Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India
Correspondence
Parthasarathi Ajitkumar
ajit{at}mcbl.iisc.ernet.in
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank accession number for the sequence reported in this paper is AF037269.
Present address: Department of Pathology, UCLA School of Medicine, Los Angeles, CA, USA.
These two authors contributed equally to this work.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
These diverse, but specific, functions of stress response protease FtsH are potentially helpful for the effective adaptation to the environment involving various stress conditions, either inside host cells, as in the case of a pathogen such as Mycobacterium tuberculosis, or in the environment, as in the case of a nonpathogenic saprophyte such as Mycobacterium smegmatis. We earlier cloned and expressed the ftsH gene of M. tuberculosis H37Rv (MtftsH) in order to understand the functional role of the protease in the mycobacterial pathogen (Anilkumar et al., 1998). In order to carry out a comparative structural and functional analysis, in this communication we report: (i) structural organization, cloning and expression of the ftsH gene of M. smegmatis SN2 (MsftsH) in E. coli cells, (ii) functional complementation by the gene, and (iii) efficient proteolytic activity of MsFtsH protease on the specific and typical FtsH substrates, namely heat shock transcription factor
32 and the protein translocase subunit SecY, in vivo in E. coli cells.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Cloning of the MsftsH gene.
The MsftsH gene was cloned from two partial genomic DNA libraries of M. smegmatis SN2. One microgram of genomic DNA was digested with PstI at an enzyme : DNA ratio of 1 : 10, overnight at 37 °C. The digested genomic DNA was fractionated and transferred from the gel onto a nylon membrane using capillary transfer. The probe used for the detection of MsftsH ORF-specific fragment in the PstI digest of genomic DNA was a 364 bp PCR product, which was amplified from the ftsH ORF of M. tuberculosis H37Rv (MtftsH) present in the SCY6F7 cosmid (a kind gift from Dr Stewart Cole, Pasteur Institute, France; Cole et al., 1998) as described in our earlier work (Anilkumar et al., 1998
). A non-radioactive labelling and detection system (Gene Images) was used for the labelling of the MtftsH PCR probe and for the detection of signals. Labelling of the PCR probe, hybridizations, and detection of signals were carried out as per the manufacturer's instructions. Southern hybridization of the PstI digest revealed a single band in the 3·2 kb region, which was eluted using low-melting-point agarose gel (Sambrook et al., 1989
). The eluted DNA fragments were ligated to PstI-digested, calf-intestinal-phosphatase-treated pBS(SK+) vector. E. coli JM109 cells were electrotransformed with the ligation mixture to obtain a PstI partial genomic DNA library, hereafter referred to as the PstI library. The transformants, which grew on agar containing ampicillin, were replica plated. The replica plates were used for colony hybridization (Sambrook et al., 1989
) using the MtftsH-specific probe. The amino acid sequence, deduced from the partial nucleotide sequence of the inserts from a few positive clones, revealed a high percentage of identity with the sequences of FtsH proteins from other bacterial systems, confirming that the clone carried the ftsH gene of M. smegmatis SN2 (MsftsH). Complete sequencing of the 3·2 kb insert showed that the fragment carried a partial MsftsH gene containing only the highly conserved AAA domain and the protease domain. The region corresponding to the N-terminal transmembrane portion of MsFtsH protein was absent from this clone obtained from the PstI library.
In order to obtain the 5' portion of the MsftsH gene, a KpnI partial genomic DNA library of M. smegmatis SN2 was constructed in pBS(SK+) vector, in a manner identical to the construction of the PstI library. A 548 bp ClaIPstI DNA fragment, which corresponded to the N-terminal transmembrane region of MtftsH gene, was used to probe the KpnI genomic DNA digest. The 548 bp fragment was obtained by PstI digestion of the pBSMtH vector (Anilkumar et al., 1998). Southern hybridization of the KpnI-digested, fractionated genomic DNA, with the 548 bp ClaIPstI DNA fragment as the probe, showed a band in the 2·9 kb region, which was used for making the second partial genomic DNA library, hereafter referred to as the KpnI library. The amino acid sequence deduced from the nucleotide sequence of the inserts from a few positive clones revealed a region corresponding to the transmembrane portion of the FtsH protein. The pBS(SK+) recombinant clone, containing the KpnI fragment, was passaged through E. coli GM 2151 (dam, dcm), to make the ClaI site on the insert sensitive to cleavage by the enzyme. It was then digested with ClaI and PstI to release a 0·576 kb fragment that represented the transmembrane region of the MsftsH gene. The PstI library was digested using PstI and XhoI enzymes to obtain the 2·3 kb DNA fragment carrying the remaining portion of the MsftsH gene, including the stop codon. The 0·576 kb ClaIPstI fragment and the 2·3 kb PstIXhoI insert were ligated to pBS(SK+) vector, which was digested with ClaI and XhoI enzymes. The three-way ligation resulted in the cloning of a 2·876 kb DNA fragment that contained the complete ORF of the MsftsH gene as a translational fusion with the lacZ' gene at the N-terminus of the MsftsH gene. This construct, pBSMsH, contained the complete MsftsH ORF of 2·31 kb.
