membrane topology of the pbr322 tetracycline … tetracycline resistance gene of pbr322 encodes a...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 267. No . 25, Issue of ’ September 5, PP , 17809-17819,1992 Printed in U.S.A.

Membrane Topology of the pBR322 Tetracycline Resistance Protein TetA-PhoA GENE FUSIONS AND IMPLICATIONS FOR THE MECHANISM OF TetA MEMBRANE INSERTION*

(Received for publication, January 29, 1992)

John D. AllardSB and Kevin P. BertrandSTIl From the Departments of $Microbiology and VBiochmistry and Biophysics, Washington State University, Pullman, Washington 99164

The tetracycline resistance gene of pBR322 encodes a 41-kDa inner membrane protein (TetA) that acts as a tetracycline/H+ antiporter. Based on hydrophobicity profiles, we identified 12 potential transmembrane segments in TetA. We used oligonucleotide deletion mutagenesis to fuse alkaline phosphatase (PhoA) to the C-terminal edge of each of the predicted periplasmic and cytoplasmic segments of TetA. In general, the PhoA activities of the TetA-PhoA fusions support a TetA topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, several TetA-PhoA fusions have unexpected properties. One PhoA fusion to a predicted cytoplasmic segment (C6) has high activity. However, previous protease accessibility studies on the related TnlO TetA protein indicated that C6 is cytoplasmically localized as predicted (Eckert, B., and Beck, C. F. (1989) J. Biol. Chem. 264,11663-1 1670). PhoA fusions to three predicted periplasmic segments (Pl, P2, and P5) have low to intermediate activity. In each case, the preceding transmembrane segment (TM1, TM3, and TM9) contains an aspartate (Asp1’, AspBe, and Aspz8’). We show that these aspartates act like signal sequence mutations for PhoA export: (i) As-Ala mutations increase the PhoA activity of fusions to P1, P2, and P6. (ii) The signal sequence mutation suppressor prlA402 increases the PhoA activity of these same fusions. We also show that the aspartates in TM1, TM3, and TM9 are critical for wild-type TetA function; they are conserved in related TetA proteins and AspdAla mutations reduce or eliminate tetracycline resistance. The properties of the anomalous TetA-PhoA fusions suggest that TetA sequences C-terminal to some cytoplasmic and periplasmic segments are required for the proper localization of those segments, i.e. long range interactions may be more important in determining the membrane topology of TetA than suggested in some general models.

Tetracycline resistance in Escherichia coli and related Gram-negative bacteria is mediated by members of a family of related tet genes. Five classes of genes (A through E) have

* This work was supported by Grant A116735 from the National Institute of Allergy and Infectious Disease. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by Predoctoral Training Grant GM08336 from the National Institute of General Medicine.

11 To whom correspondence should be addressed Dept. of Biochem-

99164-4660. Tel.: 509-335-9129: Fax: 509-335-9688. istry and Biophysics, Washington State University, Pullman, WA

been identified on the basis of DNA hybridization studies (Mendez et al., 1980; Marshall et al., 1986; for a review, see Levy, 1988). The class B genes on transposon TnlO and the class C genes on plasmid pSClOl are the most extensively studied members of this family. In each case, the resistance determinant consists of two genes, a resistance gene ( te tA) that encodes a 41-43-kDa inner membrane protein (TetA) and a repressor gene ( te tR) that encodes a 23-24-kDa regu- latory protein (TetR). Tetracycline induces transcription of both genes by binding to TetR and reducing its affinity for tandem operator sites that overlap the divergent tet promoters (for a review, see Hillen and Wissmann, 1989). The plasmid cloning vector pBR322 carries the pSClOl tetA gene, but not the pSClOl tetR gene (Bolivar et al., 1977; Unger et al., 1984). Consequently, pBR322 expresses its tetA gene constitutively.

For classes A through E, the mechanism of tetracycline resistance involves active efflux of the drug; inside-out membrane vesicles prepared from resistant bacteria concentrate tetracycline by a process that requires proton motive force (McMurry et al., 1980; Marshall et al., 1986). Recent experiments have shown that TnlO TetA transports tetracycline as a divalent metal-tetracycline complex (Yamaguchi et al., 199Oc) and that it mediates tetracycline-dependent H+ translocation (Yamaguchi et al., 1990b), i.e. TetA functions as a metal-tetracycline/H+ antiporter. Based on complementation studies with tetracycline-sensitive mutants in TnlO tetA, Cur- iale et al. (1984) suggested that TetA consists of two domains, an N-terminal LY domain and a C-terminal ,8 domain, both of which are necessary for tetracycline resistance. These and other genetic analyses also suggest that TetA exists in the membrane as a multimer (Hickman and Levy, 1988).

Several groups have developed computational methods for predicting transmembrane segments and, thereby, two-di- mensional membrane topology from amino acid sequences of membrane proteins (Kyte and Doolittle, 1982; Eisenberg et al., 1984; Engelman et al., 1986). These methods all assume that transmembrane segments are a-helices composed largely of hydrophobic amino acids; they search for hydrophobic segments of about 21 amino acids, the minimum length for an a-helix to span the apolar region of a typical phospholipid bilayer. Based on these predictive algorithms, Eckert and Beck (1989) and Henderson and Maiden (1990) proposed similar 12-transmembrane-segment models for the membrane topology of TnlO TetA and pBR322 TetA, respectively. Pro- tease accessibility studies on TnlO TetA have provided experimental support for many features of this model (Eckert and Beck, 1989).

