©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
FtsH, a Membrane-bound ATPase, Forms a Complex in the Cytoplasmic Membrane of Escherichia coli(*)

(Received for publication, May 31, 1995; and in revised form, July 13, 1995)

Yoshinori Akiyama (§) Tohru Yoshihisa Koreaki Ito

From the Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto 606-01, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(6)-Myc). When membranes prepared from cells in which FtsH-His(6)-Myc was overproduced together with the normal FtsH were treated with the cross-linker, intact FtsH and in vitro degradation products of FtsH-His(6)-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.


INTRODUCTION

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 (^1)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(6)-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.


MATERIALS AND METHODS

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^q derivative of MC4100, respectively. TYE024 (MC4100, ompT::kan/F`lacI^q) was constructed by introducing F`lacI^q of CU141 into AD202 by conjugation.

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.

Construction of the ftsH-his(6)-myc Plasmids

pSTD101 carrying ftsH-his(6)-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) . (^2)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(6)/c-Myc tag of 30 amino acid residues (EFIEGRHHHHHHIDEEQKLISEEDLLRKR) following its multicloning site. (^3)The ftsH gene and the his(6)-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(6)-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(6)-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(6)-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(6)-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-beta-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(3) (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) .


RESULTS

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.


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).



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.


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 10^3 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(6)), and a c-Myc-derived epitope at the C terminus (Fig. 3). The FtsH-His(6)-Myc protein can specifically be isolated, and detected by nickel-nitrilotriacetic acid-agarose and anti-Myc antibodies, respectively. Cells carrying pSTD101 (ftsH-his(6)-myc) were pulse-labeled, and labeled proteins were first solubilized in SDS and then precipitated with anti-FtsH or anti-Myc antibodies. (^4)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(6)-Myc was stable in vivo; no degradation was observed during a 16-min chase period examined (lanes2, 3, 5, and 6). The FtsH-His(6)-Myc protein was functional, since pSTD120 (a low copy plasmid carrying ftsH-his(6)-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(6)-Myc, like normal FtsH, was recovered in the membrane fraction (data not shown). We found that a fraction of FtsH-His(6)-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(6)-Myc tag, since FtsH` lost the Myc epitope (Fig. 5B, lane5).


Figure 3: Schematic representations of FtsH-His(6)-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(6)-Myc and EnvZ-His(6)-Myc represent His(6)-Myc tags.




Figure 4: Synthesis and stability of FtsH-His(6)-Myc. Cells of AD21/pSTD101 (ftsH-his(6)-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-beta-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(6)-Myc. A and B, cells of TYE024/pSTD113 (ftsH-his(6)-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(6)-Myc. Cells of TYE024/pSTD101 (ftsH-his(6)-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(6)-Myc.



Cells of CU141(F`lacI^q) carrying both the ftsH-his(6)-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(6)-Myc

We carried out immunoprecipitation under nondenaturing conditions (Fig. 6). Membrane fraction was prepared from FtsH-His(6)-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(6)-Myc was recovered when the membranes were first solubilized in SDS and then subjected to immunoprecipitation with anti-Myc antibodies (see Fig. 5B, lane5).

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(6)-Myc by nickel-nitrilotriacetic acid-agarose affinity column chromatography. (^5)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(6)-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(6)-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(6)-Myc has the EnvZ membrane domain followed by the tagged FtsH cytoplasmic domain.

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(6)-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(6)-myc) (lanes1 and 2 of A and lane1 of B), pSTD120 (ftsH-his(6)-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-beta-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(6)-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(6)-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(6)-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(6)-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.




DISCUSSION

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,^5 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.^5 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.


FOOTNOTES

*
This work was supported by grants from the Ministry of Education, Science, and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-75-751-4040; Fax: 81-75-771-5699 or 81-75-761-5626; yakiyama{at}virus.kyoto-u.ac.jp.

(^1)
The abbreviations used are: PhoA, alkaline phosphatase; kb, kilobase pair(s); DSP, dithiobis(succinimidyl propionate); OG, 1-o-n-octyl beta-D-glucopyranoside.

(^2)
Although we described previously that pSTD40 carried the wild-type ftsH gene(7) , we subsequently found that the ftsH gene in pSTD40 and, as a result, that in pSTD101 contained a spontaneously introduced mutation (ftsH40) that causes a Thr 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.

(^3)
T. Yoshihisa, unpublished results.

(^4)
Although we used pSTD101 in the experiments described in Fig. 2and Fig. 3, essentially the same results were obtained when pSTD113 (ftsH-his-Myc) was used.

(^5)
Y. Akiyama, unpublished results.


ACKNOWLEDGEMENTS

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.


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