Cloning of the MsftsH ORF for in vivo expression.
The pBSMsH construct was digested with XhoI, end-filled with T4 DNA polymerase, and then digested with ClaI to release the ftsH ORF as a ClaIXhoI blunt-ended fragment, which was ligated to the pT18 vector. pT18-zip is a lac promoter-based vector system, derived from pBS(KS+), containing the T18 fragment of the adenylate cyclase (cya) gene fused to the leucine zipper at the N-terminus (Karimova et al., 1998). We removed the region encoding the leucine zipper from the vector by KpnI digestion and religation to generate the pT18 vector, which was further digested with ClaI and EcoRV. The ClaIXhoI blunt-ended fragment containing the MsftsH ORF, which was obtained from the pBSMsH vector, was ligated to the ClaIEcoRV site of the pT18 vector to obtain the pT18MsH vector, wherein the MsFtsH protein is a translational fusion with the cya gene fragment.
Co-expression of MsFtsH and EcFtsH.
The vector pBHB1 (a kind gift from Dr Christophe Herman, University of California San Francisco, USA; Herman et al., 1997) was used for co-expression of EcFtsH, along with MsFtsH. E. coli JM109 cells were co-transformed with pT18MsH and pBHB1, and transformants were selected on L agar containing appropriate antibiotics. The cultures were grown in L medium and induced with 0·4 % L-arabinose and 1 mM IPTG. Cells were fixed onto poly-L-lysine-coated slides and photographed under a Zeiss microscope at x100 using a CCD camera. Cell length was calculated using ImageJ software from NIH (http://rsb.info.nih.gov/ij/).
Membrane isolation.
E. coli cells carrying appropriate constructs were grown to an OD600 of 0·50 and were induced with 1 mM IPTG for 150 min. The cells were pelleted at 5000 r.p.m. for 10 min and suspended in 100 mM phosphate buffer, pH 7·2, and sonicated in the presence of 1 mM PMSF. M. smegmatis SN2 cells, grown in YK liquid medium to an OD550 of 0·50, were sonicated in PBS, pH 7·2, in the presence of 1 mM PMSF. After removing debris by centrifugation at 12 000 r.p.m. for 15 min at 4 °C, the supernatant was used as the total-protein extract. For the protein localization studies, this supernatant was partitioned into pellet and supernatant fractions by ultracentrifugation at 40 000 r.p.m. in a type 50Ti rotor in a Beckman ultracentrifuge for 2 h. The supernatant was used as the cytosolic fraction. The pellet was suspended in 1 M NaCl solution, incubated at 4 °C for 10 min, and separated again into pellet and supernatant by ultracentrifugation using the parameters mentioned above. The pellet fraction was suspended in 50 mM phosphate buffer, pH 7·2, and used as the membrane preparation as described by Tomoyasu et al. (1993b).
Assay for proteolytic activity of MsFtsH in vivo.
The proteolytic activity of MsFtsH, which was expressed from pT18MsH, was assayed in vivo using the conventional substrates of FtsH protease, namely 32 (Ec
32) and SecY (EcSecY) proteins of E. coli. For the MsFtsH protease assay, changes in the levels of endogenous Ec
32 protein and of ectopically expressed EcSecY protein, which was induced from pKY248, were monitored independently in response to induction of MsFtsH from pT18MsH. As a positive control for proteolytic activity, degradation of endogenous Ec
32 and ectopically expressed EcSecY proteins by EcFtsH, which was induced from the pSTD113 vector, was monitored. E. coli AR 5090 (
ftsH : : kan, sfhC21) cells were co-transformed with the plasmid vector pKY248, carrying the EcsecY gene, plus pBluescript (vector control), pSTD113, pT18 (vector control) or pT18MsH, and colonies were selected on L agar containing relevant antibiotics. Exponentially growing cultures of E. coli AR 5090 cells carrying two compatible plasmid vectors, pKY248 and pBluescript, pSTD113, pT18 or pT18MsH, were induced with 2 mM IPTG for 3 h. Cells were harvested and lysed in 8 M urea in Tris/HCl, pH 8·0, containing 150 mM NaCl. The lysate was centrifuged at 12 000 r.p.m. to remove cell debris and unlysed cells. The supernatant was used for immunoblotting of Ec
32 and EcSecY proteins.
