A Small Hydrophobic Domain Anchors Leader Peptidase to the Cytoplasmic Membrane of Escherichia coZi*

Leader peptidase is an enzyme of the Escherichia coli cytoplasmic membrane which removes amino-ter-minal leader sequences from many secreted and mem- brane proteins. Three potential membrane-spanning segments exist in the first 98 amino acids of leader peptidase. We have characterized the topology of leader peptidase based on its sensitivity to protease digestion. Proteinase K and trypsin treatment of right-side-out inner membrane vesicles and spheroplasts yields protected fragments of approximately 80 and 105 amino acid residues, respectively. We have shown that both fragments are derived from the amino terminus of the protein and that the smaller protected peptide can be derived from the larger. Removal of the third potential membrane-spanning segment (residues 82-98) does not affect the size of the proteinase K- protected fragment but does reduce the size of the trypsin-protected peptide. Because the proteinase K-protected fragment is about 9000 daltons, is derived from the amino terminus of leader peptidase, and its size is not affected when amino acids 82-98 are re- moved from the protein, it must extend from the amino terminus to approximately residue 80. Likewise, the trypsin-protected fragment must extend from the amino terminus to about residue 105. These data sug- gest a model for the orientation of leader peptidase in which the second hydrophobic stretch (residues 62- 76) spans the cytoplasmic membrane and the third hydrophobic stretch resides in the periplasmic space.

Isolation of the gene which encodes leader peptidase (Date and Wickner, 1981) has facilitated the characterization of this protein and has allowed us to study the enzyme's membrane assembly requirements. These studies have shown that, like other proteins of the cytoplasmic membrane, leader peptidase is synthesized without a cleaved amino-terminal leader sequence (Wolfe et al., 1983). The assembly of this protein is dependent upon the presence of an electrochemical potential across the inner membrane and functional secA and secY gene products . Studies aimed at determining which domains of leader peptidase are essential for its insertion into the cytoplasmic membrane have demonstrated that the last 182 amino acids of the protein are required (Dalbey and Wickner, 1986). Moreover, efficient assembly requires the presence of at least two of the three hydrophobic stretches in the protein.
Mechanisms proposed for the assembly of leader peptidase must take into consideration its orientation across the cytoplasmic membrane. Previous studies on the topology of this enzyme have shown that it is anchored with a small portion of its amino-terminal domain exposed to the cytoplasmic surface and a larger polar carboxyl-terminal domain in the periplasmic space (Wolfe et al., 1983). These experiments were not, however, able to establish which of the three hydrophobic stretches (Fig. LA, boxed sequences) span the cytoplasmic membrane. Based on protease protection studies conducted on right-side-out inner membrane vesicles and spheroplasts, we report that the third hydrophobic stretch of amino acids (residues 82-98) is exposed to the periplasm and that the second hydrophobic stretch (residues 62-76) plays a critical role in the anchoring of leader peptidase to the cytoplasmic membrane. Our working model for the topology of this enzyme is depicted in Fig. 1B.

Materials-Trypsin
(tosylphenylalanyl chloromethyl ketone treated) and soybean trypsin inhibitor were from Worthington. Phenylmethylsulfonyl fluoride, chymotrypsin, proteinase K, and indoleacrylic acid were from Sigma. CNBr-activated Sepharose 4B was obtained from Pharmacia P-L Biochemicals, and Staphylococcus aureus V8 proteinase was purchased from Cooper Biomedical.
Bacteria and Plasmids-E. coli strains MC1061 (Dalbey and Wickner, 1986) and HJM114 (Wickner and Killick, 1977) have been described elsewhere. Details of the construction of pRD8 are given in Dalbey and Wickner (1985). A description of the mutagenesis procedures used to obtain pRDA82-98, pRDam79, and pRDamlO5 will be published separately (Dalbey and Wickner, 1987).
Growth and Labeling Conditions-Cells were grown at 37 "C to midlog phase in M9 minimal media (Miller, 1972) containing 0.5% fructose, 0.1 mg/ml ampicillin, and 50 pg/ml of each amino acid but methionine. Arabinose (0.2%) was added to induce synthesis of wild type or mutant leader peptidase. When appropriate, cells were pulselabeled with [3SS]methionine (1000 Ci/mmol, Amersham Corp).
