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(Received for publication, May 31, 1995; and in revised form, July 13, 1995) From the
The FtsH (HflB) protein of Escherichia coli is
integrated into the membrane with two N-terminally located
transmembrane segments, while its large cytoplasmic domain is
homologous to the AAA family of ATPases. The previous studies on
dominant negative ftsH mutants raised a possibility that FtsH
functions in multimeric states. We found that FtsH was eluted at
fractions corresponding to a larger molecular weight than expected from
monomeric structure in size-exclusion chromatography. Moreover,
treatment of membranes or their detergent extracts with a cross-linker,
dithiobis(succinimidyl propionate), yielded cross-linked products of
FtsH. To dissect possible FtsH complex, we constructed an FtsH
derivative with c-Myc epitope at its C terminus
(FtsH-His
Escherichia coli FtsH (HflB) protein belongs to a novel
ATPase family whose members are widely found among eukaryotic and
prokaryotic organisms(1) . They all have one or two copies of
the conserved regions of about 200 amino acid residues that include a
set of ATP binding consensus motifs(2) . They are suggested to
be involved in diverse cellular functions such as regulation of cell
cycle, vesicular transport in protein secretion, biogenesis of
organelles, nuclear division, regulation of transcription, and protein
degradation (2) . This protein family is called AAA (ATPases associated with a variety of cellular activities)(3) . However, their modes of involvement
in the above mentioned cellular processes are mostly unclear. Even
ATPase activities have been demonstrated only for a few of
them(4, 5, 6) . Their localizations in the
cell are also diverse; some are bound to the plasma or the organella
membrane, but many others are soluble proteins(2) . We
previously showed that mutational impairments of the ftsH gene
of E. coli caused an Std phenotype in which a normally
cytoplasmic reporter PhoA ( This study was aimed at clarifying the quaternary structure
of FtsH in the cell. We showed that FtsH in the wild-type cells exists
as a complex. Co-immunoprecipitation and cross-linking experiments
using a Myc epitope/His
L medium(12) , peptone medium(13) , and
M9 medium (10) were used. Media containing ampicillin (50
µg/ml) and/or chloramphenicol (20 µg/ml) were used for growing
plasmid-bearing strains.
Figure 1:
Size-exclusion chromatography profiles
of FtsH and SecY. A, total membranes prepared from cells of
AD202 were solubilized with OG and chromatographed through Superose 6.
Proteins in every other fractions were precipitated with
trichloroacetic acid and analyzed by SDS-polyacrylamide gel
electrophoresis followed by immunoblotting with anti-FtsH or anti-SecY. B, the positions of the major peak fractions for FtsH and SecY
determined (arrows) as well as those of molecular size markers
are shown. The markers used were as follows: thyroglobulin (670 kDa),
bovine
Figure 2:
Cross-linking of FtsH in the wild-type
cells before and after solubilization. A suspension of total membranes
prepared from cells of AD202 (A) or its OG extract (B) was treated with DSP. The samples for lanes1 and 3 received a quencher, ammonium acetate, of DSP prior
to DSP treatment. Proteins were then treated with SDS in the presence (lanes1 and 2) or absence (lanes3 and 4) of 2-mercaptoethanol and
analyzed by 4% (A) or 5% (B) polyacrylamide gel
electrophoresis followed by immunoblotting with ant-FtsH. The positions
of molecular-size standards (Kaleidoscope prestained standard, Bio-Rad)
were indicated (represented by multiples of 10
Figure 3:
Schematic representations of
FtsH-His
Figure 4:
Synthesis and stability of
FtsH-His
Figure 5:
Cross-linking of FtsH and FtsH` with
FtsH-His
Figure 6:
Co-immunoprecipitation of FtsH and FtsH`
with FtsH-His
Cells of CU141(F`lacI
These results
show that FtsH and FtsH` were co-precipitated with the epitope-tagged
FtsH. No other proteins were appreciably co-precipitated with anti-Myc
antibodies. FtsH and FtsH` were also co-purified with
FtsH-His
These
chimeric genes did not complement the ftsH1 mutation,
indicating that both the membrane-bound and the cytoplasmic regions of
FtsH are important for the FtsH functions. Cell fractionation
experiments showed that these hybrid proteins are membrane-associated
(data not shown). We then examined whether the chimeric proteins
cause a dominant Std phenotype (see Introduction). As the high level
overexpression of these proteins from the plasmids used in the
cross-linking experiments was found to be deleterious to cells, the
fusion genes were recloned into a low copy number vector that is also
