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J. Biol. Chem., Vol. 276, Issue 29, 27111-27119, July 20, 2001

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Serratia ATP-binding Cassette Protein Exporter, Lip, Recognizes a Protein Region Upstream of the C Terminus for Specific Secretion^*

Kenji Omori^§, Akiko Idei^¶, and Hiroyuki Akatsuka^¶

From the  Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., 2-50, Kawagishi-2-chome, Toda, Saitama 335-8505, and the ^¶ Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., 16-89, Kashima-3-chome, Yodogawa-ku, Osaka 532-8505, Japan

Received for publication, February 14, 2001, and in revised form, April 23, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Serratia marcescens ATP-binding cassette (ABC) exporter, the Lip system, secretes lipase (LipA_SM), metalloproteases, and a cell surface layer protein homologue but not a heme acquisition protein, HasA (HasA_SM). Secretion of HasA_SM is limited to the Has_SM system. However, HasA proteins from Pseudomonas fluorescens (HasA_PF) and Pseudomonas aeruginosa were exported through the Lip and Has_SM systems. To investigate the specificity in Lip exporter-mediated secretion, secretion analysis was performed using chimeras containing the HasA_PF and HasA_SM sequences. The segment Val-Ala-Leu (designated R1 to R3 sites), which is present close to the C terminus of HasA_PF but not HasA_SM, was revealed to be involved in the substrate specificity of the Lip exporter. Introduction of amino acid substitutions into the R1-R5 region demonstrated that R1, R3, R4, and R5 sites require some specific amino acid residues for Lip-mediated secretion. The amino acid sequence of the region was conserved considerably among the proteins secreted by the Lip exporter. On the contrary, the region was not related to HasA secretion through the Has_SM system. Interestingly, a typical C-terminal motif, so far regarded as a secretion signal, was not necessary for secretion through either the Lip or the Has_SM exporter. In LipA_SM secretion via the Lip system, the typical C-terminal motif was not essential either, but the presence of a sequence similar to Val-Ala-Leu and its location from the C terminus greatly affect the secretion level. Secretion analyses using hybrid exporters and competitors exhibited that the R1-R5 region was recognized by an ABC protein of the Lip exporter, LipB, and that the mutations aborting Lip-mediated secretion in the region resulted in a loss of the affinity to LipB. Thus, a determinant within the secretory protein for Lip-mediated secretion was fully defined.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The ATP-binding cassette (ABC)¹ exporter, which mediates translocation of proteins lacking an N-terminal signal sequence across the cell membranes, is known in Gram-negative bacteria. Secretion through the system is one step and differs from that through the sec gene-mediated pathway. The system, termed type I, is categorized as an ABC transporter family (1, 2). The transport process requires ATP hydrolysis as an energy source, and secretion through the system requires three specific components: an inner membrane protein (ABC protein), a membrane fusion protein (MFP), and an outer membrane protein (OMP).

One example of the present study is the Serratia marcescens Lip system composed of LipB (ABC protein), LipC (MFP), and LipD (OMP) (3). The other example is the S. marcescens Has_SM system, including HasD_SM, HasE_SM, and HasF_SM (4-6). HasF_SM is highly similar to the Escherichia coli OMP, TolC (6), and TolC functions instead of HasF_SM. The Lip system is involved in secretion of three unrelated proteins of S. marcescens, lipase (LipA_SM), metalloprotease (PrtA), and a cell surface layer protein homologue (3, 7), while the Has_SM system is dedicated to secretion of the HasA_SM protein, which is an extracellular polypeptide of 19 kDa exhibiting heme binding activity and facilitating iron acquisition (8, 9). Thus, two protein transport systems are present in S. marcescens, and they play a specific role for secretion of each protein in vivo. In regard to their secretion specificity, several reports were made using reconstituted exporters in the E. coli cells (10, 11).² The Lip system can promote secretion of the Erwinia chrysanthemi metalloprotease C (PrtC), Pseudomonas fluorescens lipase (LipA_PF) and alkaline protease (AprA_PF), and the Pseudomonas aeruginosa alkaline protease (AprA_PA). The Has_SM system mediates secretion of LipA_SM, S. marcescens PrtA, and the E. chrysanthemi PrtC. In contrast to the secretory proteins secreted through the Lip system, HasA_SM can be secreted only by the Has_SM system. The Lip system and the E. chrysanthemi metalloprotease exporter Prt system (PrtD-PrtE-PrtF; Ref. 12) are unable to promote HasA_SM secretion. The levels of sequence homology between each component of these transporters are considerable, and in fact, some components are exchangeable with those of other systems (10-12). Nevertheless, HasA_SM secretion is specific and limited to the Has_SM exporter.

An analysis of hybrid exporters comprising components from two distinct ABC exporters revealed that one determinant for the secretion specificity of the ABC exporter is an ABC protein (10, 12). The secretion analysis of the E. chrysanthemi metalloprotease PrtG through the Prt system has demonstrated that a secretion signal is situated at the C terminus (13). The signal does not include any common primary sequence but contains a motif consisting of a negatively charged amino acid residue followed by several hydrophobic residues. Several reports described the involvement of the C-terminal region in the secretion specificity (14-19). However, a sequence feature or protein region involved in the substrate specificity of the ABC exporter has not been investigated in detail yet.

A small and unique secretory protein, HasA_SM, is useful for the analysis of the substrate specificity. Three homologues of HasA have been identified from S. marcescens, P. aeruginosa (HasA_PA), and P. fluorescens (HasA_PF) (8, 20, 21). Two exporters, S. marcescens Has_SM and P. fluorescens Has_PF (HasD_PF-HasE_PF-HasF_PF), have been reported to be devoted to secretion of HasA proteins, hitherto. Interestingly, the Has_SM exporter is able to secrete all three HasA proteins, whereas the Has_PF exporter secretes HasA_PF and HasA_PA but not HasA_SM (21). Presently, Has_SM is a sole exporter for HasA_SM. The typical C-terminal motifs D-W-A-L-A-A, D-L-A-L-A-A, and E-L-L-A-A are found in HasA_PF, HasA_PA, and HasA_SM sequences, respectively. The motifs are analogous, and therefore, the secretion specificity of HasA_SM cannot be elucidated simply by the motif.