Preparation of Spheroplasts and Inner Membranes-Inner membranes from E. coli strain MC1061 were prepared by the method of a m Osborn et al. (1972). Spheroplasts were made from a second E. coli strain, HJM114.0.02-liter cells were grown as described above except that the period of induction in the presence of arabinose was increased to 2 h. After induction cells were chilled to 4 "C and centrifuged at 5000 X g for 5 min. (Subsequent steps were conducted at 4 "C.) Cell pH 8.0, to which 0.05 ml of 2 mg/ml lysozyme was added. After 2 min pellets were resuspended in 1 ml of 0.75 M sucrose, 10 mM Tris-C1, of incubation, 2 ml of 10 mM EDTA, pH 8.5, was added dropwise with shaking over a 5-min period. MgSO, was added to a final concentration of 20 mM.
Protease Treatment of Inner Membranes and Spheroplusts-Inner membrane vesicles and spheroplasts were incubated at 37 "C for 1 h in the presence of 0.2 mg/ml proteinase K or trypsin in 0.05 M Tris-C1, pH 8.0. Control digests were conducted in the presence of 1% Triton X-100. Reactions were stopped either by the addition of PMSF' (2 mM) or soybean trypsin inhibitor (2 mg/ml).
Construction of trpE Leader Peptidose Fusion Plasmids-Four fusion plasmids were constructed using sequences from the lep gene and the trpEZ vectors created by Koerner? These plasmids are diagrammed in Fig. 4 . 4 and have been designated pLPl-68, pLP70-141, pLP141-221, and pLP221-323. The numbers refer to amino acids in leader peptidase encoded by the plasmids. Three types of fusion vectors were used to construct these plasmids. PATH 1 was used for pLP221-323; PATH 3 for pLP70-141 and pLP141-221; and PATH 10 was used for pLP1-68. These vectors can be distinguished by the arrangement of restriction sites within the 3' end of the trpE gene. The restriction enzymes listed in Fig. 4A were used to liberate lep gene sequences from pRD8 DNA. The resulting DNA fragments were isolated from agarose gels by adsorption onto NA45 DEAE paper (Schleicher & Scbuell). Where appropriate, sticky ends of the fragments were filled in by incubation with DNA polymerase I (large fragment, New England Biolabs) and deoxynucleotide triphosphates (Maniatis et al., 1982). Ligation reactions were catalyzed by T4 DNA ligase (New England Biolabs). Recombinant plasmids were transformed into C600 cells (Cohen et al., 1973).
Preparation and Affinity Purification of Antibodies Specific for Defined Regions of Leader Peptidase-Fusion proteins between TrpE and specific regions of leader peptidase (see above) were induced by growing E. coli C600 cells which carried the appropriate fusion plasmid in the presence of indoleacrylic acid, a nonhydrolyzable analogue of tryptophan, according to the methods of Spindler et al. (1984). Each of the fusion proteins was purified'by elution from SDSpolyacrylamide gels and injected into rabbits (Wolfe et al., 1983). The resulting antibodies were purified by passing the antisera over affinity columns composed of purified leader peptidase cross-linked to cyanogen bromide-activated Sepharose 4B. Adherent antibodies were eluted with 3.5 M KSCN and dialyzed against 0.1 M NaHC03, pH 8.3.
Gel Electrophoresis and Western Blotting-SDS-polyacrylamide gel electrophoresis was performed as previously described (Ito et al., 1980) on 19% (w/v) acrylamide gels. Radioactive polypeptides were visualized by fluorography (Chamberlin, 1979). Western blotting was carried out according to the methods of Towbin et al. (1979).

RESULTS
To determine which of the three hydrophobic sequences in leader peptidase serve as membrane-spanning segments, we investigated which amino acid sequences are protected from protease added to the periplasmic surface of the membrane. We used preparations of right-side-out inner membrane vesicles and spheroplasts as our sources of membranes in these experiments. Inner membrane vesicles (IMVs) obtained from sucrose gradients of sonicated spheroplasts are known to be a composite of unsealed, sealed right-side-out, and sealed inverted vesicles. Inverted sealed membrane vesicles can be distinguished from right-side-out and leaky vesicles by the observance of a truncated form of leader peptidase in protea-* The abbreviations used are: PMSF, phenylmetbylsulfonyl fluoride; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; IMVs, inner membrane vesicles.
* trpE is a structural gene in the tryptophan operon of E. coli which encodes one of the components of the anthranilate synthetase complex. In this paper trpE will be used to signify the gene and TrpE will be used to represent the trpE gene product.