compatible with the plasmid (pKY221) carrying the reporter secY-phoA C6 gene. Extracts of cells expressing either the
chimeras, FtsH, or envZ, in addition to SecY-PhoA C6 fusion, were
treated with trypsin and analyzed by immunoblotting with anti-PhoA
antibodies (Fig. 7). The PhoA domain of SecY-PhoA C6 from the
cells expressing FtsH`-`EnvZ resisted trypsin (Fig. 7A, lanes7 and 8), indicating that it was
exported to the periplasmic space. On the other hand, expression of the
other three proteins, EnvZ`-`FtsH-His
Figure 7:
Std phenotype caused by expression of the
FtsH-EnvZ hybrid proteins. Cells of CU141 carrying pKY221 (secY-phoA C6) and either pSTD119 (envZ`-`ftsH-his
Cells
overexpressing FtsH and either FtsH`-`EnvZ,
EnvZ`-`FtsH-His
Figure 8:
Cross-linking between FtsH and the
chimeric proteins. Cross-linking experiments were carried out using
membranes from cells of TYE024/pSTD122 (ftsH`-`envZ)/pSTD401 (A), TYE024/pSTD117 (envZ`-`ftsH-his
FtsH has been implicated to have diverse cellular activities.
We suggested previously that FtsH is involved in integration/assembly
of proteins through and/or into the membrane(7, 8) .
It was also found recently that FtsH is involved in rapid degradation
of at least three short-lived proteins, cII gene product of
phage Oligomeric structure seems to be a common feature among the above
mentioned ATPase subunits as well as some other members of the AAA
family. For example, (N-ethylmaleimide-sensitive factor)
functions as a homotrimer that interacts with many other proteins
including SNAPs and SNAREs during process of vesicular transport in
eukaryotic cells(31) . p97 has also been proposed to be a
homohexamer, although its function is not known(5) . We have
shown here that FtsH is in a complex that includes more than one
molecules of FtsH. FtsH remains in high molecular mass state after
solubilization in nonionic detergent. The solubilized FtsH could be
cross-linked to form oligomeric structure and could be
co-immunoprecipitated with the epitope-tagged version of FtsH. It is
possible that the FtsH molecules are directly interacting with
themselves. The FtsH`-`EnvZ chimeric protein is cross-linkable with
FtsH and causes dominant Std phenotype. We suggest that the dominant
phenotype is at least partly a result of the formation of a
nonfunctional FtsH complex containing wild-type and mutant molecules.
The results with the hybrid proteins suggested that possible
interaction between the FtsH molecules is mediated by direct
association of their transmembrane regions. Several examples have been
reported for inter- or intramolecular association of transmembrane
segments(32, 33, 34) . The ftsH101 mutation causes a change of Val It is not known how
many FtsH molecules are present in the FtsH complex and whether any
other proteins are associated with it. The major cross-linked products
of 140 and 240 kDa might represent dimer and tetramer of FtsH. In
addition, no other major proteins were found in the preparation of FtsH
that was purified from overproducing strains (6) . These
results, however, do not exclude the possibility that the physiological
complex of FtsH contains additional components. Preliminarily, two
proteins of 27 and 16 kDa were found to co-immunoprecipitate with
anti-FtsH antibodies. Elucidation of the complete structure of the FtsH complex awaits
purification of the physiological complex from wild-type cells. The
present results showing that FtsH molecules can associate with each
other even when they are exclusively overproduced ( Fig. 5and Fig. 6) will provide an important guidance for further
biochemical characterization of this intriguing membrane protein.
Volume 270,
Number 40,
Issue of October 06, pp. 23485-23490, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Myc). When membranes prepared from cells in which
FtsH-His-Myc was overproduced together with the normal FtsH
were treated with the cross-linker, intact FtsH and in vitro degradation products of FtsH-His-Myc without the tag
were cross-linked with the tagged FtsH protein. Co-immunoprecipitation
experiments confirmed the interaction between the FtsH molecules. To
identify regions of FtsH required or sufficient for this interaction,
we constructed chimeric proteins between FtsH and EnvZ, a protein with
a similar topological arrangement, by exchanging their corresponding
domains. We found that only the FtsH-EnvZ hybrid protein with an
FtsH-derived membrane anchoring domain and an EnvZ-derived cytoplasmic
domain caused a dominant ftsH phenotype and was cross-linked
with FtsH. We suggest that the N-terminal transmembrane region of FtsH
mediates directly the interaction between the FtsH subunits.