Western blotting.
Equal amounts of total protein from the membrane and cytosol fractions of total lysates from transformed or untransformed E. coli or M. smegmatis, or from the uninduced or induced cultures of E. coli cells (25 µg and 50 µg respectively for Ec32 and EcSecY Western blots), were separated on 10 % SDS-polyacrylamide gel (Laemmli, 1970
) or on 16·1 % acrylamide0·12 % N,N'-methylene-bis-acrylamide (for SecY) and transferred onto a PVDF membrane using a semi-dry transfer apparatus, which was manufactured in the Indian Institute of Science. The membranes were blocked using 0·1 % Tween 20 and 5 % non-fat dried milk in phosphate-buffered saline (PBS). Primary antibodies against EcFtsH, EcSecY, Ec
32 and EcRRF proteins of E. coli, namely anti-EcFtsH (a kind gift from Dr Teru Ogura, Kumamoto University, Japan; Tomoyasu et al., 1993b
), anti-EcSecY (a generous gift from Dr Yoshinori Akiyama, Institute for Virus Research, Kyoto University, Kyoto, Japan; Taura et al., 1993
), anti-
32 (a kind gift from Dr Bernd Bukau, University of Heidelberg, Germany; Gamer et al., 1992
) and anti-RRF (a kind gift from Dr Umesh Varshney, Indian Institute of Science, Bangalore, India; Rao & Varshney, 2001
) were used at 1 : 5000, 1 : 5000, 1 : 4000 and 1 : 3000 dilutions respectively. The anti-MtFtsH antibody, raised against the C-terminus of FtsH protein of M. tuberculosis H37Rv, was used for the detection of MsFtsH at 1 : 5000 dilution, while protein AHRP conjugate was used at 1 : 10 000 dilution. Detection was carried out using enhanced chemiluminescence detection reagents according to manufacturer's instructions.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
While the unique domain containing 51 amino acid residues is common to all mycobacterial FtsH proteins, MsFtsH possesses, in addition, a unique stretch of sequence, which is rich in glutamine and proline residues, at the C-terminal portion of the protein (Fig. 2). While FtsH proteases of Mycobacterium avium (MaFtsH) and Mycobacterium leprae (MlFtsH) also carry a glutamine- and proline-rich stretch of residues, interestingly, MtFtsH and the FtsH protein of Mycobacterium bovis (MbFtsH) conspicuously lack the stretch of glutamine and proline residues (Anilkumar et al., 1998
). It may be noted here that glutamine-rich sequences, but not proline-rich sequences, are known to be one of the characteristic features of transcriptional activators (Wykoff et al., 1999
). However, the biological role of the glutamine- and proline-rich sequence in MsFtsH, MaFtsH and MlFtsH, and the consequent biological differences due to its absence in MtFtsH and MbFtsH, need further investigation. One potential use of this sequence might be for the construction of a phylogenetic tree among mycobacterial species.
The amino acid sequence of conserved regions of MsFtsH was aligned with the corresponding sequences of the FtsH proteases of E. coli (P28691), B. subtilis (P37476), Arabidopsis thaliana (O80860), Homo sapiens (Q9Y2Q2) and M. tuberculosis (P96942), using the CLUSTAL W program at the European Bioinformatics Institute (Thompson et al., 1994) (http://www2.ebi.ac.uk/clustalw) (Fig. 3
). The MsFtsH protein shares 44·8 %, 47·2 %, 40·2 % and 53 % identity, respectively, at the amino acid sequence level with the amino acid sequences of the FtsH proteases of E. coli, B. subtilis, A. thaliana and H. sapiens, indicating that the 2·31 kb ORF that we obtained indeed contained the MsftsH gene. Although the extent of sequence conservation between MsFtsH and FtsH molecules of the other organisms referred to above is only to the extent of about 4050 %, there is a high level of sequence conservation among mycobacterial FtsH proteases. The MsftsH ORF shares 82 % identity at the amino acid level with that of MtftsH, which has 760 amino acid residues encoding a protein of 84 kDa (Anilkumar et al., 1998
). Similarly, MsFtsH shares sequence identity of 80·2 %, 79·7 % and 79·3 %, respectively, with the FtsH proteases of M. bovis, M. leprae and M. avium.