I. J. Koerner, personal communication. lytically digested membranes. This fragment of leader peptidase is approximately 32,000 daltons or 5,000 daltons smaller than its full length counterpart. It has been designated TRF I for Trypsin-Resistant Fragment because it was first observed as a minor band in trypsin-treated sonicated spheroplasts (Wolfe et al., 1983). The inner membrane vesicles used in these studies were mostly sealed in the right-side-out orientation. Proteolytic digests of spheroplasts have been included to verify results obtained with membrane vesicles.
An inner membrane fraction was prepared from cells (pRD8/MC1061) which carry a multicopy plasmid with the leader peptidase gene under arabinose promoter regulation. Addition of arabinose to this strain induces the synthesis of leader peptidase. Induced and uninduced cells were labeled with [35S]methionine, converted to spheroplasts, sonicated, and subjected to isopycnic sucrose density centrifugation (Osborn et al., 1972). The resulting inner membrane vesicles from induced cells (Fig. 2 9). Protein-resistant fragments fell into two size classes with approximate molecular weights of 11,000 for trypsin and V8 protease ( Fig. 2A, lanes 4 and 8 ) and 9,000 for chymotrypsin and proteinase K (lanes 2 and 6). These resistant fragments were not seen in digests conducted in the presence of detergent (lanes 3,5,7, and 9). Both classes of resistant fragments were found in tight association with membrane fractions (data not shown). Studies conducted using increasing amounts of protease on these IMVs demonstrated that both the 9,000-and the 11,000-dalton protected fragments are final digestion products (data not shown). These protected fragments were not observed in digests of IMVs from uninduced cells (Fig. 2B) and, therefore, are derived from leader peptidase.
In order to test whether these two proteolytic species were Cultures were labeled for 2 min with 1 mCi of [35S]methionine and chased for 2 min with 0.2 mg/ml unlabeled methionine. These radioactive cultures were then converted to spheroplasts and sonicated. Inner membranes were isolated by sucrose gradient centrifugation. 20 pl of these IMVs were incubated in a total reaction volume of 30 pl for 1 h at 37 "C without protease (lane 1 ) or with a final concentration of 0.2 mg/ml of the following proteases: chymotrypsin (lanes 2 and 3); trypsin (lanes 4 and 5); and proteinase K (lanes 6 and 7). S. aureus V8 proteinase was used at a final concentration of 1 mg/ml (lanes 8 and 9). Lanes 3, 5, 7, and 9 represent digests conducted in the presence of 1% Triton X-100. Samples were analyzed by SDS-PAGE and fluorography as described under "Experimental Procedures." LPase, leader peptidase. related, inner membrane vesicles from induced cells (Fig. 3, lane 1 ) were treated first with trypsin (lane 2) and then subsequently with proteinase K (lane 3). The third lane shows that the trypsin-resistant fragment is converted to the proteinase K-resistant fragment when both enzymes are used. These results demonstrate that the smaller of the two fragments (the proteinase K-&sistant Fragment or KRF I) is derived from the larger fragment (the trypsin-resistant fragment or TRF 11).
To assess directly which regions of leader peptidase were yielding the proteolytic fragments TRF I1 and KRF I, we prepared antibodies which recognize residues 1-68, 70-141, 141-221, and 221-323 of leader peptidase. These antibodies were obtained from rabbits injected with TrpE-leader peptidase fusion proteins and were purified by affinity chromatography as described under "Experimental Procedures." The efficacy of this purification procedure can be seen in Fig. 4B.. (Residues 70-141 were not immunogenic, and, therefore, antibodies which recognize this region were not included in these experiments.) Identical Western blots of induced fusion proteins were probed with three affinity-purified antibodies. Each of the antibodies was able to recognize full length leader peptidase (lanes 1, 6, and 11) as well as the fusion proteins used in their preparation (lanes 2,8, and 14). However, these antibodies were not able to recognize other fusion proteins related to leader peptidase (lanes 3, 4, 7,. 9, 12, and 13) or a fusion protein consisting of the trpE gene product and an amino acid sequence unrelated to leader peptidase (lanes 5, 10, and 15).
