variety of serine, aspartate,
and cysteine proteases. We estimated, based on circular dichroism (CD)
measurements, that CII in this preparation is highly structured and is
made of 65%
helix, which is similar to the predicted
-helical
content of about 70% (31).
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Peptides recovered following proteolysis of CII. The degradation of CII by FtsH in vitro leads to the disappearance of the full-length protein. No intermediates or partially cleaved proteins were ever observed by gel electrophoresis (see also references 20 and 32). These results suggest that, once initiated, proteolysis by FtsH is rapid and complete and that substrate recognition may be the rate-limiting step. Attempts to obtain degradation intermediates by increasing the ratio of CII to FtsH or by diluting FtsH, a condition known to reduce enzyme activity (T. Ogura, personal communication), proved unsuccessful. No degradation of CII was obtained in the absence of ATP.
To identify CII degradation products, the reaction products were separated by reverse-phase HPLC and analyzed by electrospray MS analysis (liquid chromatography and MS [LC-MS]). Peptides larger than 3 residues were identified by their mass/charge ratio followed by collision-induced fragmentation (MS-MS). Peptides consisting of more than 4 residues that were found following complete digestion of CII in three independent experiments are shown in Fig. 2. These peptides account for most of CII. The peptides cluster in groups with minor differences at the N or C terminus. Only seven peptides were shorter than 10 residues, and about 65% (15 of 23) were 13 to 19 residues long (Fig. 3). We do not know why the N-terminal peptide was not obtained. Interestingly, for many of the peptides (10 of 23), the N-terminal residue is located in the highly hydrophobic region LLAVLEW. However, no obvious cleavage site consensus could be found.
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FtsH degrades
-casein.
In search of additional substrates,
we found that purified FtsH is capable of degrading
-casein. This
protein is known to be present as a noncompact, largely unstructured
protein that is highly modified at the N-terminal region by methylation
and phosphorylation (17). The proteolysis of
-casein is
ATP dependent and is inhibited by the Zn2+ chelator
o-phenanthroline (data not shown). The rate of proteolysis of
-casein is similar to that of CII (Fig.
4). In contrast, we found that FtsH is
unable to degrade bovine serum albumin (BSA) in vitro.
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Peptides recovered following proteolysis of
-casein.
As was
found with CII, no degradation intermediates of
-casein could be
detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). The modifications present in the N-terminal region of
-casein make the identification of the peptide by electrospray MS
difficult. We therefore focused on identifying the peptides generated
from the nonmodified C terminus following cleavage by FtsH. The general
profile of the peptides obtained is similar to that found with CII,
with peptide clustering and with no obvious cleavage site consensus
(Fig. 5). The size distribution of the
peptides obtained is similar to that obtained with CII; most peptides
were found to be 16 to 20 residues long (Fig.
6). About half of the peptides resulted
from cleavage in the highly hydrophobic region AFLLY.
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DISCUSSION |
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Electron microscopy revealed that purified FtsH forms ring-shaped structures with a diameter of 6 to 7 nm, suggesting that the active site is hidden in the central cavity (32). This organization of ATP-dependent proteases was also shown for ClpA/ClpP, HslU/HslV, and the 26S proteasome (see references 6 and 26 for an overview). The active site may be accessible via a narrow channel that prevents accidental protein degradation. The role of the ATPase activity in the degradation of proteins is not known. Its activity is probably utilized in providing the energy needed for conformational changes of the protease and for substrate unfolding to allow the passage of polypeptides into the catalytic chamber (6, 13).
The experiments described in this report were directed to the detailed
analysis of FtsH digestion products. Our inability to obtain
degradation intermediates may suggest that the rate-limiting step in
proteolysis is the formation of substrate-enzyme complexes. Accordingly, rapid and complete proteolysis proceeds once degradation is initiated. The generation of peptides common to the CII and
-casein substrates substantiates the hypothesis that FtsH is an
endopeptidase. For some unknown reason, the addition of tails containing His6 greatly stabilized CII against degradation
by FtsH. Unfortunately, no information on the proteolysis of other substrates by FtsH is available. It is also possible that the peptide
distribution found following proteolysis is determined by the rate of
the peptides' escape from the enzyme.
The identification of peptides following proteolysis by FtsH leads to a number of findings, set forth below.
(i) Proteolysis does not appear to be random. A number of restricted sites for degradation were observed.
(ii) Many of the peptides identified appear as clusters where the cleavage sites are separated by 1, 2, or 3 residues. This phenomenon is found at both the N and C termini of CII derivatives and was observed in all reactions, suggesting either a degree of relaxation of the cleavage sites or that the enzyme "chews" on the ends of the peptides by an auxiliary secondary activity. Similar clusters were found in the analysis of the degradation products of 20-residue-long peptides (our unpublished results).
(iii) The sites of cleavage do not reveal a consensus sequence.
Hydrophobic residues are found to be the preferred residues at the P1
site (the residue N-terminal to the cleavage site). This is especially
evident in the LLAVL cluster in CII and the AFLLY cluster in
-casein. It is possible that the presence of hydrophobic residues at
the peptide improves the stability of these peptides vis-à-vis
secondary proteolytic events.
(iv) Cleavage sites in CII were found within regions predicted to form
helices and also at the ends of the predicted helical stretches,
making it difficult to determine the importance of the secondary and
tertiary structures in determining the preferential sites of cleavage.
It is possible that CII, like
-casein, is highly unstructured during
enzymatic cleavage.
(v) We have previously found that chemical cross-linking of CII produces a collection of monomers, dimers, trimers, and tetramers. Following incubation with FtsH, the monomers and dimers were degraded, the trimeric form was partially degraded, and the tetrameric form was fully stable (31).
It is not known what determines the size distribution of the peptides
produced by FtsH. For both CII and
-casein, the most prevalent size
is 16 to 20 residues, suggesting that peptide size is determined mainly
by the enzyme. Proteolysis may be viewed as a process that occurs when
the substrate is threaded through the enzyme cavity. Alternatively, it
is possible that peptide size is determined by a "molecular ruler"
which reflects the distance between catalytic sites within the enzyme
complex. Such a model has been previously suggested for the proteasome
(36). The oligomeric state of FtsH has not been established.
However, based on the hexameric form of the highly related AAA domain
(the NSF D2 hexameric AAA motif [24, 37]), it is
possible that the protease active site of FtsH exists as a hexamer.
Proteolysis may be viewed as a concerted reaction at six catalytic
sites taking place after the substrate enters the catalytic cavity.
Both models account for the peptide size distribution and for the low
degree of neighboring-peptide overlaps observed in this work.
Additional experimental data are required to distinguish between these
models for FtsH activity.
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ACKNOWLEDGMENTS |
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We thank Yossi Shlomai and Marika Lindahl for stimulating discussions and suggestions, and we thank Teru Ogura, Bernd Bukau, and Koriaki Ito for bacterial strains, plasmids, and unpublished information. We thank Marty Gonzales for CII protein and antibodies, Hilla Giladi and Ariella Oppenheim for critical reading of the manuscript, and Susan Gottesman for stimulating discussions.
This work was supported by a grant from the Israel Science Foundation and was performed, in part, in the Irene and Davide Sala Laboratory for Molecular Genetics.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Genetics and Biotechnology, The Hebrew University-Hadassah Medical School, P.O.B. 12272, Jerusalem, Israel 91120. Phone: 972-2-6757309. Fax: 972-2-6757308. E-mail: ao{at}cc.huji.ac.il.
Present address: Washington University School of Medicine,
Department of Molecular Microbiology, St. Louis, MO 63110-1093.
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