Specific recognition by the ABC exporter is an essential process for translocation of secretory proteins, and therefore, the specificity of HasA_SM secretion is one of the important subjects for understanding the secretion mechanism of the ABC exporter. In this paper, we attempt to describe the characteristics of the sequence recognized by the ABC exporter using HasA_SM and LipA_SM mutants as a substrate and the Lip system as an exporter. We reveal involvement of a protein region close to, but not at, the C terminus in Lip-mediated secretion and recognition by an ABC protein, LipB. Furthermore, we demonstrate that a typical C-terminal motif, which is considered as a secretion signal so far, is not essential for secretion. Our findings provide new important information for understanding the secretion mechanism and protein structure involved in the secretion specificity of the ABC exporter.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Strains and Media-- E. coli K12 DH5 was used as a host, and LB medium was used as a rich medium (22). S. marcescens 413, which is a LipA_SM-deficient strain (11), was used for a host of the secretion analysis of LipA_SM mutants. Antibiotics were added at the following concentrations: ampicillin, 50 µg/ml; kanamycin, 50 µg/ml; and chloramphenicol, 20 µg/ml for E. coli and ampicillin, 1000 µg/ml for S. marcescens 413.

General Methods-- DNA manipulations were carried out according to standard procedures (21). PCR was carried out using ExTaq purchased from Takara Shuzo (Kyoto, Japan). The nucleotide sequence was determined by dideoxy chain-termination method using a BigDye Terminator Cycle Sequencing Reaction kit (Applied Biosystems) and an automated DNA sequencer ABI PRISM^TM 310. Transformation of S. marcescens 413 was done as described previously (23).

DNA Constructs-- The HasA plasmids pUC/HasA_SM (HasA_SM), pUC/HasA_PF (HasA_PF), and pSYC1000 (HasA_PA) and their chimeras (pMBF-HasA, pMBF-HasA, and pFBF-HasA) have been described previously (20, 21). HasA chimeras and their mutants were constructed based on pUC/HasA_SM by a conventional method using PCR-directed mutagenesis and synthetic oligonucleotide linkers. LipA_SM mutants were constructed from pLIPE121 (24), which is pUC19 carrying the lipA_SM gene, using PCR-directed mutagenesis and synthetic oligonucleotide linkers. Nucleotide sequences of the inserted DNAs of the resultant plasmids were confirmed by sequencing.

To produce hybrid exporters, the hasADE₈₀₀₀ genes (hasADE from S. marcescens 8000) were cloned from a S. marcescens 8000 genomic DNA library. The hasDE₈₀₀₀ genes were inserted into multiple cloning sites of pMW218 to generate pMWHasDE7. The plasmid pMW/HasE, carrying the hasE_SM gene, was created by deleting the DNA fragment coding for the hasD_SM gene from pMWHasDE7. The plasmids pACYC/LipB and pMW/LipCD carrying the lipB and lipCD genes from S. marcescens 8000 were created by deleting the lipCD and lipB fragments from pYBCD20 (7) and pMWBCD10 (25), respectively.

A FLAG-tagged HasA-VAL mutant encoded by pSTV/FLAG-HasA-VAL was generated as follows. A DNA fragment was amplified by PCR using an oligonucleotide, 5'-GGGAGCTCATTTTCAGTCAATTATGACAGC-3', the M4 primer 5'-GTTTTCCCAGTCACGAC-3', and pMFM-VAL as a template, digested by SacI and HindIII, and then inserted into the corresponding sites of pUC18 (22). After digestion with EcoRI and SacI, the resultant plasmid was ligated with a synthetic linker (5'-AATTCGGATTACAAGGACGACGATGACAAGGAGCT-3' plus 5'-CCTTGTCTACGTCGTCCTTGTAATCCG-3') and then the SacI site was disrupted using T4 DNA polymerase. The EcoRI-HindIII fragment was inserted into the corresponding sites of pSTV28, a pACYC184 (22)-derived cloning vector (Takara Shuzo), generating pSTV/FLAG-HasA-VAL, which encodes HasA-VAL containing the sequence Met-Thr-Met-Asn-Ser-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys at the N terminus.

Analysis of Protein Secretion-- The plasmids pLG575 (HlyBD/TolC; Ref. 14), pACYC/OI70 (HasDEF_PF; Ref. 21), pK150 (HasDE_SM/TolC; Ref. 10), pRUW4 (PrtDEF; Ref. 26), pAGS8 (AprDEF_PA; Ref. 19), and pYBCD20 (LipBCD; Ref. 7), which encode ABC exporters in pACYC184, were used for the secretion analysis. The E. coli DH5 cells carrying plasmids encoding the ABC exporter and the HasA derivative were cultured in LB medium at 30 °C for 28 h with vigorous shaking. The proteins in the supernatants of the cultured media were concentrated by precipitation with trichloroacetic acid at a final concentration of 10%. The proteins were subjected to SDS-PAGE. The recombinant S. marcescens 413 cells carrying the lipA_SM plasmids were cultured in the lipase medium (27) at 30 °C for 28 h, and the proteins in the supernatants were directly loaded on the gel. The precast gel PAGEL (Atto Corp., Tokyo, Japan) was used for SDS-PAGE. The proteins in the gels were stained by Coomassie Brilliant Blue G-250.