|
|
Since MsftsH was not be expressed from any of the constructs, namely pBSMsH, pGEX-4T1MsH, pRSET-AMsH, pCYB2MsH and pET20bMsH, we had no choice but to use pT18MsH for the expression of MsftsH in vivo to demonstrate proteolytic activity. Even using this vector, the protein was not detected by Coomassie blue staining, but it was detected as an 85 kDa band in Western blotting (Fig. 4b). The possible reason for the expression of MsftsH could be that the C-terminal translational fusion of the T18 fragment of the cya gene might have contributed to the stability of the protein, although the cya fragment is cleaved off in vivo, as inferred from the molecular mass of the recombinant protein observed upon immunoblotting with anti-EcFtsH and anti-MtFtsH antibodies.
The recombinant MsFtsH protein was recovered from the pellet fraction obtained after incubation with 1 M NaCl (Fig. 4c), suggesting that it was localized to the inner cell membrane (Tomoyasu et al., 1993a
, b
), as reported in the case of MtFtsH (Anilkumar et al., 1998
). The expression of MsftsH in E. coli JM109 cells was lethal and growth stopped within 1 h of induction with 1 mM IPTG (Fig. 4d
). The cells were filamentous as compared with the control cells that carried the pT18 vector alone, but they did not show any defect in nucleoid segregation (data not shown). The cells were filamentous even in the absence of IPTG. Expression of an algal FtsH has been reported to result in a similar phenotype (Itoh et al., 1999
). It is possible that sequestration of EcFtsH by MsFtsH in oligomerization, thereby making EcFtsH unavailable for biological function, could be the cause of the filamentation. In such case, overexpression of EcFtsH might rescue the cells from filamentation or toxicity caused by the expression of MsFtsH. However, co-expression of EcFtsH from pBHB1 did not suppress the toxicity to or filamentation of E. coli JM109 cells caused by the expression of MsFtsH (data not shown).
Protease activity of recombinant MsFtsH in E. coli cells
The protease activity of MsFtsH, induced from the pT18MsH construct, was tested in vivo in E. coli cells on 32 protein and SecY protein, both of which are specific substrates for EcFtsH (Tomoyasu et al., 1995
; Kihara et al., 1995
). These heterologous substrates from E. coli had to be used for the demonstration of protease activity of MsFtsH in vivo for the following reasons. A homologue for
32 protein is absent in mycobacteria, although a heat-shock-inducible sigma factor, sigH, exists (Cole et al., 1998
; Fernandes et al., 1999
). Recombinant SecY proteins of M. smegmatis or M. tuberculosis were not obtained, since expression of secY genes of both these mycobacteria in E. coli cells resulted in severe toxicity to host cells, with resultant growth arrest.
Degradation of 32 protein by MsFtsH in vivo.
Cellular levels of E. coli heat shock transcription factor 32 are controlled by the ATP-dependent proteases such as Lon, ClpXP, HslVU and FtsH (Kanemori et al., 1997
). The
32 protein is stabilized in ftsH-null strains of E. coli, and its levels are at least 20-fold higher than those in wild-type cells (Ogura et al., 1999
). One such strain, AR 5090 (
ftsH : : kan, sfhC21), offers an excellent in vivo assay system for monitoring the protease activity of FtsH (Akiyama & Ito, 2000
; Karata et al., 2001
; Akiyama & Ito, 2003
). MsFtsH was expressed from the pT18MsH recombinant vector in E. coli AR 5090 cells. Synthesis of 85 kDa MsFtsH protein, upon induction with 2 mM IPTG for 3 h, was confirmed with Western blotting using anti-EcFtsH antibodies (Fig. 5
b). Use of anti-EcFtsH antibodies also confirmed the absence of the 74 kDa EcFtsH protein from the AR 5090 lysates. Degradation of endogenous
32 protein of E. coli was monitored with Western blotting using anti-
32 antibody (a kind gift from Dr Bernd Bukau, University of Heidelberg, Germany). Recombinant MsFtsH degraded
32 protein in vivo (Fig. 5b
). Degradation of
32 by MsFtsH was not as efficient as that by EcFtsH expressed from the plasmid pSTD113 (EcftsH expressed from lac promoter; a kind gift from Dr Yoshinori Akiyama, Institute of Virus Research, Kyoto University, Japan; Akiyama et al., 1995
). About a twofold decrease in
32 protein levels was observed when MsFtsH was expressed, as compared to the fivefold decrease found in the case of degradation by EcFtsH. Reasons for the inefficient degradation of
32 could be either the differences in the expression levels of the two proteases or the possibility that
32 is a relatively poor substrate for MsFtsH. Expression levels of EcFtsH and MsFtsH could not be compared by Western blot because of possible differences in the cross-reactivity of the anti-EcFtsH antibodies to MsFtsH and EcFtsH. As an additional internal control, the lysates were immunoblotted with antibodies against ribosome recycling factor (RRF) (a kind gift from Dr Umesh Varshney, Indian Institute of Science, Bangalore, India). RRF did not show any changes in its level upon overexpression of either EcFtsH or MsFtsH (Fig. 5a, b
).
