Characterization of the hemolysin transporter, HlyB, using an epitope ...

Vol. 267, No. 6, Issue of February 25. pp, 3764-3770,1992 Printed in U. S. A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biocbemietry and Molecular Biology, Inc

Characterization of the Hemolysin Transporter, HlyB, Using an Epitope Insertion* (Received for publication, August 12, 1991)

Peter JurankaSO, Fang Zhang$, Janus KulpaS, Jane EndicottSY, Mark Blight11**, I. Barry HollandII, and Victor Ling$$$ From the $Ontario Cancer Institute andthe Department of Medical Biophysics, Universityof Toronto, Ontario M4X lK9, Canada and the IIZnstitut de Genetique et Microbiologie, URA, Centre National de la Recherche Scientifique, 01354, Uniuersite Paris-Sud, 91405 Orsay, Cedex, France

The prokaryotic hlyB gene product is a member of a superfamily of ATP-binding transport proteins that include the eukaryotic multidrug-resistance P-glycoprotein, the yeast STEG, and the cystic fibrosis CFTR gene products (Juranka, P. F., Zastawny, R. L., and Ling, V. (1989) FASEB J. 3, 2583-2592). Previous genetic studies have indicated that HlyB is involved in the transport ofthe 107-kDa HlyA protein from Escherichia coli; however, the HlyB protein has not been purified for biochemical studies due to its low abundance. In this study, we have engineered a monoclonal antibody epitope into the C-terminal end of HlyB that did not destroy its function. This has allowed us to use immunological methodsto identify and localize various molecular forms of the HlyB protein present in vivo.

The original finding that P-glycoprotein is structurallyvery similar to the Escherichia coli HlyB protein formed the basis for a proposed model for multidrug resistance in mammalian cells (1, 2). It was envisioned that, analogous to the role of HlyB in the export of a-hemolysin (HlyA) protein out of the bacterium, P-glycoprotein functions in mammalian cells as an energy-dependent pump for the efflux of various anticancer drugs (3). Subsequently, it was demonstrated that both these proteins are, in fact, part of a large superfamily of ATPdependent transport proteins involved in the translocation of diverse substrates across biological membranes in prokaryotic and eukaryotic cells. These include ions, peptides, large proteins, andsugar polymers (3).The structure of P-glycoprotein can be regarded as a tandem duplication of HlyB. HlyB is composed of two domains, a hydrophobic N-terminal half presumably comprising multiple membrane-spanning sequences and ahydrophilic C-terminal half that exhibits amino acid motifs typical of ATP-binding proteins. P-glycoprotein and HlyB haveextensive sequence conservation in the hydro-

philic domains. Although they show limited sequence homology in the hydrophobic domain, the proposed number and placement of putative transmembrane sequences are similar (1, 2). The currentmodel for secretion of HlyA in E. coli proposes that a complex of HlyB and HlyD molecules span the inner and outer E. coli membranes, possibly at junctions between these membranes (4).Energy derived from ATP hydrolysis by HlyB is likely necessary for transport (5). It has been suggested that the hlyB gene may code for two forms of the protein, a 66- and a 46-kDa polypeptide (7, 12). The relative contributions of each of these proteins to the transportprocess remains to be determined. HlyA does not have a classical N-terminal leader sequence (6). A recognition signal necessary for secretion has been localized to the27-55 amino acids at the C terminus (8, 9). Other members of the superfamily of ATP-dependent peptide transporters maybe similar in mechanism, recognizing and subsequently transporting molecules without using a N-terminal leader sequence. For example, the yeast P-glycoprotein homologue STEG transports the yeast a-type mating pheromone (a 12-amino acid peptide that does not have a N-terminal leader sequence) from MATa type cells (10).Structural and mechanistic precedents suggest that HlyB may be an excellent model system for studying P-glycoprotein and this superfamily of transport proteins. HlyB is present in very low abundance in E. coli, and ithas not been possible to purify this protein for biochemical studies. Attempts at raising antisera to HlyB have met with only limited success. We haveovercome this problem by using linker insertion and site-directed mutagenesis to engineer a monoclonal antibody epitope into the HlyB protein.This HlyB protein is functional and can be detected by immunological assays. We report here that this epitope tagging (modified form) of HlyB has allowed us to identify and localize various molecular forms of the HlyB protein in E. coli that have not been detected previously. These findings should be important for future functional studies.

* This work was supported in part by the Medical Research Council MATERIALSANDMETHODS of Canada. The costs of publication of this article were defrayed in Bacterial Strains and Plasmid-E. coli strains BW313 (dut-ung-) part by the payment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section and JM109 (dut+ung+)were used to grow M13 phage for the mutagenesis experiments. JM83 was used for cell fractionation and he1734 solely to indicate this fact. The nucleotide sequence(s)reported in thispaper has been submitted molytic assays. The complete hemolysin system is present on two totheGenBankTM/EMBLDataBankwith accession number(s) plasmids. The plasmid pLG579 expresses the HlyB protein from a 3kilobase EcoRI fragment that is present on the pACYC184 vector, M81823. which is selectable with tetracycline (11). Theother transport protein $ Recipient of a Medical Research Council of Canada fellowship. 1 Recipient of a Medical Research Council of Canada studentship. (HlyD), the structural a-hemolysin molecule (HlyA),and an activator ** Supported by a Medical Research Council (United Kingdom) protein of a-hemolysin(HlyC) are expressed from the ampicillin resistance plasmidpLG570::Tn5-32 (11). Cotransformation of studentship. $$ To whom correspondence should be addressed Structural Bi- pLG579 and pLG570::Tn5-32into competent E. coli JM83 cells yields ology, OntarioCancer Inst., 500 Sherbourne St., Toronto, Ontario hemolytic colonies on 5% sheep red blood cell agar plates containing the antibiotics ampicillin and tetracycline. M4X 1K9, Canada.

3764

Detection Immunological

of

HlyB

3765

Sequencing hlyB Gene-The hlyB gene, present on anEcoRI frag- min a t room temperature, and centrifuged at 150,000 X g for 90 min. ment in plasmid pLG579, was recloned into M13mp9 to yield the The supernatant from this final spin represents thesolubilized inner construct M13-EE8, in which the gene isantisensetothelac2 membrane proteins. The insoluble pellet contains the outer mempromoter. Fourteen 18-mer oligonucleotides complementary to M13- brane proteins and was also resuspended in NPS buffer. The total EE8 were synthesized on the basis of a previously published hlyB protein concentration was determined by the method of Lowry et al. sequence (12). These synthetic oligonucleotides,spaced -200 base (16). pairs apart, were used as primers for sequencing the entire EcoRI Western Blot Analysis-Proteins were separated by SDS-polyacrylfragment in M13-EE8 by the dideoxy chain termination method of amide gel electrophoresis according to the method of Laemmli (17) using a 3% stacking and a 11% separating gel. Prestained molecular Sanger etal. (13). LinkerInsertion Mutagenesis-PlasmidpLG579was randomly weight markers (Amersham Corp.) were included as standards. The linearized by digestion with HpaII in the presence of ethidium bro- proteins were transferredto nitrocellulose membranes by electromide (14). A KpnI linker with HpaII-compatible ends was ligated to blotting as described by Towbin et al. (18). The membranes were the plasmid. Two different sets of linkers were used. The pair of blocked overnight at 37 “C in phosphate-buffered saline containing oligonucleotides HKCl (5’-CGGGTACCGATCGAT-3’) and HKC2 3% bovine albumin, 0.1% azide. ’251-Radiolabeled C219 or C494 (3’-CCATGGCTAGCTAGC-5’) when annealedtogethercontain monoclonal antibody was prepared by the chloramine-T method (19). The nitrocellulose membranes were probedovernight at 4 “Cin both a KpnI and ClaI restriction site. The HBK oligonucleotide (5’phosphate-buffered saline containing iodinated antibody present at 5 CGCTGGTACCAG-3’) anneals to itself and forms a linkerwith relative X lo5dpm/ml, washed four times with phosphate-buffered saline at HpaII ends and a KpnI restriction site. The presence and location of the linkerwere determined by restriction enzyme analysis, room temperature, and exposed to Kodak X-Omat film a t -70 “C. and the number of copies of the linker and its orientation were determined by dideoxy double strand sequencing. RESULTS Oligonucleotide-directed Mutugenesis-Site-directed mutagenesis was undertaken according to the method of Kunkel (15). Briefly, Sequence of hlyB gene from E. coli LE2001 Cells-E. coli M13-EE8 phage was passagedtwice through E. coli BW313cells LE2001 cells were originally isolated from a human urinary grown in uridine to generate uracil-containing phage DNA. Oligonu- tract infection. The complete Hly determinant was cloned cleotides with desired mismatches (shown inFig. 1)were phosphorylA double- from the chromosome onto plasmid pLG570 (20). We have atedandannealedtotheuracil-Ml3-EE8template. sequenced the hlyB gene of this determinant (see “Materials stranded circle was synthesized in vitro by extension of the primer with Klenow polymerase, and the circle was covalently closed with and Methods” and Fig. 1) and compared it to the sequences T 4 DNA ligase. Transfectioninto E. coli JM109 cells resultsin of three other isolatesof HlyB (data not shown). Twoof the specific degradation of the wild-type strand, and mutant plaques were HlyB isolates were derived from E. coli. The hlyB gene of routinely found a t -50% frequency. Mutants were identified either by plaque hybridization with 32Pend-labeled oligonucleotide (N4A), pHly152 is a plasmid-encoded determinant, whereas pSF4000, like the presentpLG570, was chromosomal (12,21). The third by single strand sequencingusing the dideoxy chaintermination method (N57-12A) (13), orby restriction enzyme analysis (N225 and hlyB sequence was derived from Proteus vulgaris (22). All four N32). All mutations were confirmed by direct sequencing. The com- HlyB polypeptides have a high degree of amino acid conserplete EcoRI cassettes carrying the mutations were recloned into vector vation; however, the Proteus sequence is significantly differpACYC184 in the same orientationas pLG579. ent from the other three E . coli genes, with atotal of 59 amino Epitope Linker Insertion-Two complementary oligonucleotides acid differences compared to pLG579HlyB. However, the encoding the amino acid sequence of the C494 epitope, AX494 (5’majority of these differences, 51 of 59, were conserved substiCGAACACCTTGGAAGGTAACGTAC-3’) and B-C494 (3”CATGGCTTGTGGAACCTTCCATTG-5’), were synthesized and when an- tutions as determinedby the mutational data matrix(23). nealed have GTAC 3”overhangs. The unphosphorylated oligonucleInsertion of Monoclonal Antibody Epitope into HlyB-To otides were annealed together in the presenceof N32 or N225 DNA detect HlyB in uiuo, we decided to tag the protein with a that had been digested with the restriction enzyme KpnI. The DNA monoclonal antibody epitope. The epitope sequences for two solution was thentreatedwithT4 DNAligase andsubsequently monoclonal antibodies directed againstP-glycoprotein (C219 heated to 90 “C, quick-chilled on ice, and then directly transformed and C494) have been mappedby Georgeset al. (24) and shown into competent E. coli JM83 cells. Plasmids containing the C494 oligonucleotides were identified by the presence of a Sty1 restriction to be VVQAALD and PNTLEGN,respectively, located in the site, which is present in the C494 DNA sequence. The orientation of cytoplasmic ATP-binding domain. The small size of these the incorporated C494 sequence in N225 was determined by polymcontinuous epitopes and the availability of specific monoerase chain reaction of the DNA using the common 5’-HA14 oligo- clonal antibodies made these sequences ideal for the epitope nucleotide (Fig. 1) and either the AX494 orB-C494 oligonucleotide. T h e presence of a 1-kilobase polymerasechain reaction product using tagging experiments. The first approach involved an attempt to introduce the the combination HA14/A-C494 or HA14/B-C494 suggests that the epitope is present in the antisense and sense orientations, respec- C219 epitope into the HlyB protein,encoded by pLG579, by tively. All constructs were confirmed by dideoxy double strand se- conversion of the amino acid sequence IIMRNMH of HlyB quencing. (amino acids 643-649 in Fig. 1) to that of the c219 epitope The recombinant plasmid N24048,which contains both the valine sequence (VVQAALD) by oligonucleotide-directed mutagenmutation atposition 286 and theC494 epitope, was formed by placing Fig. 1. This the N57-12A mutation, presenton a HindIII/BglII fragment, into the esisusing the C219 oligonucleotideshownin position in HlyB is equivalent to the position of the C219 N225-15 plasmid. SubcellularFractionation-Cytoplasmic, innerandouter mem- epitope in P-glycoprotein (1). The mutant N4A, which conbrane proteins were prepared using a procedure similar to that de- tains the C219 epitope (see “Materials and Methods”), was scribed by Mackman et al. (7). Cells were harvested and resuspended nonhemolytic when transformed into E. coli JM83 cells harin 3 ml of 10 mM phosphate buffer, p H 7.4, 2 mM phenylmethylsul- boringthe pLG570::Tn5-32 plasmid (Fig. 2 andTableI). fonyl fluoride and thenlysed bysonication. Wholecells were removed bycentrifugation at 6000 X g for 10min. Cell membranes were Western blot analysis of membrane and soluble fractions of cells containing theN4A plasmid showed no immunoreactive sedimented at 150,000 X g for 30 min. The supernatant fraction bands when probed with ‘2sI-labeled C219 antibody (data not (cytoplasmic plus periplasmic fraction) was recovered and precipitated with 90% methanol overnight at -20 “C and centrifuged a t shown). The reason for the lack of detectable HlyB in the 20,000 X g for 90 min, and the pelletwas resuspended in NPS buffer immunoblots was not investigated further, but this experi(1% SDS,’ 10 mM phosphate, pH 7.4, 2 mM phenylmethylsulfonyl ment didsuggest that HlyBmay not easily tolerate mutations fluoride). The membrane pellet either was resuspended directly in N P S buffer to yield “total membrane” or was resuspended in 0.5% in its sequence. In a second approach, we decided to scan thehlyB gene for Sarkosyl plus 2 mM phenylmethylsulfonyl fluoride, vortexed for 90

’ The abbreviation used is: SDS, sodium dodecyl sulfate.

any region where insertions of KpnI restriction sites would not disrupt the function of HlyB, reasoning that such sites


Mutant

Phenotype"

++++

~

++++

N32

+++

N57-12A

+++

N225-15

++

N225-4

+

v v w P

SInsertion of C494 epitope DNA sequence in sense orientation into KpnI site of N225

Insertion of C494 epitope DNA sequence in antisense orientation into KpnI site of N225

N240-S8 N32-L5

P

N32-L3

P

Combination of N57-12A Met to Val mutant and N225-15 C494 epitope in sense orientation C of C494 epitope DNA seInsertion quence in sense orientation into KpnI site of N32 C Insertion of C494 epitope DNA sequence in antisense orientation into KpnI site of N32

703 Q V CAGGTAC CGTCA CAGGTACCGAACACCTTGGAAGGT~CGTACCGTCA Q V P N T L E G N V P S 703 Q v P S CAGGTAC CGTCA CAGGTACGTTACCTTCCAAGGTGTTCGGTACCGTCA Q V R Y L P R C S V P S

1 M

1

N4A

Conversion to C219 epitope sequence by oligonucleotide-directed mutagenesis

624

F77

Generation of KpnI site by site-directed insertion of HBK linker at HpaII site

423

F198


462

F119


473

F55

Generation of KpnI site by site-directed insertion of two head-tohead HKC linkers at HpaII site

482

FlOO


607

F185

Generation of KpnI site by site-directed insertion of HBK linker a t HpaII site

694

Generation of KpnI site by site-directed insertion of HKC linker at HpaII site

694

F61

-

3767

TABLEI Description of HlyB mutants Genotype* Description of mutant Wild type 703 Q L Q S Creation of KpnI site at C terminus CAGTTACAGTCA by oligonucleotide-directed mutaCAGGTACCGTCA genesis Q V P S 1 M D S C Creation of KpnI site at N termiATGGATTCTTGT nus by oligonucleotide-directed ATGGTACCTTGT mutagenesis M V P C 285 V M W Conversion of Met to Val by oligoGTAATGTGG nucleotide-directed mutagenesis GTAGTGTGG

~

pLG579 N225

of HlyB

V ATGGTAC CTTGT ATGGTACCGAACACCTTGGAAGGT~CGTACCTTGT M V P N T L E G N V P C M V ATGGTAC CTTGT ATGGTACGTTACCTTCCAAGGTGTTCGGTACCTTGT M V R Y L P R C S V P C H I I M R N M H K CATATCATCATGCGCAATATGCACAAA CATGTCGTCCAGGCCGCTCTGGACAAA H V V Q A A L D K P V C CGGTT CCGCTGGTACCAGCGGTT P L V P A V P E C CGGAA CCGCTGGTACCAGCGGAA P L V P A E I R ATC CGG ATCCGCTGGTACCAGCGG I R W Y Q R V P C CGGTT CCGATCGATCGGTACCCGGGTACCGATCGATCGGTT P I D R Y P G T D R S V s G TC CGGA TCCGCTGGTACCAGCGGA S A G T S G P E C CGGAA CCGCTGGTACCAGCGGAA P L V P A E P E C CGGAA CCGGGTACCGATCGATCGGAA P G T D R S E

"The relative size of hemolytic zones around colonies plated on blood agar plates is graded from the largest zone (++++) to thesmallest (+) and also no detectable zone (-) as described for Fig. 2. The various HlyB mutant plasmids were transfected into JM83 cells harboring plasmid pLG570:Tn5-32 (HlyA/HlyC/HlyD). bThe DNA sequence of the mutant and its amino acid translation in single-letter code are given below the corresponding wild-type DNA and amino acid sequences. The position of the amino acid in the wild-type sequence is written to the left of the sequence and follows the numbering system in Fig. 1.

3768


of HlyR

structs were studied by immunoblot technique. 1 2 3 4 5 ' 6 7 8 9 1011 Irnrnunodetection of HlyB Proteins-Conventionalmembrane purification methods and immunoblotting using monoclonal antibody C494 were used to localize the epitope-tagged 2: hlyB product in E. coli cells harboring plasmid N225-15. The maximum codingcapacity of the hlyB gene has been estimated as 79.9 kDa (12).As can be seen in Fig. 3, the inner membrane contains a major polypeptide of -66 kDa. The size of HlyB detected here is smaller than the predicted79.9 kDa, but this may be due to the fact that highly hydrophobic membrane proteinscanmigrateanomalouslyonSDS-polyacrylamide gels. Other explanations are also possible. The 66-kDa protein, however, is likely the authentic HlyB protein since a A of a T n 5 insertioninto hlyB leadstothedisappearance 1 2 3 4 5 6 component of this size (7), and this is also the size of the major gene product detected for pLG579 in minicells (7, 25). Purification of the epitope-tagged HlyB protein and deter66 561 mination of its sequence will ultimately be required to define the exact relationship of this product with the total coding capacity of the hlyB gene. - 38 In addition to the major 66-kDa polypeptide, three minor E:; C494 reactive bands of 33, 32, and 28 kDa are frequently observedin theinnermembranefractions of JM83 cells containing N225-15(Fig. 3, A, lanes 4-6; and C, lane 1 ) . Densitometry measurements revealed that the relative amount of the 66-kDapolypeptidewastwice that of the combination of 33- and 32-kDa bands(Fig. 3A, lane 6). These B three polypeptides are HlyB-specific since they are present in 1 2 E. coli cells carrying N225-15 but absent in the inner membrane fractions of strains with N225-4 (Fig. 3A, lane 7),E. coli cells without a plasmid, andcells containing N32-L5 (data not shown). It should be noted that cells carrying plasmid N225-4 are thenegative control for N225-15 since it contains the epitope sequences in the reverse orientation that would not be recognized by monoclonal antibody C494 (see Table I). The stained polypeptide a t 54 kDa is not HlyB-specific since it is also present in inner membranes of E. coli cells containing N32, N32-L5, or N225-4 as well as E. coli cells without a plasmid. It is noteworthy that a 46-kDa component observed in previous in uitro and minicell studies (7) is not e detected in this system. The origin of the minor HlyB polypeptides of the inner C membrane is unknown. Both the total membrane and inner FIG.3. Immunoblot analysis of subcellular fractions of E. membrane fractionsshow the 66-kDa HlyB protein; however, coli c e l l s c a r r y i n gpLG679 (HlyR) mutants probed withC494 the pattern of the low molecular mass species appears to monoclonal antibody. Suhcellular fractions were prepared as described under "Materials and Methods." Samples were run on SDSdependontheextraction procedure. The 33- and32-kDa polyacrylamides gels, blotted to the nitrocellulose memhranes. and polypeptides of the inner membrane are more prominently probed with "51-laheled C494 monoclonal antibody. A. immunoblot observed duringtheSarkosylextraction procedure,which memhrane (lanes I - : ] ) , inner of 100 pg ofproteinoftheouter involves a 90-min room temperature Vortex step (compare membrane (lanes 4-7), andcytoplasmic plus periplasmic fraction inner membrane (Fig. 3A, lane 5 ) with total membrane (Fig. (lanes 8-11) of N225-4 (lanes 3 . 7, and 1 1 ) and N225-15 (lanes I . 2. 3B, lane 3 ) ) . It is possible that these low molecular mass 4-6, and 8-10). Cells were fractionated at different stages of exponential growth: N225-15 grown to Am., = 0.07 (lanes 4 and 8).0.47 species are degradation productsof larger precursors. Three strongly immunoreactive polypeptides are detected (lanes I. 5,and 9 ) ,and 1.0 (lanes 2. 6, and 1 0 ) and N22.5-4 cells grown in the soluble fraction of cells with N225-15 and are present to Amnm = 0.47 (lanes 3, 7, and 1 I ). R. immunohlot of 150 pg of total membrane protein (lanes 1 , 3. and 5 ) and 100 pg of cytoplasmic plus in approximately equal concentrations(Fig. 3, A, lanes 8-10; periplaamic protein (lanes 2, 4, and 6 ) from N225-4 (lanes f and 2). and C, lane 2). These proteins migrate on SDS-polyacrylN225-15 (lanes 3 and 4), and N240-SR (methionine tovaline mutant) amide gels with apparent molecular masses of 65, 32, and 31 (lanes 5 and 6 ) cells grown to A m n r n= 0.5. C. immunohlot of 1 0 0 pg kDa. It is not clear a t present how these proteins relate to the of inner membrane protein (lane I ) and cytoplasmic plusperiplasmic 66-, 33-, and 32-kDa HlyB peptides found in the inner mem- protein (lane 2) of N225-15 cells grown to Am,, = 0.5. brane. However, initial pulse-chase experiments using ["'SS] methionineindicatethat radiolabeled immunoprecipitable noreactive bands seen in the soluble fractions of cells with peptides of approximatelythese molecular massescan be N225-15(Fig. 3A, lanes 8-10) are not HlyR since they are also presentin the negative controls (including the weak band chased to higher molecular mass forms in the inner membrane.' Further studieswill be required to elucidate the naturemigrating near 65 kDa in Fig. 3A, lane 1 1 ). The presenceof a significant amountof HlyB in the soluble of this post-translational conversion. The other C494 immufraction has notbeen reported previously and was unexpected. It cannot be explained as contamination of this fraction by M. S. Poruchynsky and V. Ling, unpublished data.

i,

Immunological Detectionof HlyB

3769

the inner membrane since the pattern of polypeptides detected the soluble fractions were not tested in the above report, we by monoclonal antibody C494 is clearly different in the two detected the presence of a major proportion of HlyB in the fractions. More important, calculationssuggest that the total soluble(cytoplasmic plus periplasmic) fraction of normal, amount of HlyB in thesolubIe fraction is equal to or greaterexponentially growing cultures of E. coli cells. Interestingly, than the amount in the inner membrane. Equal amounts of the major nonmembrane form of HlyB appears to be 1 kDa protein were analyzed from both fractions;however, the dis- smaller than themajor inner membrane form of this protein. tribution of total cellular protein in E. coli cells was 90% in Initial pulse-chase experiments indicate that the newly synthe cytoplasm plus periplasm,7% in the inner membrane, andthesized HlyB protein may be post-translationally modified 3%in the outer membrane under growth the conditions used.3 to ahighermolecular mass form. Furtherstudies will be The level of HlyB in this system in both the membrane and required to determine the nature of this modification and soluble fractions varies