)domain of a model membrane
protein (SecY-PhoA) was exported to the periplasmic
space(7, 8) . Since the Std phenotype signifies
insufficient anchoring of the transmembrane segment that precedes the
reporter domain, we suggested that FtsH is involved in the process of
protein assembly into the membrane. We also found that a decreased
cellular content of the FtsH protein resulted in a strong Std phenotype
and an impaired translocation of some secreted proteins (Sec
phenotype)(7) . Therefore, FtsH might have a role in protein
export as well. Additionally, we found that the expression of
C-terminally truncated forms of FtsH or ATP binding site mutants of
FtsH from a plasmid caused the Std and Sec phenotypes
dominantly(8) . The existence of dominant negative alleles of ftsH raises a possibility that FtsH may function in multimeric
states.-tagged FtsH revealed that the FtsH
molecules interact with each other. A series of chimeric proteins
between FtsH and EnvZ were constructed, and cross-linking experiments
using them showed that the FtsH-FtsH association required the
N-terminal membrane association region but not the cytoplasmic domain.
Bacterial Strains and Media
E. coli K12
strains AD21 (9) and MC4100 (10) were described
previously. AD202 (11) was a ompT::kan derivative of
MC4100, and CU141 (7) was an F`lacI
derivative of MC4100, respectively. TYE024 (MC4100, ompT::kan/F`lacI) was constructed by
introducing F`lacI of CU141 into AD202 by
conjugation.Construction of the ftsH-his
pSTD101 carrying ftsH-his-myc
Plasmids-myc was constructed as follows.
pSTD40 in which a mutant ftsH gene (the ftsH40 allele) was placed under the lac promoter/operator was
described previously (7) . (
)The 2.7 kb EcoRI-PstI fragment of pSTD40 was blunt-ended by
treatment with T4 polymerase and cloned into SmaI site of a
pBlueScript SK(-) (Stratagene) derived vector, pTYE007, which
carried a sequence encoding a bipartite His/c-Myc tag of 30
amino acid residues (EFIEGRHHHHHHIDEEQKLISEEDLLRKR) following its
multicloning site. (
)The ftsH gene and the his-myc sequence on the resulting plasmid
were fused in frame by site-directed mutagenesis according to Kunkel et al.(14) using a mutagenic primer
(5`-TGTCAGAGCAGTTAGGCGACAAGGAATTCATCGAAGGCCGTCACCA-3`).pSTD113
(carrying ftsH-his
-myc)
was constructed by replacing the 1.5-kb SphI fragment of
pSTD101 by that of pSTD401, which had the same structure as pSTD40
except that it carried the wild-type ftsH gene. pSTD120 was
constructed by inserting the 2.8-kb XbaI-KpnI
fragment that contained the entire region of ftsH-his
-myc into the XbaI-KpnI site of pMW119 (Nippon gene),
a pSC101-derived low copy number vector.Constructions of Hybrid Genes between ftsH and
envZ
pSTD117 that carried an envZ`-`ftsH hybrid gene
was constructed by site-directed mutagenesis as follows. First, a
0.8-kb XbaI-EcoRV fragment of pAT2005S (15) carrying the envZ gene was ligated with pSTD113
that had been digested with BamHI, blunt-ended by treatment
with T4 polymerase, and then digested with XbaI. Then, the
region encoding the membrane anchoring domain (from the amino terminus
to the 179th amino acid residue) of EnvZ and the region encoding the
cytoplasmic domain (from the 121th amino acid residue of FtsH to the
carboxyl terminus) of FtsH-His-Myc were fused in frame
according to the method of Kunkel et al.(14) using a
mutagenic primer
(5`-TAGGCGGGGCGTGGCTGTTTATTCGTCAAATGCAGGGCGGCGGTGG-3`). pSTD122 that
carried the ftsH`-`envZ hybrid gene was constructed similarly.
An about 2-kb HpaI-NruI fragment of pAT2005S was
ligated with pSTD113 that had been digested with SmaI and EcoRV, and the region encoding the membrane anchoring domain
(from the amino terminus to the 120th amino acid residue) of FtsH and
the region encoding the cytoplasmic domain (from the 180th amino acid
residue to the carboxyl terminus) of EnvZ were fused in frame using a
mutagenic primer
(5`-TTGGTGTCTGGATCTTCTTCATGCGTATCCAGAACCGACCGTTGGT-3`). pTYE030 was
constructed as follows. The envZ open reading frame was
amplified by polymerase chain reaction with primers of
5`-GCTCTAGAATAAGGAGGCTCTAAAGCATGAGGC-3` and
5`-CGGGATCCCCCTTCTTTTGTCGTGCC-3`. The amplified fragment was subcloned
into pBluescript SK(-) using an XbaI site and a BamHI site introduced by the polymerase chain reaction. While
a central part of the insert (a 0.98-kb MunI-BglII
fragment) was replaced by that of pAT2005S, the remaining part of it
was confirmed by sequencing. Low copy number plasmids carrying envZ`-`ftsH (pSTD119), ftsH-envZ-his-myc (pSTD125), or envZ (pSTD124) were constructed by inserting a 2.2-kb XbaI-SmaI fragment of pSTD117, a 1.6-kb XbaI-EcoRI fragment of pSTD122, or a 1.7-kb XbaI-HpaI fragment of pAT2005S into the multicloning
region of pMW119, respectively.Fractionation of Membrane Proteins by Size-exclusion
Chromatography
Cells of AD202 were grown to a mid-log phase in L
medium, collected, and washed with buffer C (50 mM Hepes-KOH,
pH 7.0, 50 mM KCl, 1 mM dithiothreitol, 20%
glycerol)(15) . Total membrane fraction was prepared by
disruption of cells by sonication followed by ultracentrifugation
essentially as described previously(9) . Membranes were
suspended in buffer C and solubilized with OG in the presence of E.
coli phospholipids as described previously(16) . After
removal of insoluble materials by centrifugation, proteins were mixed
with molecular-size standards (obtained from Bio-Rad), loaded to
Superose 6 column, and developed with 50 mM, 150 mM NaCl, 1.25% OG, 10% glycerol. Proteins in each fraction were
precipitated with 5% trichloroacetic acid, separated by 15% acrylamide,
0.12% N,N`-methylenebisacrylamide polyacrylamide gel
electrophoresis (9) and subjected to immunoblotting with
anti-FtsH (17) or anti-SecY(18) .Pulse-Chase Experiments and Immunoprecipitation of
Denatured Proteins
Cells were grown in M9 medium supplemented
with 18 amino acids (20 µg/ml) other than Met and Cys, thiamine (2
µg/ml), 0.4% glucose, and appropriate antibiotics. After 10 min of
induction with isopropyl-1-thio-
-D-galactopyranoside (1
mM) and cAMP (5 mM), cells were pulse-labeled for 30
s with about 0.37 MBq/ml [S]methionine followed
by chase with 200 µg/ml of nonradioactive L-methionine.
100 µl of samples were removed at intervals and mixed with an equal
volume of 10% trichloroacetic acid. Immunoprecipitation with anti-FtsH (17) or anti-c-Myc (Ab-1) (Oncogene Science, Inc) was carried
out as described previously(7) . Proteins were separated by 10%
polyacrylamide gel electrophoresis(19) .
Immunoprecipitation Under Nondenaturing
Conditions
Cells were grown in M9 medium and pulse-labeled for 5
min. Membrane proteins were solubilized with OG and phospholipids as
described above, diluted with 1.5 volumes of IP buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1.25% OG, 40% glycerol,
1.5 mg/ml E. coli phospholipids) (16) and incubated at
0 °C for 2 h with anti-FtsH, anti-Myc, or normal serum in the
presence or absence of the FtsH (TNRPDVLDPALLRPGR) (17) or c-Myc (AEEQKLISEEDLLRKRREQLKHKLEQLRNSCA) (Oncogene
Science, Inc. ) epitope peptides (10 µg/ml), followed by the
addition of protein A- or protein G-Sepharose (Pharmacia Biotech Inc.)
and further a 2 h of incubation. Immunocomplexes were isolated by
centrifugation, washed 3 times with IP buffer with 0.5 M urea,
IP buffer with 0.5 M NaCl, and rinse buffer (50 mM Tris-HCl, pH 8.0, 20% glycerol, 1.25% OG, 0.5 mg/ml E. coli phospholipids)(16) . Cross-reacting proteins were
separated by 15% acrylamide, 0.12% N,N`-methylenebisacrylamide polyacrylamide gel.
Cross-linking of Membrane Proteins with DSP
Total
membranes were prepared as above. For solubilization, total membranes
were treated with OG as described above except that Tris was not
included and that pH of Hepes-KOH was 7.5 instead of 7.0. For
cross-linking, either total membranes or their OG extracts were treated
with 0.25 mg/ml DSP, a membrane permeable cross-linker, at 4 °C for
1 h, and the reaction was terminated by the addition of 0.2 M ammonium acetate followed by incubation at 4 °C for 10 min.
Control samples received 0.2 M ammonium acetate prior to the
addition of DSP. Samples were adjusted to 1% SDS, incubated at 37
°C for 10 min and subjected to immunoprecipitation as described
above. Precipitated proteins were dissolved in SDS sample buffer (20) without 2-mercaptoethanol at 37 °C for 10 min before
electrophoresis. For cleavage of the cross-linker, 10%
2-mercaptoethanol was included in SDS sample buffer.Trypsin Digestion and Immunoblotting
Cells were
grown in peptone medium supplemented with appropriate antibiotics;
rapidly chilled by mixing with NaN (0.02%), chloramphenicol
(100 µg/ml), and a small piece of ice; and disrupted by lysozyme
freezing-thawing(7) . The cell lysates were treated with
trypsin as described previously(7) . Proteins were separated by
10% SDS-polyacrylamide gel electrophoresis and analyzed by
immunoblotting with anti-PhoA (obtained from 5 Prime 3 Prime,
Inc.), anti-FtsH or anti-EnvZ as described previously(7) .
Size-exclusion Chromatography of the FtsH
Protein
Our previous findings that ftsH can be mutated
to dominant negative with respect to the Std and Sec phenotypes (8) suggested that FtsH may function in multimeric states. To
directly examine higher order structures of FtsH, we solubilized the
cytoplasmic membrane with OG and subjected the solubilized proteins to
size-exclusion chromatography using Superose 6. FtsH was eluted with a
peak at fractions 45-47 that corresponded to a molecular mass of
about 280 kDa (Fig. 1, A and B), while its
monomeric molecular mass should be 71 kDa. Although the value of 280
kDa determined by the calibration using soluble proteins should not be
regarded as accurate, FtsH was eluted far earlier than SecY, a major
part of which was eluted at the position of about 50 kDa (Fig. 1, A and B). This form of SecY could
either be a monomer (the molecular mass is 49 kDa) or in a form of
SecY-SecE-SecG complex of estimated molecular mass of about 74 kDa.
-globulin (158 kDa), chicken ovalbumin (44 kDa), equine
myoglobin (17 kDa), and vitamin B
(1.35
kDa).
Cross-linking of FtsH in Membranes and in Detergent
Extracts
We addressed the subunit structure of FtsH by
cross-linking experiments. The membranes prepared from wild-type cells
were treated with DSP and analyzed by SDS-polyacrylamide gel
electrophoresis and immunoblotting with anti-FtsH. Treatment with DSP
yielded products with molecular masses of about 240 and 140 kDa (Fig. 2A, lane4) that were not
observed without DSP treatment (lane3) or after
cleavage of the cross-linker with 2-mercaptoethanol (lane2). Cross-linked products of FtsH were also generated
when solubilized membrane proteins were treated with DSP (Fig. 2B, lane4). Under the latter
condition, however, the intensity of the 240-kDa species was much less
than when intact membranes were cross-linked.
of molecular
weights) on the leftsides of gels. Filledarrowheads indicate cross-linked
products.
Cross-linking between the FtsH Proteins with and without
an Epitope Tag
To dissect the putative FtsH complex, we
constructed an FtsH derivative with two tandemly located molecular
tags, oligohistidine residues (His), and a c-Myc-derived
epitope at the C terminus (Fig. 3). The FtsH-His-Myc
protein can specifically be isolated, and detected by
nickel-nitrilotriacetic acid-agarose and anti-Myc antibodies,
respectively. Cells carrying pSTD101 (ftsH-his-myc) were pulse-labeled, and
labeled proteins were first solubilized in SDS and then precipitated
with anti-FtsH or anti-Myc antibodies. (
)Anti-FtsH serum
brought down two species of proteins (Fig. 4, lane2). The upperband represented the
tagged FtsH, since it was precipitated by anti-Myc antibodies as well (Fig. 2, lane5). The lowerband represented the normal FtsH, since it comigrated with the
chromosomally encoded FtsH (lane1) and did not
cross-react with anti-Myc (lanes4 and 5).
FtsH-His-Myc was stable in vivo; no degradation
was observed during a 16-min chase period examined (lanes2, 3, 5, and 6). The
FtsH-His-Myc protein was functional, since pSTD120 (a low
copy plasmid carrying ftsH-his-myc)
complemented the temperature-sensitive ftsH1 mutation(20) . It did not interfere with the cell growth.
When pulse-labeled cells were disrupted by sonication and fractionated,
most of FtsH-His-Myc, like normal FtsH, was recovered in
the membrane fraction (data not shown). We found that a fraction of
FtsH-His-Myc was cleaved in vitro by unknown
proteases to a product (FtsH`) slightly smaller than the authentic FtsH
during the process of membrane preparation (see Fig. 5and Fig. 6). The cleavage seemed to occur around the junction
between FtsH and the His-Myc tag, since FtsH` lost the Myc
epitope (Fig. 5B, lane5).
-Myc and the hybrid proteins between FtsH and EnvZ.
The regions derived from the FtsH and EnvZ sequences are represented by open or shadedrectangles. Transmembrane
segments of FtsH (amino acid residues 5-24 and 96-120) (1) and EnvZ (amino acid residues 16-46 and
162-179) (21) are indicated by hatchedboxes. Filledboxes at the C terminus
of FtsH-His-Myc and EnvZ-His-Myc represent
His-Myc tags.
-Myc. Cells of AD21/pSTD101 (ftsH-his-myc) were grown in minimal
medium and pulse-labeled with [S]methionine for
30 s before (lanes1 and 4) or after (lanes2 and 5) a 10-min induction with 1
mM isopropyl-1-thio-
-D-galactopyranoside and 5
mM cAMP. After pulse labeling, induced cells were chased in
the presence of unlabeled methionine for 16 min (lanes3 and 6). Proteins were precipitated with trichloroacetic
acid, subjected to immunoprecipitation with anti-FtsH (lanes1-3) or anti-Myc (lanes4-6), and analyzed by SDS-polyacrylamide gel
electrophoresis.
-Myc. A and B, cells of
TYE024/pSTD113 (ftsH-his-myc)/pSTD401 (ftsH) were grown, induced for 10 min and pulse-labeled with
[S]methionine for 5 min. Total membrane
fractions were treated (A and lanes1-4 of B) or not treated (lanes5 and 6 of B) with DSP. The samples for lane2 of A and lanes3 and 4 of B received ammonium acetate prior to DSP treatment. Proteins
were treated with SDS, and subjected to immunoprecipitation with
anti-FtsH antibodies (A and lanes2, 4, and 6 of B) or anti-Myc serum (lanes1, 3, and 5 of B).
Immunoprecipitates were solubilized in SDS sample buffer with (B) or without (A) 2-mercaptoethanol, and separated
by 15% acrylamide-0.12% N,N`-methylenebisacrylamide
gel electrophoresis. C, cross-linked products that were
precipitated with anti-Myc (lane1) were dissociated
with SDS and subjected to the second immunoprecipitation with anti-FtsH
serum (lane2). Precipitated proteins were
solubilized in SDS sample buffer with
2-mercaptoethanol.
-Myc. Cells of TYE024/pSTD101 (ftsH-his-myc) were grown in minimal
medium, induced for 10 min, and pulse labeled with
[S]methionine for 5 min. Membrane proteins were
solubilized under a nondenaturing condition and precipitated with anti
FtsH serum (lanes1 and 2), anti-Myc
antibodies (lanes3 and 4), or normal serum (lane5) in the presence or absence of the FtsH (lane2) or Myc (lane4) epitope
peptides. Proteins were separated by SDS-polyacrylamide gel. FtsH` indicates the C-terminally-cleaved product of
FtsH-His
-Myc.
) carrying both the ftsH-his-myc plasmid (pSTD113) and the ftsH plasmid (pSTD401) were induced and pulse-labeled, and
total membrane fractions were prepared. To minimize possible artificial
effects resulting from overaccumulation of plasmid-encoded proteins,
their synthesis was induced only for a short period (10 min) before
pulse labeling in this and the following experiments. Membranes were
treated with DSP, solubilized with SDS, and subjected to
immunoprecipitation using anti-Myc or anti-FtsH antibodies. Samples
were analyzed by SDS-polyacrylamide gel electrophoresis without (Fig. 5A) or following (Fig. 5B)
cleavage of the cross-linker by 2-mercaptoethanol. Treatment of the
membranes with DSP yielded high molecular weight cross-linked products
that were immunoprecipitated with anti-FtsH (Fig. 5A, lane1). Such cross-linked products were not detected
when the cross-linker had been quenched by ammonium acetate (lane2). When DSP was cleaved by 2-mercaptoethanol before
electrophoresis, FtsH and FtsH` were recovered with anti-Myc antibodies (Fig. 5B, lane1), whereas they were
never recovered with anti-Myc without cross-linking (lane3). The identities of FtsH and FtsH` were confirmed by
recovery of these proteins by the second immunoprecipitation with
anti-FtsH serum (Fig. 5C). These results suggested that
more than two molecules of FtsH form a complex.Coimmunoprecipitation of FtsH with
FtsH-His
We carried out
immunoprecipitation under nondenaturing conditions (Fig. 6).
Membrane fraction was prepared from FtsH-His-Myc-Myc
overproducing cells that had been pulse-labeled for 5 min and
solubilized with OG, and proteins were immunoprecipitated with anti-Myc
or anti-FtsH antibodies. Anti-FtsH precipitated the tagged FtsH, intact
FtsH, and FtsH` (lane1), whereas normal serum did
not (lane5). Anti-Myc antibodies also precipitated
all of these proteins (lane3). Inclusion of the FtsH
peptide (lane2) or the Myc peptide (lane4) during immunoprecipitation abolished the precipitation
of all of these proteins. When the anti-Myc-precipitates were
dissociated with SDS and subjected to reaction with anti-FtsH serum,
all three proteins were precipitated, confirming their identities (data
not shown). In contrast, only FtsH-His-Myc was recovered
when the membranes were first solubilized in SDS and then subjected to
immunoprecipitation with anti-Myc antibodies (see Fig. 5B, lane5).-Myc by nickel-nitrilotriacetic acid-agarose
affinity column chromatography. (
)Cross-linking (Fig. 2) and co-immunoprecipitation (Fig. 6) after
solubilization preclude the possibility that the cross-linking of these
proteins in the membrane was caused by artificial proximity resulting
from their overaccumulation in the membrane.Identification of the FtsH-FtsH Interaction Region Using
Chimeras between ftsH and envZ
We then examined the roles of the
two regions, the membrane-associated N-terminal region and the
cytoplasmic C-terminal region, in the FtsH-FtsH interaction. We
previously showed that an N-terminal fragment of FtsH caused a dominant
Std effect. Thus, the N-terminal region of FtsH may be important for
the subunit interaction of FtsH. To examine this possibility, we
constructed chimeric genes between ftsH-his-myc and envZ. The EnvZ
protein is an E. coli inner membrane protein with FtsH-like
topology(21) . We constructed two kinds of chimeric genes
encoding FtsH`-`EnvZ and EnvZ`-`FtsH-His-Myc (Fig. 3). The FtsH`-`EnvZ chimeric protein consists of the
FtsH-derived transmembrane domain and the EnvZ-derived cytoplasmic
domain, whereas EnvZ`-`FtsH-His-Myc has the EnvZ membrane
domain followed by the tagged FtsH cytoplasmic domain.-Myc (lanes1 and 2), FtsH (lanes3 and 4), or EnvZ (lanes5 and 6) did not
cause the Std phenotype. All of the above proteins evidently
accumulated in the cells as shown by Western blotting with anti-FtsH or
anti-EnvZ (Fig. 7B). These results suggested that among
the above proteins, only FtsH`-`EnvZ could interact with the
chromosomally-encoded FtsH to interfere with its function.
-myc) (lanes1 and 2 of A and lane1 of B), pSTD120 (ftsH-his-myc) (lanes3 and 4 of A and lane2 of B), pSTD124 (envZ) (lanes5 and 6 of A and lane3 of B),
pSTD125 (ftsH`-`envZ) (lanes7 and 8 of A and lane4 of B) or
pHSG575 (vector) (lanes9 and 10 of A and lane5 of B) were grown in peptone
medium containing 1 mM
isopropyl-1-thio--D-galactopyranoside and appropriate
antibiotics. A, cells were disrupted by lysozyme
freezing-thawing and treated with 50 µg/ml trypsin as indicated.
After separation by 10% polyacrylamide gel electrophoresis, proteins
were visualized by anti-PhoA immunoblotting. PhoA* indicates
the trypsin-resistant PhoA moiety that is expected if it is exported to
the periplasmic space. B, cultures were directly mixed with
trichloroacetic acid, and total proteins were separated by 10%
polyacrylamide gel and visualized by immunoblotting using antisera
against FtsH (upperpart) or EnvZ (lowerpart).
-Myc, or EnvZ were pulse-labeled, and
membranes were treated with DSP. Cross-linked products were examined by
immunoprecipitation. Fig. 8A shows results of an
experiment with FtsH`-`EnvZ. The anti-FtsH serum used in this study had
been directed against a sequence in the cytoplasmic domain of FtsH (17) . Thus, without cross-linking, the FtsH`-`EnvZ protein was
immunoprecipitated with anti-EnvZ serum but not with anti-FtsH serum (lanes5 and 6). The anti-EnvZ antibodies
did not cross-react with FtsH (lane6). After
cross-linking with DSP, FtsH`-`EnvZ was recovered with anti-FtsH (lane1), and FtsH was recovered with anti-EnvZ (lane2). Quenching of DSP before cross-linking
abolished these cross-reactions (lanes3 and 4). On the other hand, EnvZ`-`FtsH-His-Myc was not
cross-linked with FtsH, since FtsH was not precipitated with anti-Myc
antibodies even after DSP treatment (Fig. 8B). As
expected, no cross-linking was observed between FtsH and EnvZ (Fig. 8C). These results confirmed that FtsH`-`EnvZ can
interact with FtsH but EnvZ`-`FtsH-His-Myc cannot. The
interaction between FtsH molecules is likely to be mediated by its
membrane-associated region.
-myc)/pSTD401 (B), or TYE024/pTYE030 (envZ)/pSTD401 (C) as
described in the legend to Fig. 5B, except that
anti-FtsH, anti-Myc or anti-EnvZ was used for precipitation of the
cross-linked products as indicated.
(22) , the heat shock sigma factor, RpoH
(
)(6, 23) , and uncomplexed forms
of SecY(18) . From an in vitro study using purified
FtsH and RpoH, FtsH was suggested to have a proteolytic
activity(6) . Yta10, a mitochondrial inner membrane protein,
which is closely related to FtsH, was also suggested to participate in
degradation of abnormal proteins in the mitochondrial matrix
space(24, 25) . How can these diverse apparent
functions of FtsH be reconciled? The E. coli ClpA and ClpX
proteins, regulatory subunits of the Clp protease and distantly related
to FtsH, do not have any proteolytic activity themselves. Instead, they
are proposed to target substrate proteins for ATP-dependent
degradation(26, 27, 28) . It was also shown
that ClpA functions as a molecular chaperone in replication of P1
plasmid or in in vitro protein folding
reactions(28, 29) . Similarly, the AAA family includes
some of regulatory ATPase subunits of proteasomes. They have been
proposed to function in presentation of substrate proteins to the
protease subunits, the process in which energy of ATP hydrolysis is
somehow used (30) . FtsH may be a multifunctional protein that
exerts chaperone-like activities in the assembly or translocation of
some cell surface proteins and degradation of some unstable proteins.
to Met in the
periplasmic region of FtsH(7) . It did not affect the
interaction between the FtsH molecules,
implicating that
the membrane domain is not only important for the oligomerization but
may itself have some role in the FtsH functions. The 27-kDa protein was
co-immunoprecipitated even after treatment of the membrane with urea.
)-D-glucopyranoside.)
Ala substitution. The ftsH40 form of the gene can complement ftsH&cjs0453;kan(7) but not ftsH1(Ts)(20) , suggesting
that this mutation lowers the FtsH activity to a small extent.
))-his
-Myc)
was used.
)
We thank T. Ogura for a gift of anti-FtsH serum and
helpful discussion, T. Mizuno for a gift of pAT2005S and anti-EnvZ
serum, A. Kihara for discussion, and K. Mochizuki and K. Ueda for
technical and secretarial assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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