|
Nevertheless, in order to further substantiate the protease activity of recombinant MsFtsH in E. coli cells, EcSecY, the degradation of which is not dependent upon a classical stress response, was used for the in vivo assay. Moreover, FtsH is the only protease hitherto known to be involved in the degradation of SecY. Therefore, degradation of EcSecY was monitored in response to induction of MsFtsH. Thus, any reduction in the levels of SecY protein, upon induction of MsFtsH in the EcFtsH-null AR 5090 strain, would be due solely to MsFtsH protease expressed from the plasmid.
Degradation of EcSecY by MsFtsH in vivo.
SecY encodes an integral transmembrane protein, which spans the cytoplasmic membrane ten times (Akiyama & Ito, 1987) and plays an essential role, in conjunction with SecE and SecG, in forming the peptide translocase complex (Brundage et al., 1990
; Ito, 1992
). In E. coli, moderately overexpressed and unassembled SecY is rapidly degraded with a half-life of 2 min (Taura et al., 1993
). The degradation of SecY that failed to associate with its partner SecE was dependent upon FtsH (Akiyama et al., 1996a
), and mutations that resulted in the loss of function or underexpression of the ATP-dependent FtsH protease stabilized the overexpressed SecY (Kihara et al., 1995
). Overexpression of FtsH accelerated the degradation of unassembled SecY (Kihara et al., 1995
). Therefore, the protease activity of FtsH on the membrane-bound substrates can be assessed by its ability to degrade overexpressed and uncomplexed SecY.
Exponentially growing cultures of E. coli AR 5090 (EcftsH null) carrying two compatible plasmids, pKY248 with pBluescript, pSTD113, pT18 or pT18MsH, were induced with 2 mM IPTG for 3 h. Western blotting with anti-SecY antibodies (a generous gift from Dr Akiyama, Institute of Virus Research, Kyoto University, Japan) was used to monitor SecY levels in the induced cultures. Co-expression of MsFtsH resulted in the degradation of SecY molecules that were produced in excess (Fig. 6b). The level of expression achieved for SecY, when MsFtsH was co-expressed, was 10-fold less than that achieved in an ftsH-null strain carrying the vector alone. EcFtsH, expressed from pSTD113, was used as the positive control for the degradation of SecY (Fig. 6a
). As an internal control, immunoblotting with anti-RRF antibodies showed no changes in the levels of RRF, indicating that the degradation of SecY by FtsH was specific. Densitometric comparison of the levels of SecY in the absence of FtsH, and in the induced presence of FtsH, showed that the degradation of EcSecY by MsFtsH was twofold less efficient than that by EcFtsH. The degradation of EcSecY by MsFtsH demonstrated that the recombinant MsFtsH protein, expressed in E. coli cells, was proteolytically active. This observation alludes to the possibility of the existence of conserved mechanisms of protein translocation and their control across bacterial genera. It indirectly supports the contention that degradation of
32 protein, concomitant with the induction of MsFtsH, might also have been due to MsFtsH protease itself, and not to some other classical stress-response-specific protease.