Secretion Analysis with Hybrid Exporters-- The HasA plasmid (pUC/HasA_SM, pMFM-VAL, or pMFM-VAA; ampicillin-resistant), the ABC protein plasmid (pACYC/LipB or pACYC/HasD; chloramphenicol-resistant), and the MFP-OMP or MFP plasmid (pMW/LipCD or pMW/HasE; kanamycin-resistant) were introduced into the E. coli cells. The resultant recombinant cells were cultured in LB medium at 30 °C for 40 h with vigorous shaking. After trichloroacetic acid precipitation, the proteins in the supernatants of the cultured media were loaded on the gel as described above and then electrophoretically transferred to an Immobilon-P filter (Millipore). The blots were blocked by soaking in Block Ace (Dainippon Pharmaceutical, Osaka, Japan) overnight at 4 °C and incubated with an anti-HasA_SM antibody (a kind gift from Dr. Cecile Wandersman) at room temperature for 2 h (diluted 1:5000 in PBS containing 0.1% Tween 20). They were washed and incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G, and the bound antibody was detected with the enhanced chemiluminescence systems (Amersham Pharmacia Biotech).

Secretion Competition Analysis-- The plasmid pSTV/FLAG-HasA-VAL coding for a FLAG-tagged HasA-VAL mutant as a secretion substrate and the plasmids encoding HasA mutant proteins as a competitor were introduced into the E. coli cells carrying pFBCD1 (lipBCD in the mini-F derivative pKPT1124 (28)).³ The recombinants were cultured in LB medium at 30 °C for 24 h with vigorous shaking. The proteins in the supernatants of the cultured media were subjected to SDS-PAGE and immunoblot analysis with anti-FLAG monoclonal antibody (Sigma) and anti-HasA_SM antibody. In all experiments, protein analysis was carried out two or three times independently, and similar results were obtained. To access cell lysis, -galactosidase activity was measured using o-nitrophenyl--D-galactoside as a substrate according to the method described in Ref. 29. The levels of the extracellular -galactosidase activity of the cells were <2% compared with those of cell lysates, indicating no significant cell lysis in all experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Secretion of the HasA_PF Protein through the Lip Exporter-- It has been reported that HasA_SM secretion is confined to the Has_SM exporter, whereas HasA_PF secretion has not been examined except for the Has_PF and Has_SM exporters. HasA_PF secretion was investigated using the recombinant E. coli cells expressing several ABC exporters. The proteins in the cultured supernatants were analyzed by SDS-PAGE (Fig. 1A). Interestingly, the Lip exporter, which is unable to mediate HasA_SM secretion (11), secreted HasA_PF at a high level as well as Has_SM (TolC was utilized as OMP in the E. coli cells), compared with a genuine exporter for HasA_PF, Has_PF. Prt and Apr_PA (an exporter for AprA_PA) also promoted HasA_PF secretion at low and very low levels, respectively. The E. coli hemolysin exporter, Hly, did not let the cells secrete HasA_PF at a detectable level. Thus, the secretion capability of the Lip exporter for a protein categorized as HasA was demonstrated. Secretion of three HasA homologues, HasA_SM, HasA_PA, and HasA_PF, were tested (Fig. 1B). The Has_SM exporter allowed secretion of all three HasA proteins as described previously (20, 21), whereas the Lip exporter secreted both HasA_PF and HasA_PA but not HasA_SM. It is intriguing that secretion of HasA_PF and HasA_PA was achieved by an unrelated exporter, the Lip system.

View larger version (66K): [in this window] [in a new window]   Fig. 1.   Secretion of HasA proteins via several ABC exporters. A, E. coli DH5 cells carrying pUC/HasAPF and ABC exporter plasmids were cultured at 30 °C for 40 h. The polypeptides in the supernatants of the cultured media (1.5 OD equivalents) were subjected to 12.5% SDS-PAGE and stained by Coomassie Brilliant Blue G-250. The position of HasAPF is shown by an arrowhead. Lane 1, molecular mass markers; lane 2, pACYC184 (a vector); lane 3, pLG575 (HlyBD/TolC); lane 4, pACYC/OI70 (HasDEFPF); lane 5, pK150 (HasDESM/TolC); lane 6, pRUW4 (PrtDEF); lane 7, pAGS8 (AprDEFPA); lane 8, pYBCD20 (LipBCD). B, E. coli DH5 cells harboring HasA plasmid (pUC/HasASM, pUC/HasAPF, or pSYC1000) and ABC exporter plasmid (pACYC184, pK150, or pYBCD20) were cultured at 30 °C for 40 h. The polypeptides in the media were analyzed as described above. The positions of molecular mass markers are shown on the left. The positions of HasA proteins are shown by arrowheads on the right. The exporters used are indicated above. Lane 1, pUC/HasASM; lane 2, pUC/HasAPF; lane 3, pSYC1000.

Involvement of the HasA_PF C-terminal Segment in Secretion through the Lip Exporter-- The HasA_PF and HasA_PA proteins were suspected to contain a sequence necessary for Lip-mediated secretion but needless for secretion through the Has_SM exporter. The most notable difference in the amino acid sequences among three HasA homologues is the presence of segments, composed of 12 and 14 amino acid residues, close to but not at the C termini of HasA_PF and HasA_PA, respectively (Fig. 2A). Involvement of the segment in Lip-mediated secretion was tested using HasA chimeras containing the HasA_SM and HasA_PF sequences (Fig. 2B). Secretion of all HasA chimeras through the Has_SM exporter has already been confirmed (21). As shown in Fig. 2C, the Lip system secreted HasA_PF and a chimera carrying the HasA_PF C terminus (pMBF-HasA) but not HasA_SM nor chimeras containing the HasA_PF C terminus without the segment of 12 amino acid residues (pMBF-HasA and pFBF-HasA). HasA chimera, including the 12-amino acid segment in the HasA_SM sequence (pMFM-HasA; Fig. 3A), was secreted by both Has_SM and Lip (Fig. 3B). Thus, it was concluded that the HasA_PF C-terminal segment is involved in secretion through the Lip exporter but not required for Has_SM-mediated secretion.