To confirm that TRF I1 and KRF I are derived from the amino terminus of leader peptidase, Western blots of IMVs digested with trypsin and proteinase K were probed with  IgG (lanes 11-15). Purified leader peptidase (500 ng, Wolfe et al., 1983) was included in lanes 1, 6, and 11 as a control. Lunes 2, 7, and 12 represent total proteins from cells carrying pLP1-68. In lanes 3,8, and 13, lysates from pLPl41-221/C600 are shown. Lunes 4,9, and 14 represent pLP221-323/C600 cell lysates. Total proteins from C600 cells which overproduce a trpE fusion protein containing amino acids not related to leader peptidase can be seen in lanes 5,10, and 15.
affinity-purified antibodies which recognize residues 1-68, 141-221, and 221-323 (Fig. 5, A, B, and C , respectively). Both resistant fragments can only be recognized by those antibodies directed against the amino-terminal region of leader peptidase (Fig. 5A, lanes 2 and 4). Antibodies which react with carboxylterminal sequences are only able to recognize leader peptidase from IMVs which have not been subjected to proteolysis (Fig.  5, B and C , lane 1).
To verify that the inner membrane vesicles described in the previous experiments were sealed in the right-side-out orientation, we performed proteolytic digests on spheroplasts. pRDS/HJM cells were grown in the presence of arabinose, converted to spheroplasts, and digested with increasing amounts of trypsin in the absence (Fig. 6A, lanes 1-4) or presence (lane 5 ) of detergent. Digested and undigested spheroplasts were subjected to .Western blot analysis using the antibody directed against residues 1-68 of leader peptidase as a probe. Incubation of spheroplasts with 0.02 mg/ml trypsin for 1 h at 37 "C was sufficient to convert all of the full length leader peptidase to the trypsin-resistant form (lane 3). Digestions conducted using a higher concentration of enzyme (0.2 mg/ml) did not result in further digestion of this resistant fragment (lane 4 ) .
Similar experiments were performed to check whether the proteinase K-protected fragment obtained from spheroplasts was the same KRF I seen in the digests of IMVs. Spheroplasts were treated with proteinase K and subjected to Western blot analysis using the antibody directed against residues 1-68 of leader peptidase as a probe. The resulting proteinase Kresistant fragment (Fig. 6B, lane 4 ) has the same electrophoretic mobility as the one generated from proteinase K digestion of IMVs (lane 2). This observation is confirmed by the fact that protected peptides from the two samples (KRF I from spheroplasts and IMVs) migrate as one band when mixed (lane 3). The same kind of mixing experiment was conducted with KRF I from spheroplasts (lane 9) and TRF I1 from inner membrane vesicles (lane 7). The difference in the apparent molecular weights of these two peptide species can be seen in lane 8.
To determine whether the third hydrophobic stretch of amino acids in leader peptidase is exposed to the periplasm, inner membrane vesicles were prepared from a strain (pRDA82-98) which overproduces a mutant leader peptidase missing residues 82-9tL4 These pRDA82-98/MC1061 vesicles were digested with trypsin and proteinase K and compared with identical digests of wild type pRD8/MC1061 IMVs. Western blot analysis, again using affinity-purified antibodies ' R. E. Dalbey and W. Wickner, submitted for publication.

peptidase.
which recognize residues 1-68 of leader peptidase as a probe, shows that this mutant protein (Fig. 7, lane 2) migrates slightly faster than its wild type counterpart ( l a n e I ) . Bands smaller than the full length pRDA82-98-encoded leader peptidase ( l a n e 2) represent membrane-associated in vivo generated degradation products? As predicted from the model in Fig. lB, the size of the trypsin-resistant fragment is smaller in the pRDA82-98/MC1061 vesicles (Fig. 7, lane 4 ) than in the pRD8/MC1061 vesicles ( l a n e 3). When both types of vesicles were treated with proteinase K, protected fragments with identical electrophoretic mobilities were observed (lanes 5 and 6). Therefore, the size of the proteinase K-protected fragment has not been affected by the removal of the third hydrophobic stretch of amino acids whereas the size of the trypsin-protected fragment is reduced. Since KRF I is about 9,000 daltons, reacts with antibody to residues 1-68, and yet is not altered by removal of residues 82-98, it must extend from within a few amino acids of the amino terminus in leader peptidase to approximately residue 80. By the same logic, TRF I1 must extend from very close to the amino terminus to approximately residue 105.