|
Complementation of E. coli strain AR 423 (ftsH : : kan/pAR171) with MsftsH
E. coli AR 423 (ftsH : : kan/pAR171; Akiyama et al., 1994
; a kind gift from Dr Teru Ogura, Kumamoto University, Japan) was used for the complementation studies. The pAR171 plasmid, which has a ts ori, is defective for replication at 42 °C, and carries the esssential EcftsH gene under its own promoter and a chloramphenicol resistance (CmR) marker. Complementation assays were carried out exactly as described by Nilsson et al. (1994)
, using several constructs, namely pBSMsH, pGEX-4T1MsH, pCYB2MsH and pT18MsH, all of which carried an ampicillin resistance (ApR) marker. E. coli AR 423 (
ftsH : : kan/pAR171) was transformed with the constructs, and the transformants were selected for CmR, KmR and ApR at 30 °C. These transformants were grown in LB at 42 °C for 6 h and then plated on LB agar at 30 °C. The colonies that were obtained were all KmR and ApR, indicating that they had retained the MsftsH-containing constructs and the mutation
ftsH3 : : kan. All the colonies obtained were also CmR, showing that there was no loss of pAR171. However, unlike the case of complementation by Lactococcus lactis ftsH (LlftsH), CmS colonies were not obtained. Thus, none of these constructs were able to complement E. coli AR 423 (
ftsH : : kan/pAR171).
A possible reason for lack of complementation could be that none of the constructs could give an optimum level of MsFtsH comparable to that of the endogenous EcFtsH protein. However, this could not be ascertained since expression from most of these constructs was not detected, even with Western blotting. Another reason for the inability of MsFtsH to complement could be a probable deficiency in the protease activity of the recombinant protein, as it is known that the proteolysis of LpxC by FtsH is essential for viability (Ogura et al., 1999). It has also been shown that a mutant EcFtsH that retains ATPase activity, but lacks protease activity, does not complement the growth defect of an FtsH-depleted strain (Jayasekera et al., 2000
). Although the MsFtsH protein, expressed from pT18MsH, showed protease activity in vivo against two known substrates of EcFtsH, namely Ec
32 and EcSecY in E. coli cells, determination of proteolytic activity of MsFtsH against LpxC would ascertain the reason for the inability of MsftsH to complement the EcftsH mutant.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akiyama, Y. & Ito, K. (2000). Roles of multimerization and membrane association in the proteolytic functions of FtsH (HflB). EMBO J 19, 38883895.
Akiyama, Y. & Ito, K. (2003). Reconstitution of membrane proteolysis by FtsH. J Biol Chem 278, 1814618153.
Akiyama, Y., Ogura, T. & Ito, K. (1994). Involvement of FtsH in protein assembly into and through the membrane. I. Mutations that reduce retention efficiency of a cytoplasmic reporter. J Biol Chem 269, 52185224.
Akiyama, Y., Yoshihisa, T. & Ito, K. (1995). FtsH, a membrane-bound ATPase, forms a complex in the cytoplasmic membrane of Escherichia coli. J Biol Chem 270, 2348523490.
Akiyama, Y., Kihara, A., Tokuda, H. & Ito, K. (1996a). FtsH protease is an ATP-dependent protease selectively acting on SecY and some other membrane proteins. J Biol Chem 271, 3119631201.
Akiyama, Y., Kihara, A. & Ito, K. (1996b). Subunit a of proton ATPase F0 sector is a substrate of the FtsH protease in Escherichia coli. FEBS Lett 399, 2628.[CrossRef][Medline]
Anilkumar, G., Chauhan, M. M. & Ajitkumar, P. (1998). Cloning and expression of the gene coding for FtsH protease from Mycobacterium tuberculosis H37Rv. Gene 214, 711.[CrossRef][Medline]
Beyer, A. (1997). Sequence analysis of the AAA protein family. Protein Sci 6, 20432058.
Brundage, L., Fimmel, C. J., Mizushima, S. & Wickner, W. (1990). SecY, SecE, and band 1 form the membrane-embedded domain of Escherichia coli preprotein translocase. J Biol Chem 267, 41664170.
Caldas, T., Binet, E., Bouloc, P., Costa, A., Desgres, J. & Richarme, G. (2000). The FtsJ/RrmJ heat shock protein of Escherichia coli is a 23S ribosomal RNA methyltransferase. J Biol Chem 275, 1641416419.
Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537544.[CrossRef][Medline]
Cserzo, M., Wallin, E., Simon, I., von Heijne, G. & Elofsson, A. (1997). Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the Dense Alignment Surface method. Protein Eng 10, 673676.[CrossRef][Medline]
Cutting, S., Anderson, M., Lysenko, E., Page, A., Tomoyasu, T., Tatematsu, K., Tatsuta, T., Kroos, L. & Ogura, T. (1997). SpoVM, a small protein essential to development in Bacillus subtilis, interacts with the ATP-dependent protease FtsH. J Bacteriol 179, 55345542.[Abstract]
Deuerling, E., Paeslack, B. & Schumann, W. (1995). The ftsH gene of Bacillus subtilis is transiently induced after osmotic and temperature upshift. J Bacteriol 177, 41054112.[Abstract]
Deuerling, E., Mogk, A., Richter, C., Purucker, M. & Schumann, W. (1997). The ftsH gene of Bacillus subtilis is involved in major cellular processes such as sporulation, stress adaptation, and secretion. Mol Microbiol 23, 921933.[Medline]
Fernandes, N. D., Wu, Q. L., Kong, D., Puyang, X., Garg, S. & Husson, R. N. (1999). A mycobacterial extracytoplasmic sigma factor involved in survival following heat shock and oxidative stress. J Bacteriol 181, 42664274.
Fischer, B., Rummel, G., Aldridge, P. & Jenal, U. (2002). The FtsH protease is involved in development, stress response, and heat shock control in Caulobacter crescentus. Mol Microbiol 44, 461478.[CrossRef][Medline]
Gamer, J., Bujard, H. & Bukau, B. (1992). Physical interaction between heat shock proteins DnaK, DnaJ, and GrpE and the bacterial heat shock transcription factor sigma 32. Cell 69, 833842.[Medline]
Ge, Z. & Taylor, D. E. (1996). Sequencing, expression, and genetic characterization of the Helicobacter pylori ftsH gene encoding a protein homologous to members of a novel putative ATPase family. J Bacteriol 178, 61516157.[Abstract]
Goff, S. A. & Goldberg, A. L. (1985). Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes. Cell 41, 587595.[Medline]
Guelin, E., Rep, M. & Grivell, L. A. (1994). Sequence of the AFG3 gene encoding a new member of the FtsH/Yme1/Tma subfamily of the AAA-protein family. Yeast 10, 13891394.[Medline]
Herman, C., Ogura, T., Tomoyasu, T., Hiraga, S., Akiyama, Y., Ito, K., Thomas, R., D'Ari, R. & Bouloc, P. (1993). Cell growth and lambda phage development controlled by the same essential Escherichia coli gene, ftsH/hflB. Proc Natl Acad Sci U S A 90, 1086110865.[Abstract]
Herman, C., Thevenet, D., D'Ari, R. & Bouloc, P. (1995). Degradation of 32, the heat shock regulator in Escherichia coli, is governed by HflB. Proc Natl Acad Sci U S A 92, 35163520.[Abstract]
Herman, C., Thevenet, D., D'Ari, R. & Bouloc, P. (1997). The HflB protease of Escherichia coli degrades its inhibitor lambda cIII. J Bacteriol 179, 358363.[Abstract]
Ito, K. (1992). SecY and integral membrane components of the Escherichia coli protein translocation system. Mol Microbiol 6, 24232428.[Medline]
Ito, K., Akiyama, Y., Yura, T. & Shiba, K. (1986). Diverse effects of theMalELacZ hybrid protein on Escherichia coli cell physiology. J Bacteriol 167, 201204.[Medline]
Itoh, R., Takano, H., Ohta, N., Miyagishima, S.-Y., Kuroiwa, H. & Kuroiwa, T. (1999). Two ftsH-family genes encoded in the nuclear and chloroplast genomes of the primitive red alga Cyanidioschyzon merolae. Plant Mol Biol 41, 321337.[CrossRef][Medline]
Jayasekera, M. M., Foltin, S. K., Olson, E. R. & Holler, T. P. (2000). Escherichia coli requires the protease activity of FtsH for growth. Arch Biochem Biophys 380, 103107.[CrossRef][Medline]
Kanemori, M., Nishihara, K., Yanagi, H. & Yura, T. (1997). Synergistic roles of HslVU and other ATP-dependent proteases in controlling in vivo turnover of sigma32 and abnormal proteins in Escherichia coli. J Bacteriol 179, 72197225.[Abstract]
Karata, K., Inagawa, T., Wilkinson, A. J., Tatsuta, T. & Ogura, T. (1999). Dissecting the role of a conserved motif (the second region of homology) in the AAA family of ATPases. Site-directed mutagenesis of the ATP-dependent protease FtsH. J Biol Chem 274, 2622526232.
Karata, K., Verma, C. S., Wilkinson, A. J. & Ogura, T. (2001). Probing the mechanism of ATP hydrolysis and substrate translocation in the AAA protease FtsH by modelling and mutagenesis. Mol Microbiol 39, 890903.[CrossRef][Medline]
Karimova, G., Pidoux, J., Ullmann, A. & Ladant, D. (1998). A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A 95, 57525756.