View larger version (58K): [in this window] [in a new window]   Fig. 2.   Secretion of HasA chimeras composed of the HasASM and HasAPF sequences via the Lip and HasSM exporters. A, comparison of the predicted amino acid sequences among HasA proteins. The entire amino acid sequences are presented in one-letter code. To maximize homology, gaps (dashes) were introduced into the sequences. Identical amino acid residues are shown in an outline typeface with black background. The C-terminal segments present in HasAPF and HasAPA are boxed. B, HasA chimeras are schematically illustrated. The closed and open boxes represent amino acid sequences of HasASM and HasAPF, respectively. The HasAPF C-terminal segment (amino acid residues 181-192) is indicated by a cross-hatched box. Amino acid residue numbers of the HasAPF sequence are printed in italics. The BglII site is shown. C, the plasmids encoding HasASM, HasAPF, and HasA chimeras were introduced into the E. coli DH5 cells carrying the plasmids pACYC184 and pYBCD20. Secretion of these HasA chimeras via the HasSM exporter has been confirmed (21). The polypeptides in the supernatant of the media cultured at 30 °C for 40 h (1.5 OD equivalents) were subjected to 12.5% SDS-PAGE and then stained with Coomassie Brilliant Blue G-250. The positions of the molecular mass markers are shown on the left. The positions of HasA proteins are shown by arrowheads. The exporters used are indicated above. Lane 1, pUC/HasAPF; lane 2, pUC/HasASM; lane 3, pMBF-HasA; lane 4, pMBF-HasA; lane 5, pFBF-HasA.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Construction of HasA mutants and their secretion through the Lip and HasSM exporters. A, the HasAPF C-terminal segment of 12 amino acid residues and its derivatives were inserted into the HasASM sequence (between amino acid residues 174 and 175). The inserted sequences are presented in one-letter designations. Gaps (dashes) indicate the deletions, and amino acid replacements are shown in an outline typeface with black background. The numbers indicated below the sequences correspond to amino acid residue numbers of the HasAPF C-terminal segment. The C-terminal segments of HasAPF and HasAPA are shown at the bottom. B, the HasA mutant plasmids were introduced into the E. coli DH5 cells carrying pACYC184, pK150, or pYBCD20. The polypeptides in the supernatants of the media cultured at 30 °C for 28 h were subjected to 12.5% SDS-PAGE. The protein amounts equivalent for optical densities of 3, 1.5, and 1.5 were loaded for none, HasSM, and Lip, respectively. The gels were stained by Coomassie Brilliant Blue G-250. The positions of molecular mass markers are shown on the left. The exporters used are indicated above each gel. Lane 1, pUC/HasASM; lane 2, pMFM-HasA; lane 3, pMFM-182A; lane 4, pMFM-184A; lane 5, pMFM-189A; lane 6, pMFM-(190-191); lane 7, pMFM-(190-191)G; lane 8, pMFM-ADVAL; lane 9, pMFM-AVAL; lane 10, pMFM-VAL; lane 11, pMFM-AL; lane 12, pMFM-ATDVL.

Analysis of the Amino Acid Sequence Related to Secretion by the Lip Exporter-- The C-terminal segments of HasA_PF and HasA_PA contain a couple of common features: the sequence Ala-His-Ala-Thr, a Thr/Ala cluster, and a negatively charged residue (Asp or Glu) followed by three hydrophobic amino acid residues, in this order (Fig. 2A). To investigate a minimum segment and amino acid residues necessary for secretion via the Lip exporter, secretion of HasA_SM mutants containing derivatives of the HasA_PF segment in the C-terminal region (Fig. 3A) was examined in the E. coli cells carrying the Lip and Has_SM exporters (Fig. 3B). These alterations did not affect Has_SM-promoted secretion, indicating that all HasA mutants are functional for secretion.

Ala substitutions at His¹⁸², Thr¹⁸⁴, and Asp¹⁸⁹ of the C-terminal segment (pMFM-182A, pMFM-184A, and pMFM-189A, respectively) and deletion of a Thr cluster and its flanking region (pMFM-ADVAL, pMFM-AVAL, and pMFM-VAL) did not alter the levels of Lip-mediated secretion. The levels are the same as that of the HasA chimera encoded by pMFM-HasA. However, other deletion mutants (pMFM-AL, pMFM-ATDVL, and pMFM--(190-191)) and a mutant with Gly replacements at Val¹⁹⁰ and Ala¹⁹¹ (pMFM-(190-191)G) reduced secretion through the Lip exporter to undetectable levels. Finally, the minimum segment Val¹⁹⁰-Ala¹⁹¹-Leu¹⁹² (carried by HasA_SM-VAL) was demonstrated to be closely associated with secretion promoted by the Lip exporter.

Roles of Amino Acid Residues in the C-terminal Segment in Secretion via the Lip System-- Roles of amino acid residues, Val¹⁹⁰, Ala¹⁹¹, and Leu¹⁹² (designated R1, R2, and R3 sites, respectively), in Lip exporter-mediated secretion were examined by introducing the amino acid substitutions. Secretion of HasA mutants that are HasA_SM containing a segment, Ala-Val-Ala-Leu or Val-Ala-Leu, with amino acid substitutions (Val, Leu, Ile, Phe, Met, Ala, Gly, Arg, His, Glu, Gln, Thr, and Pro) at R1, R2, and R3 sites (Fig. 4, A and B), were tested in the E. coli cells carrying exporters. The Has_SM exporter secreted these HasA mutants but at various levels, indicating that mutations introduced did not cause a severe conformational change leading to abolishment of secretion.

View larger version (54K): [in this window] [in a new window]   Fig. 4.   Secretion of HasA mutants with amino acid substitutions at R1 to R6. Substitutions at R1 and R2 (A) were introduced into HasASM-AVAL encoded by pMFM-AVAL, and substitutions at R3, R4, R5, and R6 (B) were introduced into HasASM-VAL encoded by pMFM-VAL (consult Fig. 3A). The primary structures of these HasA mutants are schematically illustrated above. Bars present HasASM sequence, and the amino acid sequences of the inserted segments are boxed. The HasASM sequences surrounding the segments are shown in one-letter designations with an outline typeface. The HasA mutant plasmids were introduced into the E. coli DH5 cells carrying pACYC184, pK150, or pYBCD20. The polypeptides in the supernatants of the media cultured at 30 °C for 28 h (1.5 OD equivalents) were subjected to 15% SDS-PAGE and then stained by Coomassie Brilliant Blue G-250. The exporters used are shown in the left of each gel. Amino acid substitutions are indicated in one-letter designations above each lane. No extracellular HasA mutant proteins were detected in the cultured media of the E. coli cells carrying pACYC184 (data not shown).

Secretion of these HasA mutants through the Lip exporter provided us several interesting observations. First, the importance of the R1 residue for secretion was exhibited (Fig. 4A). Replacement with Ile did not alter the secretion level, whereas other substitutions, including hydrophobic residues, Leu, Phe, and Met, reduced it to undetectable levels. These observations implied that the presence of a hydrophobic residue at R1 is necessary, but not sufficient, for Lip-promoted secretion. Val and Ile at R1 are likely to play a specific role for secretion. The Lip exporter secreted HasA mutants with replacements at R2 except for Pro (Fig. 4A), indicating that the Ala residue at R2 is not essential. Deletion of the R2 residue (pMFM-ATDVL), however, disturbed Lip-mediated secretion (Fig. 3B), suggesting that the residue functions as a spacer to allocate the hydrophobic R1 at a proper position necessary for the secretion. The R3 residue was replaceable with Ile (Fig. 4B). A decrease in the secretion levels (but at a detectable level) was observed by replacement with Val at R3. Thr substitution allowed secretion of the mutants at a low level. HasA mutants containing other residues at R3 were not secreted via the Lip system at a detectable level.

Thus, the (Val/Ile)-X-(Leu/Ile/Val) sequence upstream of the C terminus was demonstrated to be essential for secretion through the Lip exporter. Because the sequence was unrelated to Has_SM-mediated secretion, the R1-R3 region was suggested to be involved in the recognition or specific transport process by the Lip exporter.

Comparison of the C-terminal Sequences of the Proteins Secreted by the Lip Exporter-- Fig. 5 shows the presence of a consensus similar to (Val/Ile)-X-(Leu/Ile/Val) in the R1-R3 region of Lip-secreted proteins. The following sequence of the consensus also contained several conserved amino acid residues Val/Ile/Thr, Gly, and Val/Gln at R4, R5, and R6, respectively (Fig. 5). Furthermore, HasA mutants with substitutions at R4, R5, and R6 were generated to access a function of each residue in secretion via the Lip and Has_SM exporters (Fig. 4B).

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Comparison of the C-terminal amino acid sequences of the proteins secreted through the Lip exporter. The amino acid sequences at the C termini of HasASM and the proteins secreted through the Lip exporter are shown in one-letter designations. Gaps (dashes) are introduced into the sequences to maximize homology. The C-terminal hydrophobic amino acid residues, and negatively charged residues are shown in an outline typeface with black background, and the negatively charged amino acid residues are boxed. The amino acid residues of the region R1 to R6, valine, alanine, leucine, valine, glycine, and valine at the positions 175, 176, 177, 178, 179, and 180 of the HasASM-VAL sequence, are boxed.

In regard to R4 mutants, secretion of HasA mutants with replacements at R4 was achieved by the Has_SM exporter at various levels. The Lip exporter secreted a HasA mutant with Ile at an equal level to that with Val. HasA mutants with Met and Thr were secreted via the Lip exporter at a reduced level. Replacements with other hydrophobic amino acid residues, such as Leu and Phe, reduced the secretion to undetectable levels. Amino acid residues Val/Ile/Thr/Met at R4 allowing Lip-mediated secretion agreed with those found in the sequences of the proteins secreted by Lip (Fig. 5). The conserved amino acid residue Gly at R5 was essential for secretion through the Lip and Has_SM exporters. Immunoblot analysis with anti-HasA_SM antibody demonstrated production of HasA mutant proteins at equal levels in the cell extracts of E. coli without ABC exporter (data not shown). The amino acid residue at R6 was replaceable with several other amino acid residues, although the secretion levels were various. Gln and Val conserved at R6 were dispensable to secretion.

In conclusion, the amino acid residues at R1, R3, R4, and R5 were strongly related to Lip-mediated HasA secretion. The residue Gly at the R5 site also played an important role in high level secretion through Has_SM.

ABC Protein LipB Is Responsible for Lip-mediated Secretion of HasA Mutants-- Hybrid exporter analysis using Has_SM and Lip exporter components revealed that a hybrid, LipB-HasE_SM-TolC, was functional for secretion of HasA_SM-VAL (Fig. 6). However, neither HasA_SM nor HasA_SM-VAA (a HasA_SM-VAL variant with Ala substitution at R3) was secreted by the Lip and hybrid exporters. HasD_SM-LipC-LipD has already been known to be inactive for secretion (11), and an exporter lacking the ABC protein did not direct secretion. Thus, the sequence Val-Ala-Leu was essential for secretion through exporters, including the ABC protein LipB, confirming that LipB is a determinant of the substrate specificity of the exporter. In other words, LipB might recognize the R1-R3 region of the secretion protein.

View larger version (46K): [in this window] [in a new window]   Fig. 6.   Secretion of HasA mutants through hybrid exporters. The plasmids encoding HasA mutants (HasASM, HasASM-VAL, and HasASM-VAA; consult Fig. 4B) were introduced into the E. coli DH5 cells carrying pACYC184 (none) plus pMW/LipCD, pACYC/LipB plus pMW/LipCD, pACYC/HasDSM plus pMW/HasESM, and pACYC/HasDSM plus pMW/HasESM. The polypeptides in the supernatants of the media cultured at 30 °C for 40 h (1.5 OD equivalents) were subjected to 15% SDS-PAGE. Proteins were subjected to immunoblot analysis using anti-HasASM antibody. Lane 1, pUC/HasASM; lane 2; pMFM-VAL; lane 3, pMFM-VAA. Combinations of the hybrid exporter are shown above each gel.

C-terminal Secretion Signal Is Not Essential for Secretion via ABC Exporters-- Proteins secreted by the Lip exporter also contain a conserved motif consisting of a negatively charged amino acid residue followed by several hydrophobic residues at the C terminus (Fig. 5). Involvement of the motif in secretion was investigated using HasA_SM-VAL mutants (Fig. 7A). Interestingly, Ala substitutions of Glu¹⁸⁴ or/and Asp¹⁸¹ allowed secretion through the Lip and Has_SM systems, although dual Ala replacement of HasA_SM-VAL (pMFM-A1A2) reduced the secretion level via the Lip exporter (Fig. 7B). Furthermore, each C-terminal hydrophobic amino acid residue in the motif was replaceable with a negatively charged residue, Glu (Fig. 7, A and B). In the Prt exporter, which secretes metalloproteases and lipases but not HasA_SM as well as the Lip exporter, the Val-Ala-Leu segment, but not the C-terminal motif, participated in secretion (Fig. 7C). These findings indicated that the C-terminal motif, so far considered as a secretion signal, is not essential for secretion through the ABC exporter. Although the motif may have a role for something other than secretion, the importance of the R1-R5 region in secretion via ABC exporters except for Has_SM was generally recognized.

View larger version (50K): [in this window] [in a new window]   Fig. 7.   Secretion analysis of HasA mutants carrying amino acid substitutions at the C terminus. A, substitutions introduced into the C-terminal sequence of HasASM-VAL encoded by pMFM-VAL (consult Fig. 4A) are shown at the top in one-letter designations with amino acid residue numbers. The conserved C-terminal motif is shown in an outline typeface with black background, and the negatively charged amino acid residue is boxed. The inserted segment is boxed. An asterisk indicates a termination codon. The amino acid residues substituted are printed in an outline typeface with black background. B, the HasA mutant plasmids were introduced into the E. coli DH5 cells carrying pACYC184, pK150, or pYBCD20. The polypeptides in the supernatants of the media cultured at 30 °C for 28 h were concentrated (1.5 OD equivalents), subjected to 15% SDS-PAGE, and then stained by Coomassie Brilliant Blue G-250. The positions of molecular mass markers are shown on the left. The exporters used are indicated above each gel. Lane 1, pUC18; lane 2, pMFM-VAL; lane 3, pMFM-A1; lane 4, pMFM-A2; lane 5, pMFM-A1A2; lane 6, pMFM-E4; lane 7, pMFM-E3; lane 8, pMFM-E2; lane 9, pMFM-E1; lane 10, pMFM-A1E4; lane 11, pMFM-A1E3; lane 12, pMFM-A1E2; lane 13, pMFM-A1E1; lane 14, pMFM-A2E4; lane 15, pMFM-A2E3; lane 16, pMFM-A2E2; lane 17, pMFM-A2E1. C, the HasA plasmids (pUC/HasASM, pMFM-VAL, pMFM-A1, pMFM-E1) were introduced into the E. coli DH5 cells carry-ing pACYC184, pK150, pYBCD20, or pRUW8. The polypeptides in the supernatants of the media cultured at 30 °C for 28 h (1.5 OD equivalents) were analyzed as described above. The exporters used are indicated above each gel. Lane 1, pUC/HasASM; lane 2, pMFM-VAL; lane 3, pMFM-A1; lane 4, pMFM-E1.

Involvement of the R1-R5 Region and C-terminal Motif in Lip-mediated LipA_SM Secretion in Vivo-- Involvement of the R1-R5 region in secretion through the Lip exporter in vivo was tested. S. marcescens LipA_SM mutants with Ala substitutions at R1 to R6 (amino acid residues 596-601) were created and introduced into the S. marcescens 413 cells deficient in LipA_SM (Fig. 8, A and B). The secretion level of the LipA_SM mutant with Ala at R3 was greatly reduced. Ala substitution at R1 or R4 decreased the secretion level of the mutant to some extent compared with that of the wild-type LipA_SM (pLIPE121). Replacement of Gly at R5 with Ala slightly affected LipA_SM secretion in S. marcescens, although the Gly residue was essential for HasA mutant secretion through both Lip and Has_SM exporters in E. coli. Further mutations were introduced into the LipA_SM C-terminal sequence (Fig. 8C). As shown in Fig. 8D, Ala substitution of the negatively charged residue Asp⁶⁰⁹ (pLIPA-609A) and introduction of a charged or a bulky amino acid residue at the C terminus did not change the secretion level except for Lys replacement at the C terminus (pLIPA-K1). In the same way as demonstrated in secretion of HasA mutants, a typical C-terminal motif was not essential for LipA_SM secretion via the Lip system in S. marcescens either.

View larger version (68K): [in this window] [in a new window]   Fig. 8.   Secretion analysis of LipASM mutants in S. marcescens. A, Ala substitutions were introduced at the positions R1 to R6 of LipASM. The C-terminal sequence of LipASM is shown at the top in one-letter designations with amino acid residue numbers. A C-terminal motif is shown in an outline typeface with black background, and a negatively charged amino acid residue is boxed. The amino acid residues at the positions R1 to R6 are boxed. The amino acid residues substituted are printed in an outline typeface with black background. B, secretion of LipASM mutants from the S. marcescens 413 cells. The polypeptides in the supernatants of the lipase media cultured at 30 °C for 28 h (0.24 OD equivalents) were subjected to 12.5% SDS-PAGE and stained by Coomassie Brilliant Blue G-250. The positions of molecular mass markers are shown on the left. The positions of LipASM (L), PrtA (P), and flagellin (F) are indicated with an arrowhead on the right. Lane 1, pUC19; lane 2, pLIPE121; lane 3, pLIPA-596A; lane 4, pLIPA-597A; lane 5, pLIPA-598A; lane 6, pLIPA-599A; lane 7, pLIPA-600A; lane 8, pLIPA-601A. C, LipASM mutants carrying Ala substitutions and deletions in the C-terminal sequence are schematically illustrated as described above. To maximize homology, gaps (dashes) were introduced into the sequences. Spans between Gly at R5 and the negatively charged Asp in the C-terminal motif and distances of the R5 Gly from the C terminus are indicated on the right. D, secretion of LipASM carrying mutations at the C terminus from the S. marcescens 413 cells. The polypeptides in the supernatants of the lipase media cultured at 30 °C for 28 h (0.24 OD equivalents) were subjected to 12.5% SDS-PAGE as described above. The positions of molecular mass markers are shown on the left. The positions of LipASM (L), PrtA (P), and flagellin (F) are indicated with an arrowhead on the right. Lane 1, pUC19; lane 2, pLIPE121; lane 3, pLIPA-609A; lane 4, pLIPA-S4; lane 5, pLIPA-S5; lane 6, pLIPE121E; lane 7, pLIPA-S10; lane 8, pLIPA-S12; lane 9, pUC19; lane 10, pLIPE121; lane 11, pLIPA-PA2; lane 12, pLIPA-PA1; lane 13, pLIPA-M1; lane 14, pLIPA-M2; lane 15, pLIPA-M3; lane 16, pLIPA-F1, lane 17, pLIPA-K1; lane 18, pLIPA-E1.

Influence of the location of the R1-R5 region from the C terminus on secretion was examined (Fig. 8C). As shown in Fig. 8D, LipA_SM mutants, having Gly (R5) 13-17 amino acid residues upstream from the C terminus, were secreted, but at various levels, whereas the secretion levels were low on mutants containing Gly (R5) 11-12 and 19 amino acid residues upstream from the C terminus. All LipA_SM mutants expressed in the E. coli cells without exporter exhibited intracellular lipase activity, indicating production of lipase proteins (data not shown). The position of the R1-R5 region, but not a specific sequence of the C-terminal tail, was demonstrated to be critical for Lip-mediated secretion based on the similarity of the C-terminal tails of proteins secreted by the Lip system and the results from the secretion analysis of LipA_SM mutants.

Function of the R1-R5 Region in Lip-mediated Secretion-- Secretion competition analysis was performed to determine whether the inability of the Lip exporter to secrete HasA_SM and secretion-deficient HasA-VAL mutants is due to a lack of their affinity to the ABC protein or a defect in the secretion process. FLAG-tagged HasA-VAL (pSTV/FLAG-HasA-VAL) was secreted in the E. coli cells carrying a mini-F derived low copy number (one to two copies per cell) Lip exporter plasmid, pFBCD1. HasA_SM (pUC/HasA_SM), HasA-VAA (pMFM-VAA), and HasA-R5A (pMFM-R5A), which are not secreted through the Lip exporter, and HasA-VAL (pMFM-VAL), a substrate of the Lip system, were employed as a competitor. As shown in Fig. 9, a FLAG-tagged HasA-VAL protein of 22 kDa was secreted through the Lip system as well as HasA-VAL without a tag (21 kDa). Immunoblot analysis demonstrated reduction of the secretion levels of the 22-kDa FLAG-tagged HasA-VAL when a competitor, HasA-VAL of 21 kDa, was co-expressed and secreted, indicating secretion competition between HasA-VAL proteins. Interestingly, the presence of pUC18, pUC/HasA_SM, pMFM-VAA, and pMFM-R5A did not affect the secretion levels of FLAG-tagged HasA-VAL. Under these conditions, neither HasA_SM nor other HasA-VAL mutants exhibited secretion competition with HasA-VAL and disturbed the secretion.

View larger version (32K): [in this window] [in a new window]   Fig. 9.   Secretion competition analysis of HasA-VAL through the Lip exporter in E. coli. Secretion of FLAG-tagged Has-VAL was tested in the E. coli cells carrying pSTV/FLAG-HasA-VAL, pFBCD1, and competitor plasmids. The polypeptides in the supernatants of the cultured media at 30 °C for 24 h (2 OD equivalents for lanes 1-5; 1.5 OD equivalents for lane 6) were subjected to 15% SDS-PAGE and stained by Coomassie Brilliant Blue G-250 (upper). The positions of molecular mass markers are shown on the left. Proteins (1 OD equivalent) were also analyzed with anti-FLAG (middle) and anti-HasASM (lower) antibodies. The positions of FLAG-HasA-VAL and HasA-VAL proteins are indicated with an arrowhead on the right. Lane 1, pUC18 plus pSTV/FLAG-HasA-VAL plus pFBCD1; lane 2, pUC/HasASM plus pSTV/FLAG-HasA-VAL plus pFBCD1; lane 3; pMFM-VAL plus pSTV/FLAG-HasA-VAL plus pFBCD1; lane 4, pMFM-VAA plus pSTV/FLAG-HasA-VAL plus pFBCD1; lane 5, pMFM-R5A plus pSTV/FLAG-HasA-VAL plus pFBCD1; lane 6, pUC/HasA-VAL plus pYBCD20.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Lip system exports several types of secretory proteins, whereas HasA_SM is a poor secretion substrate of the system. One possible explanation for this is a lack of a protein region necessary for Lip-mediated secretion in the HasA_SM sequence. Interestingly, a C-terminal motif, so far regarded as a secretion signal, was not involved in secretion through the Lip and Has_SM exporters. The same was the case with LipA_SM secretion via the Lip system in vivo. Secretion analysis using HasA chimeras and their mutants revealed a protein region, including a new motif (Val/Ile)-X-(Leu/Ile/Val)-(Val/Ile/Met/Thr)-Gly (designated the R1-R5 region) close to, but not at, the C terminus. The motif was involved in secretion through the Lip or Prt system, whereas the motif was not necessary for Has_SM-mediated secretion. Similar sequences corresponding to the R1-R5 region were found in Lip-secreted proteins, including LipA_SM. A component within the Lip exporter, determining the secretion specificity, was LipB, an ABC protein of the Lip system, coinciding with our previous observations (11). No competition for Lip-mediated HasA-VAL secretion with competitors HasA_SM and HasA-VAL derivatives (HasA-VAA and HasA-R5A) was observed, that is, proteins retaining a motif inactive for Lip-mediated secretion at R1-R5 did not cause a blockade of secretion. These findings indicate that proteins, including the inactive motif, do not have high affinity to the ABC protein, which leads to an impediment to the secretion process. The protein region carrying an active motif at R1-R5 is recognized by the ABC protein with higher affinity than a protein region containing an inactive one. Secretion competition between LipA_SM and PrtA in S. marcescens, which has been reported previously in Ref. 25, is explained by competition in the process of recognizing the R1-R5 regions of these proteins by the Lip system. Thus, a new determinant within secretory proteins, which enables secretion through the Lip system, in other words, which the Lip system recognizes, was revealed. Secretion profile of LipA_SM mutants with Ala replacements differed from that of HasA_SM mutants; for example, a conserved Gly residue at R5 was demonstrated necessary for HasA mutant secretion via the Has_SM and Lip exporters but not essential for LipA_SM secretion via the Lip system. These observations indicate that a motif in the region is not rigorous and that each amino acid residue in the R1-R5 region possibly affects secretion through the Lip exporter. In addition, LipA_SM mutants containing Gly (R5) at positions 14 to 17 from the C terminus were secreted through the Lip exporter, whereas those with a longer or shorter C-terminal tail were a poor substrate. No specific feature was found in the sequence between R5 and the C terminus, and the alteration of the C-terminal tail sequence by insertion or deletion of one or two amino acid residues did not change the secretion level severely, indicating that location of the R1-R5 region from the C terminus is another important factor.

One report (13) supports this idea. In E. chrysanthemi PrtG, the C-terminal 15-amino acid sequence Val-Asn-Ile-Val-Gly-Ala-Ala-Leu-Gln-Pro-Ser-Asp-Val-Ile-Val-COOH, which includes the R1-R5 region (underlined) at position 11 from the C terminus, has been shown to be a minimum functional part for Prt-mediated secretion. Furthermore, the PrtG mutant lacking the C-terminal 14 amino acid residues, but exhibiting a newly created typical C-terminal motif, was secreted, and therefore, a conserved C-terminal motif was regarded as a secretion signal. The deletion, however, located the sequence Leu-His-Leu-Ser-Gly, which is similar to the R1-R5 region, at position 9. These previous findings might suggest the importance of the R1-R5 region at a proper position in secretion.

What is the function of the conserved C-terminal motif? At the least, the motif is not necessary for secretion through the Lip, Prt, and Has_SM exporters. Considering that the motif is conserved among proteins secreted through ABC exporters, including highly conserved repeat toxin family exporters, the motif may be necessary for protein folding after secretion through ABC exporters. However, its real function has not been defined yet.

To clarify the recognition mechanism by the Lip system, the analysis of structural features in the R1-R5 region is necessary. Although the three-dimensional structure of LipA_SM is unknown presently, crystallographic analyses of AprA_PA and PrtA, both of which are secreted by the Lip system and possess the sequence homologous to the R1-R5 region, show very similar C-terminal structure (30, 31). The conserved C-terminal motif and a protein region corresponding to the R1-R5 region are -sheet structured. On the contrary, conformation of the HasA_SM C terminus produced in E. coli, which is the last 15 residues containing the R4-R5 region, has been studied and was revealed to be highly flexible and unstructured (32), suggesting that the C-terminal structure of HasA_SM-VAL having the R1-R5 region is unstructured. Apparently, C-terminal tails of secretory proteins are unstructured during a process of secretion, and therefore, no rigid conformation seems to be necessary for secretion through the Lip system, although the R1-R5 region plays an important role for the secretion.

Our findings here opened a new avenue for investigating a secretion mechanism of the ABC exporter. Besides the crystallographic analysis of LipA_SM mutants or HasA chimeras, studies on a protein region of the ABC protein LipB, which is involved in the specific recognition by the Lip system, are intriguing. The three-dimensional structure of the E. coli outer membrane protein TolC, which demonstrates that a tunnel connected to the external environment is constituted with trimeric TolC protein, has been reported recently (33, 34). However, the three-dimensional structure of the ABC protein is still unknown, and the mechanism of secretion is not fully understood. Chimeric ABC proteins containing the LipB and HasD_SM sequences or mutants of ABC proteins will be helpful and informative for the exploration of the secretion specificity of the Lip and Has_SM exporters. Through these approaches, a mechanism of the substrate recognition by the ABC exporter will be totally understood.

	ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. C. Wandersman for her valuable advice and generous gifts of the plasmids encoding the S. marcescens and P. aeruginosa HasA proteins and the S. marcescens Has_SM and E. chrysanthemi Prt exporters, and the anti-HasA antibody. The P. aeruginosa aprDEF_PA genes were kindly provided by Dr. M. Murgier. We thank Dr. I. B. Holland for his kind gift of the plasmid pLG575.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: 81-48-433-8068; Fax: 81-48-433-8159; E-mail: k-omori@tanabe.co.jp.

Published, JBC Papers in Press, May 2, 2001, DOI 10.1074/jbc.M101410200

² K. Omori, A. Idei, E. Kawai, and H. Akatsuka, unpublished data.

³ A. Idei, H. Matsumae, E. Kawai, H. Akatsuka, and K. Omori, unpublished data.

	ABBREVIATIONS

The abbreviations used are: ABC, ATP-binding cassette; MFP, membrane fusion protein; OMP, outer membrane protein; Has, heme-acquisition; PCR polymerase chain reaction, PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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