Amber mutations in the leader peptidase gene which encode truncated versions of leader peptidase were used to estimate the sizes of TRF I1 and KRF I. Two amber mutants were made available for our studiess which encode the first 78 and 104 amino acids in leader peptidase. Plasmids which carry these two mutants, pRDam79 and pRDaml05, were transformed independently into MC1061, grown in the presence of 0.2% arabinose, and labeled for 1 min with [3sSS]methionine. Total cell lysates from induced cells were electrophoresed either separately (Fig. 8, lanes 3 and 6,  104 and 78 amino acids long, respectively) or in mixtures (lanes 2 and 5) with protease-protected fragments from 35Slabeled IMVs (lanes 1 and 4). Lane 2 represents a mixture of total proteins from pRDam105/MC1061 and TRF I1 from IMVs digested with trypsin. Lane 5 represents a mixture of cell lysates from pRDam79/MC1061 and KRF I from IMVs. These results indicate that the observed length of TRF I1 is very close to 104 amino acids and KRF I is slightly larger than 78 amino acids in length.

DISCUSSION
It has not been established how proteins such as leader peptidase, synthesized without cleavable leader sequences, are able to assemble into the inner membrane of E. coli. It has been postulated that internal uncleaved signal sequences are required for translocation to occur (Blobel, 1980). It has also been suggested (Wickner and Lodish, 1985) that spontaneous insertion domains may be responsible for the integration of these proteins into the lipid bilayer. All hypotheses which attempt to explain the assembly of a given protein into a lipid bilayer must, however, take into consideration its final orientation in the membrane.
Studies described by Wolfe et al. (1983) demonstrated that leader peptidase is anchored to the cytoplasmic membrane by a segment near the amino-terminal end of the polypeptide. This conclusion was based on an experiment in which inverted vesicles, prepared by the sonication of spheroplasts, yielded a smaller form of leader peptidase upon trypsin digestion. (While these vesicles were obtained by sonicating spheroplasts, the sealed inverted vesicles represented only about 10% of the total population of vesicles.) The authors postulated that this trypsin-resistant fragment (TRF I) resulted A B 1 2 3 4 5 6 7 ' 8 9 1 0 I 2 3 4 5 LPase -

TRFE-
FIG. 6. Proteolytic treatment of spheroplasts and IMVs yield the same protected fragments. Cells (pRD8/HJM) were grown to Am = 0.2 at 37 "C and incubated with 0.2% arabinose for 2 h. Spheroplasts were prepared as described under "Experimental Procedures." 20 ml of cell culture yielded 1.5-ml spheroplasts. A, 30-p1 spheroplasts were treated at 37 "C with no ( l a n e I ) , 0.002 mg/ml ( l a n e 2), 0.02 mg/ml (lanes 3 and 5), and 0.2 mg/ ml trypsin ( l a n e 4 ) . Lane 5 represents spheroplasts digested in the presence of 1% Triton X-100. B, after digestion of 1.5 ml of spheroplasts with proteinase K and addition of PMSF (2 mM) to the reaction mixture, total membranes were isolated. Digested spheroplasts were passed through the French press one time a t 8000 p.s.i. and centrifuged for 1 h at 37,000 X g, 0 "C. The membrane fraction was resuspended in 0.01 M Tris-C1, pH 8.0, and subjected to acid precipitation and gel electrophoresis. Lanes 1 and 2 represent untreated and proteinase K-treated (0.2 mg/ ml) pRD8/MC1061 IMVs, respectively. Lanes 4 and 5 show spheroplasts treated with ( l a n e 4 ) and without ( l a n e 5) proteinase K at a final concentration of 0.2 mg/ml. Samples from lanes 2 and 4 were mixed in lane 3. Untreated IMVs can be seen in lane 6 and trypsin-treated (0.2 mg/ml) membranes in Lane 7. lane 8 represents a mixture of trypsin-treated IMVs and proteinase K-treated spheroplasts. Spheroplasts treated with proteinase K and analyzed separately can be seen in lane 9. Again, lane 10 represents undigested spheroplasts. Reactions were stopped by the addition of PMSF (2 mM) or soybean trypsin inhibitor (2 mg/ml). Samples were subjected to SDS-PAGE and Western blot analysis. Nitrocellulose filters were probed with antibodies specific for residues 1-68 of leader peptidase (LPose).
from the loss from one end of the protein of a peptide approximately 5000 daltons in molecular mass. Two-dimensional thin layer chromatography was used to establish that this peptide was derived from the amino terminus of leader peptidase. However, these studies did not establish which regions of the protein span the cytoplasmic membrane. Three potential membrane-spanning regions are predicted from analysis of the amino acid sequence of leader peptidase (Eisenberg et al., 1984, Fig. lA, boxed sequences). We have shown here that the second hydrophobic stretch (residues 62-76) spans the cytoplasmic membrane and that the third hydrophobic sequence of amino acids (residues 83-98) resides in the periplasmic space. This model is based on the following findings. 1) Right-side-out membrane vesicles and spheroplasts protect a 9000-dalton peptide from complete digestion when treated with proteases which exhibit wide substrate specificities (ie. proteinase K and chymotrypsin); 2) this protected fragment is derived from the amino terminus of the protein; and 3) its apparent size is not affected by the removal of amino acid residues 82-98. This hydrophobic stretch of 15 amino acids is shorter than those generally expected for membrane-spanning regions. Our data indicate that the proteinase K-protected fragment is several amino acids longer than the 78-amino acid amber fragment, suggesting that some of the residues between amino acids 77 and 82 are included in the membrane-spanning region. However, we cannot be sure whether the residues Pro-Gly-Trp-Leu-Glu between amino acids 57 and 61 or the amino acids between residues 76 and 83, Arg-Ser-Phe-Ile-Tyr-Glu, are included in the membrane anchor region. At this time little is known about the inclusion of ions or ion pairs in membrane-spanning domains. This is due to the fact that the topology of few transmembrane proteins is known with accuracy.
The asymmetric orientation of leader peptidase offers a unique opportunity for assessing the orientation of sealed vesicles. Kaback and co-workers have shown that the majority Topological localization of the third hydrophobic stretch of amino acids in leader peptidase (LPase). IMVs were made from an E. coli strain pRDA82-98/MC1061. Proteolytic digests of these vesicles were compared with digests of pRD8/MC1061 vesicles. Odd-numbered lanes represent IMVs from pRD8 vesicles, and even-numbered lanes show IMVs from pRDA82-98 vesicles. Undigested membranes can be seen in lanes I and 2. Vesicles treated with 0.2 mg/ml trypsin for 1 h at 37 "C are shown in lanes 3 and 4. Proteinase K-digested vesicles (0.2 mg/ml, 1 h, 37 "C) are seen in lanes 5 and 6. PMSF (2 mM) or soybean trypsin inhibitor (2 mg/ml) was used to stop proteolytic reactions. These samples were subjected to Western blot analysis using affinity-purified antibodies which recognize residues 1-68 of leader peptidase as a probe. of the markers for the inner membrane are largely exposed on the cytoplasmic surface of the bilayer (Owen and Kaback, 1979). Leader peptidase, as presented here, exhibits characteristic digestion patterns when sealed vesicles of either orientation are subjected to treatment with trypsin. Inverted vesicles yield a 32,000-dalton protected fragment (TRF I), and right-side-out vesicles yield a protected fragment of 11,000 daltons (TRF 11). To date, only the coat protein of M13 phage (Chang et al., 1979;Wickner, 1976) has afforded similar opportunities for studying membrane orientation. The orientation of leader peptidase also poses interesting questions of how the structure of the cytoplasmic domain may influence the enzymatic activity of the periplasmic domain (Zimmerman et al., 1982). Studies conducted in uitro using protease-treated inverted membrane vesicles have shown that removal of the amino terniinus of leader peptidase inactivates leader peptide-processing activity (Ohno-Iwashita et al., 1984). In addition, mutants in leader peptidase which are missing residues 4-50 of the mature protein are able to assemble into the inner membrane but fail to cleave precursor proteins to their mature forms in uitro.4 The function of this amino-terminal domain on the opposite side of the membrane from the catalytic domain is not known. The results described in this paper are consistent with the observation (Dalbey and Wickner, 1987) that residues 62-76 are essential for the assembly of leader peptidase into the inner membrane. When these amino acids are removed from the polypeptide using genetic techniques, the mutant protein  1 and 4 represent IMVs digested with trypsin (0.2 mg/ ml) and proteinase K (0.2 mg/ml), respectively. Lane 2 is a mixture of the samples run in lanes 1 and 3. Lane 5 represents a mixture of the samples run in lanes 4 and 6. Samples were subjected to SDS-PAGE and visualized by fluorography. a.a., amino acids. fails to insert across the bilayer. In addition, a fusion protein composed of residues 51-82 of leader peptidase and the mature part of OmpA is capable of translocation across the inner membrane? Therefore, it appears that the second hydrophobic domain functions both as a signal and a membrane anchor sequence for leader peptidase, as is the case for influenza virus neuraminidase (Blok et al., 1982).