Kihara, A., Akiyama, Y. & Ito, K. (1995). FtsH is required for proteolytic elimination of uncomplexed forms of SecY, an essential protein translocase subunit. Proc Natl Acad Sci U S A 92, 45324536.[Abstract]
Kihara, A., Akiyama, Y. & Ito, K. (1999). Dislocation of membrane proteins in FtsH-mediated proteolysis. EMBO J 18, 29702981.
Kunst, F., Ogasawara, N., Moszer, I. & 148 other authors (1997). The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390, 249256.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Lindahl, M., Tabak, S., Cseke, L., Pichersky, E., Andersson, B. & Adam, Z. (1996). Identification, characterization, and molecular cloning of a homologue of the bacterial FtsH protease in chloroplasts of higher plants. J Biol Chem 271, 2932929334.
Lysenko, E., Ogura, T. & Cutting, S. M. (1997). Characterization of the ftsH gene of Bacillus subtilis. Microbiology 143, 971978.[Medline]
Messing, J., Crea, R. & Seeburg, P. H. (1981). A system for shotgun DNA sequencing. Nucleic Acids Res 9, 309321.[Abstract]
Miroux, D. & Walker, J. E. (1996). Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260, 289298.[CrossRef][Medline]
Nilsson, D., Lauridsen, A. A., Tomoyasu, T. & Ogura, T. (1994). A Lactococcus lactis gene encodes a membrane protein with putative ATPase activity that is homologous to the essential Escherichia coli ftsH gene product. Microbiology 140, 26012610.[Medline]
Ogura, T. & Wilkinson, A. J. (2001). AAA+ superfamily ATPases: common stucture diverse function. Genes Cells 6, 575597.
Ogura, T., Tomoyasu, T., Yuki, T., Morimura, S., Begg, K. J., Donachie, W. D., Mori, H., Niki, H. & Hiraga, S. (1991). Structure and function of the ftsH gene in Escherichia coli. Res Microbiol 142, 279282.[CrossRef][Medline]
Ogura, T., Inoue, K., Tatsuta, T. & 10 other authors (1999). Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli. Mol Microbiol 31, 833844.[CrossRef][Medline]
Rao, A. R. & Varshney, U. (2001). Specific interaction between the ribosome recycling factor and the elongation factor G from Mycobacterium tuberculosis mediates peptidyl-tRNA release and ribosome recycling in Escherichia coli. EMBO J 20, 29772986.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Shotland, Y., Koby, S., Teff, D. & 8 other authors (1997). Proteolysis of the phage CII regulatory protein by FtsH (HflB) of Escherichia coli. Mol Microbiol 24, 13031310.[Medline]
Taura, T., Baba, T., Akiyama, Y. & Ito, K. (1993). Determinants of the quantity of the stable SecY complex in the Escherichia coli cell. J Bacteriol 175, 77717775.[Abstract]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Tomoyasu, T., Yuki, T., Morimura, S., Mori, H., Yamanaka, K., Niki, H., Hiraga, S. & Ogura, T. (1993a). The Escherichia coli FtsH protein is a prokaryotic member of a protein family of putative ATPases involved in membrane functions, cell cycle control, and gene expression. J Bacteriol 175, 13441351.[Abstract]
Tomoyasu, T., Yamanaka, K., Murata, K., Suzaki, T., Bouloc, P., Kato, A., Niki, H., Hiraga, S. & Ogura, T. (1993b). Topology and subcellular localization of FtsH protein in Escherichia coli. J Bacteriol 175, 13521357.[Abstract]
Tomoyasu, T., Gamer, J., Bukau, B. & 9 other authors (1995). Escherichia coli FtsH is a membrane-bound, ATP-dependent protease, which degrades the heat-shock transcription factor 32. EMBO J 14, 25512560.[Abstract]
Wykoff, D. D., Grossman, A. R., Weeks, D. P., Usuda, H. & Shimogawara, K. (1999). Psr1, a nuclear localized protein that regulates phosphorus metabolism in Chlamydomonas. Proc Natl Acad Sci U S A 96, 1533615341.
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103119.[CrossRef][Medline]
Zellmeier, S., Zuber, U., Schumann, W. & Wiegert, T. (2003). The absence of FtsH metalloprotease activity causes overexpression of the W-controlled pbpE gene, resulting in filamentous growth of Bacillus subtilis. J Bacteriol 185, 973982.
Received 10 February 2004;
revised 9 April 2004;
accepted 18 May 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |