(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-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.
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 ()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.
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-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.
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 -globulin (158 kDa), chicken ovalbumin (44 kDa), equine
myoglobin (17 kDa), and vitamin B
(1.35
kDa).
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 of molecular
weights) on the leftsides of gels. Filledarrowheads indicate cross-linked
products.
Figure 3:
Schematic representations of
FtsH-His-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.
Figure 4:
Synthesis and stability of
FtsH-His-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.
Figure 5:
Cross-linking of FtsH and FtsH` with
FtsH-His-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.
Figure 6:
Co-immunoprecipitation of FtsH and FtsH`
with FtsH-His-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.
Cells of CU141(F`lacI) 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.
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-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.
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-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.
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-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).
Cells
overexpressing FtsH and either FtsH`-`EnvZ,
EnvZ`-`FtsH-His-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.
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-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.
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 (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.
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 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.
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. The 27-kDa protein was
co-immunoprecipitated even after treatment of the membrane with urea.
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.