Alkaline phosphatase (PhoA) gene fusions have been used to analyze the membrane topology of a number of E . coli inner membrane proteins (Manoil and Beckwith, 1986; for a review, see Manoil et al., 1990). PhoA has the important property

17809

17810 Membrane Topology of the pBR322 Tetracycline Resistance Protein

TABLE I Bacterial strains, phages, ana' plasmids

Becteria/phage/plasmid Genotype/description Source (ref.) Bacteria

TG1 CJ236

A(lac-pro), supE, thi, hsdA5 (F' traD36, proAB, ladq, lacZAM15) Amersham Corp. (Gibson, 1984)

MV1190 dut, ung, thi, relAl (pCJ105); Cm' Bio-Rad A(1ac-pro), thi, supE, A(srl-recA)306::TnlO (F' traD36,proAB, ladq, lacZaM15) Bio-Rad

MC4100 AlacU169, araD139, rpsL150, thi, flb5301, deoC7,ptsF25, relA1, rbsR RL402 MC4100; malEA12-18,lamBS6O,prlA402 L. Randall (Bankaitis and Bassford,

L. Randall (Casadaban, 1976)

1985)

Phages mpll, mp18 M13; lacZ"

R408 fl; IR1, A packaging signal, g t r d rvl M13; interference resistant mutant

Plasmids pBR322 Ap', Tc'

pZ150 pBR322 derivative; Ap', Tc', M13 intergenic region

pBST324 pACYC177 derivative; Km', pSClOl tetR+ ~KP105 ~BR322 derivative: AD'. tetA::TnDhoA

J. Messing (Yanisch-Perron et al.,

Stratagene (Russell et al., 1986) M. Berman (Levinson et al., 1984)

1985)

Laboratory stock (Bolivar et al., 1977)

M. Berman (Zagursky and Berman, 1984)

T. Nguyen (Nguyen, 1987) K. Postle

that it is enzymatically active only when it is exported across the inner membrane into the periplasmic space. In general, PhoA fusions to periplasmic segments of inner membrane proteins show high PhoA enzyme activity, while fusions to cytoplasmic segments show low activity. Several groups have described PhoA fusions to pBR322 TetA (Manoil and Beck- with, 1985; Hoffman et al., 1987) and TnlO TetA (Hickman end Levy, 1988). However, none of these studies focused on TetA membrane topology.

In this paper, we describe the systematic application of the PhoA gene fusion approach to analyzing the membrane topology of pBR322 TetA. Overall, our results support the 12- transmembrane-segment model. However, the properties of several TetA-PhoA fusion proteins gre inconsistent with this model. We speculate that the properties of these anomalous fusions reflect a requirement for TetA sequences C-terminal to some cytoplasmic and periplasmic segments for the proper localization of those segments. Finally, we discuss these results in terms of general models for the organization of topogenic sequences in membrane proteins.

EXPERIMENTAL PROCEDURES

Bacteria, Phages, Plasmids, ana' Growth Media-Table I lists the bacterial strains, phages, and plasmids used in this study. LB medium (Miller, 1972) contains 10 g/liter Bacto tryptone (Difco), 10 g/liter NaCl, and 5 g/liter Bacto yeast extract (Difco). LB plates contain, in addition, 15 g/liter Bacto agar (Difco). The chromogenic substrates 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (XP,' Sigma) and 5-bromo-4-chloro-3-indolyl P-D-galactopyranoside (XG, Sigma) were dissolved in dimethylformamide and added to media at 40 pg/ ml. Antibiotics (Sigma) were added to media at the following concentrations: 100 pg/ml ampicillin (Ap), 50 pg/ml kanamycin (Km), 25 pg/ml chloramphenicol (Cm) and 10 pg/ml tetracycline. 6a,6-Anhy- drotetracycline (lot no. 4967-2610-3) was kindly provided by N. Belcher, Pfizer. All cultures were grown aerobically a t 37 "C.

Construction of tetA-phoA Plasmids"PlasmidpKP105 is a pBR322 derivative with a tetA::TnphoA fusion generated by transposition from X::TnphoA (Manoil and Beckwith, 1985); the TnphoA fusion is to TetA amino acid 17. To make the starting plasmid for constructing tetA-phoA fusions, the 1680-bp BamHI-BstEII fragment spanning the phoA gene was isolated from pKP105, treated with DNA poly-

' The abbreviations used are: XP, 5-bromo-4-chloro-3-indolyl phosphate; XG, 5-bromo-4-chloro-3-indolyl galactoside; Ap, ampicillin; Km, kanamycin; Cm, chloramphenicol; bp, base pairs; SDS, sodium dodecyl sulfate; RF, replicative form; MIC, minimum inhibitory concentration.

merase I Klenow fragment (Amersham Corp.) to fill-in ends, and cloned into the PuuII site downstream of the tetA gene in pZ150, creating plasmid pJA100. Then the 1930-bp HincII fragment spanning the 3' end of tetA and the 5' end of phoA was isolated from pJAlOO and cloned into the HincII site in M13 mpll, creating phage mJA100. The initial fusion ofphoA to the 3' end of tetA (TetA amino acid 396) was constructed in mJAlOO by oligonucleotide-directed deletion mutagenesis. The 5' half of the mutagenic primer (JA2:

GGCTCCA-3') is complementary to IS50L sequences at the left end of TnphoA (underlined) and the 3' half is complementary to the 3' end of tetA. A modification of the mutagenesis procedures described by Boyd et al. (1987a) and Craik et al. (1985) was used. Mutagenic primer (-9 pmol) was phosphorylated and annealed to single- stranded mJAlOO DNA (-0.5 pmol). Complementary strand DNA was synthesized in a 100-pl reaction containing annealed primer- template, 10 pg/ml T4 DNA polymerase (Boehringer Mannheim), 100 pg/ml T4 gene 32 protein (Boehringer Mannheim), 5 units of T4 DNA ligase (Bethesda Research Laboratories), 0.2 mM deoxynucleo- side triphosphates (Pharmacia LKB Biotechnologies Inc.), 0.15 mM ATP (Sigma), 10 mM Mg(OAc),, 66 mM KOAc, 33 mM Tris-OAc, pH 7.8, and 5 mM dithiothreitol; the reaction was incubated for 2.5 h at 37 "C. Following DNA synthesis and ligation, 25 units of mung bean nuclease (New England Biolabs) was added to digest single-stranded DNA. TG1 was transformed with the mutagenized DNA, phage plaques were picked, and replicative form (RF) DNA was digested with HincII to screen for the deletion; 60% of the progeny phage contained the desired deletion. One of these phage was designated mJAlO2.

To construct a plasmid containing the full-length tetA-phoA fusion, the 988-bp HincII fragment spanning the tetA-phoA fusion junction was isolated from mJAlO2 RF DNA and ligated to the 4605-bp HincII fragment from pJAlOOH, creating plasmid pJAlOl. Plasmid pJAlOOH is a derivative of pJAlOO lacking the HincII site in the Ap' gene. When single-stranded pJAlOl DNA was used as the template to create a phoA fusion to TetA amino acid 364, cells harboring the resulting plasmid produced very small dark blue colonies on LB-Ap- XP plates, suggesting that the TetA-PhoA fusion protein is toxic. Since the region of pZ150 between the 3' end of tetA and the PuuII site was deleted during the construction of pJAlOl, pJAlOl is rom- and has an increased copy number relative to pZ150 (Twigg and Sherratt, 1980). To construct a rom+ homolog of pJAlOl, the 2769- bp HindIII-BstEII fragment spanning the tetA-phoA region was isolated from pJAlOl and ligated to the 3446-bp HindIII-BstEII fragment from pZ150B, creating plasmid pJA102. pZl5OB is a pZ150 derivative in which the BalI site immediately 3' to tetA has been converted into a BstEII site. When pJAlO2 was used as the template to create aphoA fusion to TetA amino acid 364, the resulting plasmid again produced small colonies, suggesting that the lower plasmid copy number did not eliminate the toxicity of this fusion protein.

To minimize expression of toxic TetA-PhoA fusion proteins during

5"CGCTACTTGTGTATAAGAGTCAGAGGTCGAGGTGGCCC-

Membrane Topology of the pBR322 Tetracycline Resistance Protein 17811

plasmid constructions, mutagenesis reactions were transformed into TG1 harboring the plasmid pBST324 which expresses the pSclOl tetR repressor and therefore represses the tetA promoter in pJAlO2 and other pBR322 derivatives. pBST324 was constructed by ligating the 732-bp Clal-NdeI pSClOl tetR fragment (S1 nuclease trimmed) into the HincII site of pACYC177, such that tetR is expressed constitutively from the pACYC177 Ap’ promoter (Nguyen, 1987). Single- stranded pJAlO2 DNA was prepared by infecting TG1 harboring pJAlO2 with either rv1 or R408 helper phage at a multiplicity of infection of 10-50; the infected culture was diluted 1:lOO into LB-Ap- Km and grown for 6 h. Deletion mutagenesis followed the same procedure as described above. All of the mutagenic primers (45-mers) used to create tetA-phoA fusions have the same 23-nucleotide IS50L sequence at their 5’ end as the primer (JA2) used to create the full- length fusion in pJAlO2. As with TnphoA fusions generated by transposition (Manoil and Beckwith, 1985), the first amino acid encoded by the ISSOL linker sequence varies (Ser, Pro, Thr, or Ala). Mutagenized DNA was transformed into TG1 harboring pBST324, transformants were selected on LB-Ap-Km plates, and then screened on LB-Ap-Km plates containing tetracycline (10 pglml). The parent plasmid (pJA102) confers resistance to tetracycline; the tetA deletion plasmids do not. Plasmids from tetracycline-sensitive transformants (5-40% of all transformants) were further screened by digestion with EcoRI.

Construction of A s p A l a Mutations-Asp-+Ala mutations in tetA were created by oligonucleotide mutagenesis using the dut ung method (Kunkel, 1985) and reagents and protocols in the Mutagene kit (Bio- Rad). To construct the template for making the D17A and D86A mutations, the 622-bp HindIII-Sal1 fragment spanning the 5‘ half of tetA was isolated from pBR322 and cloned between the Hind111 and Sal1 sites of M13 mp18, creating phage mJA622. To construct the template for making the D287A mutation, the 793-bp Sun-BalI fragment spanning the 3‘ half of tetA was isolated from pZ150 and cloned between the SalI and SmaI sites of M13 mp18, creating phage mJA793. Uracil-containing single-stranded DNA was prepared by infecting the dut ung strain CJ236. The mutagenic primers for D17A, D86A and D287A were JA16 (5’-CACCCTGGcTGCTGTAG-3’), JA17 (5’-CACTATCGCCTACGC-GA-3’), and JA20 (5”GGCGGC- CGcCGCGCTGG-3’), respectively. Mutagenized DNA was transformed into MV1190, phage plaques were picked, and single-stranded DNA was sequenced to screen for the tetA mutations. To transfer the mutations into pZ150 or tetA-phoA fusion plasmids, tetA fragments spanning the mutations were isolated from phage RF DNA and ligated to appropriate fragments isolated from the parent plasmids. For D17A, the 156-bp HindIII-EcoRV fragment was transferred; for D86A, the 190-bp EcoRV-BamHI fragment was transferred; and for D287A, the 321-bp SalI-NruI fragment was transferred.

DNA Sequencing-The tetA-phoA fusion junctions were sequenced by the dideoxy method (Sanger et al., 1977) using [35S)a-thio-dATP (1300 Ci/mmol, Du Pont-New England Nuclear) and Sequenase v.1.0 (United States Biochemicals). Overnight cultures of TG1 harboring pBST324 and the fusion plasmid were infected with either rvl or R408 helper phage at a multiplicity of infection of 10-50; the infected cultures were diluted 1:lOO into LB-Ap-Km and grown for 6 h. Single- stranded plasmid DNA was extracted and used as template. The tetA- phoA fusion junctions were sequenced using a primer that anneals to phoA sequences near the left end of TnphoA (L276 5”AGCCCG-

For D17A and D86A, the sequencing primer was JA18 (5”GCAGT- Point mutations in tetA were sequencedusing tetA-specific primers.

CAGGCACCGTGT-3’); for D287A, the primer was JA21 (5’- CGGCCTGTCGCTTGCGG-3’). All three mutations were sequenced in the final plasmid constructs as well as in the M13 phages in which they were isolated. Double-stranded plasmid DNA was isolated as described by Del Sal et al. (1988), treated with RNase, extracted with phenol, and sequenced using Sequenase. Mutagenic primers and custom sequencing primers were synthesized by Louise Schmidt (University of California, San Diego) or Operon Technologies.

Enzyme Assays-Alkaline phosphatase was assayed as described by Michaelis et al. (1983). Overnight cultures of TG1 harboring pBST324 and tetA-phoA fusion plasmids were diluted 1:125 into LB- Ap-Km, grown until the optical density a t 550 nm reached 0.15-0.20, and then induced for 90 min with 0.2 pg/ml5a,6-anhydrotetracycline. The values reported in the tables are means of duplicate assays on two to four independent cultures. In general, standard errors were less than 10% of the means. TG1 is phoA+ and expresses 1 unit of alkaline phosphatase activity under these growth conditions; this

GTTTTCCAGAACAGG-3’).

background has been subtracted from the values reported in the tables.

Toxicity of Fusion Proteins-To determine the relative toxicities of the TetA-PhoA fusion proteins, plasmid DNA samples containing both the tetA-phoA plasmid (Ap’) and the tetR+ plasmid pBST324 (Km’) were used to transform TG1. Transformants were selected on LB-Ap-XP plates and then screened on LB-Ap-Km-XP plates. In general, about 10% of the transformants were Km’, indicating they contained both plasmids. If the Km” (tetR-) colonies were smaller than the Km’ (tetR+) colonies, we concluded that constitutive expression of the fusion protein inhibited cell growth. For all but the least active fusions, this comparison could be made directly on LB-Ap-XP plates since the Km” transformants formed light to dark blue (PhoA+) colonies while the Km‘ transformants formed white (PhoA-) colonies.

Radiolabeling of Fusion Proteins-Overnight cultures of TG1 harboring pBST324 and tetA-phA fusion plasmids were diluted 1:250 into M9 minimal medium (Miller, 1972) supplemented with 0.2% glucose, 1 mM thiamine HCl, 1 mM MgSO,, 0.01 mM CaC12, 4 rg/ml of each L-amino acid except methionine, Ap and Km. Cultures were grown until the optical density at 550 nm reached 0.4, induced for 10 min with 0.2 pg/ml5a,6-anhydrotetracycline, and then 1 ml of culture was pulse-labeled for 2 min with 50-100 pCi/ml [35S]methionine (1100 Ci/mmol, Du Pont-New England Nuclear). Labeling was stopped by adding 0.4 ml of 17.5% trichloroacetic acid and incubating on ice for at least 1 h. For pulse-chase experiments, 2 ml of culture was labeled and incorporation of [35S]methionine was stopped after 2 min by adding a 10,000-fold excess of unlabeled methionine (150 wg/ml). Immediately after and 5 , 10, 20, 30, 60, and 90 min after addition of the unlabeled methionine, 0.2-ml aliquots of the culture were re- moved, added to 0.08 ml of 17.5% trichloroacetic acid, and incubated on ice. Protein precipitates were collected by centrifuging for 30 min, washed twice with 1 ml of ice-cold acetone, air dried, and resuspended in 0.1 volume of sodium dodecyl sulfate (SDS) buffer (1% SDS, 10 mM Tris-HC1 pH 8.0).

Immunoprecipitation of Fusion Proteins-Immunoprecipitations were performed as described by Ito et al. (1981). Samples were heated for 5-10 min at 55 “C and centrifuged to remove insoluble material; 0.02 ml of the supernatant was diluted into 0.65 ml of ice-cold Triton buffer (2% Triton X-100, 50 mM Tris-HC1, pH 8.0, 0.15 mM NaCl, and 0.1 mM EDTA) and again centrifuged to remove insoluble material. Rabbit antiserum to bacterial alkaline phosphatase (2 p l , 5 Prim-3 Prime) was added and the sample incubated on ice for 16- 20 h. Protein A-Sepharose CL-4B (0.1 ml of a 10% suspension in Triton buffer, Sigma) was added and the sample incubated on ice for 2-4 h with occasional mixing. Protein A-Sepharose-immunoprecipi- tate complexes were collected by centrifuging for 1 min, washed twice with Triton buffer, and once with 10 mM Tris-HC1 pH 8.0. To dissociate the complexes, 40 pI of gel sample buffer (2% SDS, 62.5 mM Tris-HC1, pH 6.8, 5% 2-mercaptoethanol, 10% glycerol, and 0.002% bromphenol blue) was added; the sample was heated for 2 min at 55 “C and then centrifuged. Immunoprecipitates were electrophoresed 16-20 h at 10 mA in 15 cm X 30 cm X 0.75 mm Tris-glycine- buffered SDS-polyacrylamide gels (Laemmli, 1970) containing 11% acrylamide and 0.29% bisacrylamide. Protein molecular weight markers (Pharmacia) were: phosphorylase b (94,000), bovine serum albu- min (67,000) and ovalbumin (43,000). Gels were processed for fluo- rography (Autofluor, National Diagnostics). For quantitation, x-ray films (XAR, Kodak) were preflashed (Laskey and Mills, 1975), exposed, and then scanned using an LKB Ultroscan XL densitometer.

Immunoblot Detection of Fusion Proteins-Overnight cultures of TG1 harboring pBST324 and tetA-phoA fusion plasmids were diluted 1:125 into LB-Ap-Km, grown until the optical density at 550 nm reached 0.15-0.20, and then induced for 90 min with 0.2 pg/ml 5a,6- anhydrotetracycline. Approximately 5 X 10’ cells were pelleted, resuspended in 0.05 ml of gel sample buffer, heated for 15 min at 85 “C, centrifuged to remove any insoluble material, and then 0.04-ml aliquots were electrophoresed in SDS-polyacrylamide gels as described in the preceding section. Proteins were transferred to Immobilon-P filters (Millipore) by electrophoresis for 16 h at 175 mA in a Tran- sphor apparatus (Hoeffer Scientific). Filters were blocked by soaking 1 h in 2% powdered milk and 0.3% Tween-20 in TBS (20 mM Tris- HCl, pH 7.4, 0.9% NaCl) and then rinsed with TBS. Blocked filters were incubated with rabbit anti-alkaline phosphatase serum (20 pl in 100 ml of TBS) for 2 h at room temperature, washed five times in TBS (10 min/wash), incubated with goat anti-rabbit IgG-horseradish peroxidase conjugate (25 pl in 100 ml TBS, TAG Inc.) for 1 h, and again washed five times in TBS (5 min/wash). 0-Dianisidine (30 mg in 10 ml of methanol, Sigma) and 50 ml of TBS were added to the

17812 Membrane Topology of the pBR322 Tetracycline Resistance Protein filter; 50 p1 of 30% H202 was added to start the peroxidase reaction.

Tetracycline Resistance-Minimum inhibitory concentrations (MICs) of tetracycline were determined as described by Moyed et al. (1983). Overnight cultures were diluted 1:25 into LB-Ap, grown until the optical density at 550 nm reached 0.7-0.8, diluted 1:lOO into LB- Ap, and spotted (3 pl) on LB-Ap plates containing varying concentrations of tetracycline. Plates were incubated 18-20 h, and the lowest drug concentration that prevented confluent growth was taken as the MIC. The following concentrations were tested 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40,45, 50, 60, 70, 80, and 90 pg/ml.

RESULTS

Working Model of TetA Membrane Topology-We used the hydrophobicity scale and criteria of Eisenberg et al. (1984) to identify potential transmembrane segments in pBR322 TetA. All non-overlapping, 21-residue segments with mean hydrophobicity >0.42 were considered candidates. There are 12 such segments in pBR322 TetA (Table 11). A parallel analysis using the hydrophobicity scale and criteria of Engelman et al. (1986) also identified 12 potential transmembrane segments. Moreover, the boundaries for all but two of these segments (TM5 and TM7) are essentially the same whether the Eisen- berg or Engelman scale is used. An analysis of the TnlO TetA sequence using the Engelman hydrophobicity scale also identified 12 potential transmembrane segments having boundaries very similar to those of pBR322 TetA (data not shown). The deduced amino acid sequences of pBR322 TetA (Sutcliffe, 1979; Pedan, 1983) and TnlO TetA (Hillen and Schollmeier, 1983; Nguyen et al., 1983) show 45% identity and 67% simi- larity. It seems reasonable, therefore, to assume that the two proteins have fundamentally similar structures (Chothia and Lesk, 1986).

Our initial working model of pBR322 TetA membrane topology was based on the 12 transmembrane segments predicted by the Eisenberg hydrophobicity scale (Fig. 1). As discussed below, we subsequently adjusted the boundaries of TM7 to better fit the experimental data. The model has the following additional features: (i) The N and C termini are both located on the cytoplasmic side of the membrane. (ii) The distribution of basic amino acids near the ends of transmembrane segments generally follows the “positive-inside rule” (von Heijne, 1986); that is, there are more basic amino acids near the cytoplasmic ends of transmembrane segments. (iii) The periplasmic segments are generally somewhat shorter than the cytoplasmic segments. (iv) Finally, there are only four charged amino acids within transmembrane segments

TABLE I1 Potential transmembrane segments in pBR322 TetA

Potential transmembrane segments (TM) are the 12 most hydrophobic, non-overlapping, 21-amino-acid segments of pBR322 TetA (Sutcliffe, 1979; Pedan, 1983) based on the hydrophobicity scales of Eisenberg et al. (1984) and Engelman et al. (1986). Hydrophobicity values vary from 1.38 (Ile) to -2.53 (Arg) in the Eisenberg scale, and from -3.7 (Phe) to 12.3 (Arg) in the Engelman scale.

Eisenberg Engelman

Amino acids Hydrophobicity Amino acids Hydrophobicity TM

1 7-27 0.74 7-27 2 46-66

-1.64 0.70 46-66

3 80-100 0.61 79-99 -1.35 -1.78

4 104-124 0.60 104-124 5 139-159 0.70 133-153 -1.94

-1.19

6 161-181 0.66 161-181 7 216-236

-1.60 0.71 208-228

8 247-267 -1.87

0.67 246-266 -1.74 9 278-298 0.60 278-298

10 300-320 0.72 300-320 -1.18 -2.02

338-358 12 365-385 0.65 365-385 -1.52

-1.73 11 340-360 0.58

and they are all aspartates. Even if the key features of this model are correct, the assumption that all 12 transmembrane segments are precisely the same length is almost certainly an over-simplification (Deisenhofer et al., 1985; Henderson et al., 1990). Moreover, the length of 21 amino acids used for a- helical transmembrane segments does not take into account residues in contact with the polar headgroups of membrane phospholipids. Consequently, this model and others like it may systematically overestimate the lengths of short periplasmic and cytoplasmic segments.

Construction of tetA-phoA Gene Fusions-To test the TetA topology model, we constructed a series of tetA-phoA gene fusions. Initially, PhoA was fused to the C-terminal edge of the six periplasmic segments (Pl-6) and seven cytoplasmic segments (Cl-7) predicted by the Eisenberg hydrophobicity scale. All of the fusions were constructed in vitro by oligonucleotide-directed deletion mutagenesis as described in “Ex- perimental Procedures.” The fusion junctions are like those generated by TnphoA transposition (Manoil and Beckwith, 1985). In particular, the resulting fusion proteins contain a 17-amino-acid linker sequence derived from the IS50 sequence at the left end of TnphoA. Plasmid pJAlO2 (Fig. 2), which encodes the C-terminal TetA-PhoA fusion protein, served as the starting point for constructing the other fusions. All of the fusion plasmids were constructed in a background containing a second plasmid (pBST324, Fig. 2) that expresses the pSClOl tetR repressor and, therefore, represses transcription from the pJAlO2 tetA promoter. Addition of the gratui- tous inducer 5a,6-anhydrotetracycline induces maximal expression from the tetA promoter without inhibiting protein synthesis (Moyed et al., 1983; Nguyen, 1987).

Alkaline Phosphatase Activities of Fusion Strains-The PhoA enzyme activities of the fusion strains were assayed 90 min after induction (Table 111). Among the initial seven fusions to predicted cytoplasmic segments, five (fusions to C1, C2, C3, C5, and C7) have PhoA activities (123 units) that are at least 15-fold lower than the most active fusion (359 units). The activities of these fusions are clearly consistent with the topology model shown in Fig. 1. However, one of the initial fusions to a predicted cytoplasmic segment (C4-215) has intermediate PhoA activity (45 units) and one (C6-339) has high activity (287 units). In the case of the (24-215 fusion, the following transmembrane segment (TM7) is situated in an unusually long hydrophobic sequence (TetA amino acids 208-236). In fact, this is the transmembrane segment for which the boundaries based on the Eisenberg and Engelman scales are most different (Table 11). To test the influence of the position of the PhoA fusion junction prior to TM7, we constructed an additional fusion to TetA amino acid 210. The PhoA activity of this fusion (C4-210) is over 20-fold lower than that of the C4-215 fusion (2 versus 45 units). Apparently, the eight amino acids that follow ArgZo7 (Gly-Met-Thr-Ile- Val-Ala-Ala-Leu) are sufficient to constitute a weak (-10%) export signal for PhoA. Along this same line, Calamia and Manoil (1990) showed that the N-terminal 11 amino acids of two predicted transmembrane segments in Lacy are sufficient for efficient (>50%) PhoA export. We changed the boundaries of TM7 in our model to reflect the lower PhoA activity of the C4-210 fusion.

Positive charge appears to be important for anchoring cytoplasmic segments of inner membrane proteins on the cytoplasmic side of the membrane (Boyd and Beckwith, 1989; von Heijne, 1989; Nilsson and von Heijne, 1990). The C3 and C6 segments are the only predicted cytoplasmic segments that do not have a net positive charge; they have net charges of -1 and -2, respectively. To investigate the role of charge in


PERIPLASM 71 22 44 380(D17A) 300(D86A) 207 332 405(D287A) 359

FIG. 1. Proposed membrane topology of the pBR322 tetracycline resistance protein (TetA). Trans- membrane segments (TMI-12) are shown as open rectangles; periplasmic segments (Pl-6) and cytoplasmic segments (Cl-7) are shown as curved lines; charged amino acids are indicated by 0 (Argand Lys) and 0 (Asp and Glu). The boundaries of the 21-amino-acid transmembrane segments are based on the predictive algorithm of Eisenberg et al. (1984). Alkaline phosphatase activities of selected TetA-PhoA fusions and their mutant derivatives (D17A, D86A, and D287A) are shown above and below the model; the properties of these fusions are described more fully in Tables 111 and IV.

RI 5 RV-

CMOPLASM \ < 1

RI N N

M13 P

Or' pJA102 pBST324 phoA

RI Km'

RI

i s N FIG. 2. Structure of tetA-phoA plasmid pJAlO2 and tetR

plasmid pBST324. pJAlO2 is a pBR322 derivative; it encodes the C-terminal TetA-PhoA fusion protein (C7-396). pBST324 is a pACYC177 derivative; it is compatible with pJAlO2 and it encodes the pSClOl tetR repressor, which represses transcription from the pJAlO2 tetA promoter. The pBST324 tetR gene is expressed constitutively from the Ap' promoter. M13 ori, M13 origins of replication; Ap', ampicillin resistance gene; Km', kanamycin resistance gene; arrows, location and orientation of relevant promoters. Restriction sites: RZ, EcoRI; H, HindIII; RV, EcoRV; Ba, BamHI; N , NruI; Bs, BstEII; P, PstI.

the case of the anomalously active C6 fusion, we constructed an additional fusion to TetA amino acid 328. This fusion deletes the three negative charges (Asp3", Asp33o, and that follow the single positive charge (Arg326) in the C6 sequence. In fact, the C6-328 fusion actually has a somewhat higher PhoA activity than the C6-339 fusion (406 versus 287 units). Thus, the net negative charge of the C6 sequence is not the major determinant of the high PhoA activity of the C6-339 fusion protein.

Aspartates in TMl, TM3, and TM9 Act Like Signal Se- quence Mutations-PhoA fusions to three of the six predicted periplasmic segments (P3, P4, and P6) have high PhoA activities (207-359 units) consistent with the topology model in Fig. 1. However, the fusions to P1 and P5 have intermediate activities (71 and 44 units) and the fusion to P2 has low activity (22 units). In each of these last three cases, there is an aspartate residue near the middle of the transmembrane segment immediately preceding the fusion junction (Asp17 in TM1, AspR6 in TM3, and Aspza7 in TM9). By analogy with signal sequence mutations in wild-type PhoA (Michaelis et

2 2 3 w 2 12 287 \ 2

TABLE 111 Properties of TetA-PhoA fusion proteins

Fusion junctions are designated by the TetA cytoplasmic or periplasmic segment as defined in Fig. l , followed by the C-terminal TetA amino acid in the fusion protein. Alkaline phosphatase activities are for TG1 derivatives containing the tetR' plasmid pBST324 and the indicated tetA-phoA fusion plasmid, 90 min after induction. Relative toxicities are based on the colony size of TG1 derivatives containing the fusion plasmid alone compared to bacteria containing both the fusion plasmid and pBST324: -, no effect; +, ++, increasingly smaller colonies; +++, no visible colony. Relative rates of fusion protein synthesis are based on experiments like the one shown in Fig. 3A.

Alkaline Fusion Fusion phosphatase ~~~~~~~ protein

Relative

plasmid junction activitv svnthesis

pJA114 pJAll3 pJA112 pJAll l pJAllO pJAlO9 pJA123 pJA108 pJA107 pJAlO6 pJA105 pJA130 pJA104 pJAlO3 pJAlO2

C1-6 P1-45 C2-79 P2-103 C3-138 P3-160 C4-210 C4-215 P4-246 C5-277 P5-299 C6-328 C6-339 P6-364 C7-396

units <1 71

2 22 23

207' 2

45 332

12 44

406 287 359

2

- - - +

++ +++ ++ ++ + + + +

++

-

-

4.7 1.7 0.8 0.5 0.6 1.6

1.7 1.6 1.2

1.1 1.7 1.0

a No fusion protein detected. 'Extrapolated from the activity 30 min after induction; see text

for details.

al., 1983) and in other periplasmic and outer membrane proteins (for a review, see Benson et al., 1985), it seemed likely that the aspartates in TM1, TM3, and TM9 could act to block export of the PhoA moieties in the P1, P2, and P5 fusion proteins. To test this idea, we used oligonucleotide- directed mutagenesis to change the Asp codons (GAT/C) to Ala codons (GCT/C) and then transferred the mutations into the relevant fusion plasmids. In each case, the Asp-Ala mutation increases the PhoA activity of the fusion to the following periplasmic segment (Table IV). In fact, the resulting PhoA activities are comparable to those of the most active


TABLE IV Effects of A s p A l a mutations on TetA-PhoA fusion proteins

Alkaline phosphatase activities are for TG1 derivatives containing pBST324 and the indicated tetA-phoA fusion plasmid, 90 min after induction. Relative rates of fusion protein synthesis are based on experiments like the one shown in Fig. 4A.

Fusion plasmid junction

Fusion Alkaline Phosphatase Relative protein activity synthesis

units

pJA113 P1 71

pJAll2 c2 2

pJAl l l P2 22

pJA113-Dl7A P1 2.9

380 (5.4)" 3.6 (1.2)'

pJA112-Dl7A C2 2 (1.0)

pJAlll-Dl7A P2 47 (2.1) pJAlll-DS6A P2 300 (13.6) 1.6 (2.3)

0.7

pJAllO c 3 23

pJA105 P5 44 1.0

pJAlO4 C6 287

a Ratio of alkaline phosphatase activities in the A s p A l a mutant

Ratio of relative protein synthesis in the A s p A l a mutant and

pJA110-D86A C3 63 (2.7)

pJA105-D287A P5 405 (9.2) 1.3 (1.3)

pJA104-D287A C6 88 (0.31)

and the Asp-containing parent.

the Asp-containing parent.

fusions. This effect is most dramatic for the P2 and P5 fusions, where the D86A and D287A mutations increase PhoA activities 9.2- and 13.6-fold, respectively.

To determine if the effects of the Asp-Ala mutations are specific for PhoA fusions to the following periplasmic segments, we also transferred the mutations into fusions to the following cytoplasmic segments. The result is somewhat different in each case (Table IV). The D17A mutation in TM1 has no effect on the PhoA activity of the C2 fusion. The D86A mutation in TM3 increases the PhoA activity of the C3 fusion, but not to the same extent as it increases the activity of the P2 fusion (2.7- uersus 13.6-fold). Finally, the D287A mutation in TM9 actually reduces the PhoA activity of the anomalously active C6 fusion (3.3-fold). In one case, we also determined the effect of an Asp+Ala mutation on PhoA fused to a distal periplasmic segment; the D17A mutation in TM1 increases the PhoA activity of the P2 fusion, but only slightly compared to the effect of the D86A mutation in TM3 on this same fusion (2.1- uersus 13.6-fold). Taken together, the data in Table IV suggest that the effects of the A s p A l a mutations are relatively specific for PhoA fusions to the following periplasmic segments and do not result in a general increase in PhoA activity for all TetA-PhoA fusion proteins.

To further test the idea that the aspartates in TM1, TM3, and TM9 act like signal sequence mutations, we examined the effects of a signal sequence mutation suppressor. The prlA402 allele of prlA/secY was isolated as a dominant suppressor of signal sequence mutations in MalE and LamB; it is a strong suppressor of signal sequence mutations in these and other secreted proteins (Bankaitis and Bassford, 1985). We introduced fusion plasmids into the prlA402 strain RL402 and its prlA' parent MC4100. Although PhoA activities are somewhat higher in MC4100 (Table V) than in TG1 (Tables I11 and IV), the relative activities in the two backgrounds are similar. As shown in Table V, the pr01402 suppressor significantly increases the PhoA activity of the P1, P2, and P5 fusion proteins (2.5-4.6-fold), but has little or no effect on these same fusion proteins when they contain an Asp+Ala mutation in the transmembrane segment preceding the fusion junction. In addition, prlA402 has essentially no effect on the low PhoA activity of the C4 and C7 fusions. These results

TABLE V Effect of prlA402 suppressor on activity of TetA-PhoA fusion proteins

Alkaline phosphatase activities are for MC4100 (prZA+) and RL402 (prlA402) derivatives containing pBST324 and the indicated tetA- phoA fusion plasmid, 90 min after induction.

Fusion Fusion Alkaline phosphatase activity plasmid junction prlA+ prlA402 prlA402/prlA+

units pJAll3 P1 191 487 pJA113-Dl7A P1

2.5 650 612

pJAll1 P2 0.9

51 215 pJA111-D86A

4.2 P2 340 433

pJA123 c 4 2 2 1.3

pJA105 P5 99 451 4.6 1.0

pJA105-D287A P5 494 439 pJAlO2

0.9 c 7 2 2 1.0

clearly support the idea that TMl, TM3, and TM9 are defec- tive signals for PhoA export.

The observation that the PhoA activity of some TetA-PhoA fusion proteins is elevated in prlA402 strains suggests that these fusion proteins interact with wild-type PrlA/SecY and presumably with other components of the protein secretory pathway as well. However, the conclusion that insertion of wild-type TetA into the inner membrane is SecY dependent is not warranted based on these results alone. Since translocation of wild-type PhoA across the cytoplasmic membrane is itself a SecY-dependent process, it is possible that the effect of prlA402 on TetA-PhoA fusion proteins is specific for the PhoA moiety. Akiyama and Ito (1989) raised this same point in discussing their observation that membrane insertion of SecY-PhoA fusion proteins is SecY-dependent.

Toxicity of Fusion Proteins-Although neither wild-type TetA nor the C-terminal TetA-PhoA fusion protein (C7-396) is toxic when expressed constitutively from the tetA promoter, 10 of the 15 TetA-PhoA fusions that we constructed are toxic to some extent (Table 111). The P3 fusion protein is the most toxic; TG1 harboring pJAlO9 alone is not viable, and growth of TG1 harboring both pJAlO9 and pBST324 is completely inhibited 30 min after induction. In fact, the PhoA activity of this strain levels off 30 min after induction and actually declines at later times. The activity reported for the P3 fusion (Table I11 and Fig. 1) is based on a comparison with the P4- 246 and P6-364 fusions 30 min after induction, and has been normalized to the activities of these two reference fusions 90 min after induction. Calamia and Manoil (1990) noted a general correlation between toxicity and PhoA activity for Lacy-PhoA fusions. To some extent, we observed a similar pattern. None of the TetA-PhoA fusions with very low PhoA activities (12 units) are toxic and, with one exception, all of the fusions with higher activities are toxic. However, among the toxic fusions, the correlation between PhoA activity and degree of toxicity is less obvious. Along this same line, the D86A mutation substantially increases the toxicity of the P2 fusion, but the D17A and D287A mutations have no effect on the toxicity of the P1 and P5 fusions, respectively (data not shown). To minimize complications related to the constitutive expression of toxic fusion proteins, we constructed and ma- nipulated the fusion plasmids under conditions where the tetA promoter is repressed. In retrospect, this was an important feature of the experimental design.

Synthesis of Fusion Proteins-To visualize the fusion proteins and determine their relative rates of synthesis, fusion strains were induced for 10 min, pulse-labeled for 2 min with [35S]methionine, and the fusion proteins were immunoprecipitated with anti-PhoA antiserum (Fig. 3A). We could not detect the C1 fusion protein, even on overexposed films of

Membrane Topology of thepBR322 Tetracycline Resistance Protein 17815 A

C7 P6 C6 P5 C5 P4 C4 P3 C3 P2 C2 P1

43-

FIG. 3. Synthesis and accumulation of TetA-PhoA fusion proteins. Panel A , derivatives of TG1 containing the tetR+ plasmid pBST324 and the indicated t e tA-phA fusion plasmid were induced for 10 min with 5a,6-anhydrotetracycline and then pulse-labeled for 2 min with [”S]methionine. Fusion proteins were precipitated from equal amounts of solubilized cells using anti-PhoA serum and protein A-Sepharose beads as described under “Experimental Procedures” and then electrophoresed in an SDS-polyacrylamide gel. From left to right: C7, pJAlO2; P6, pJA103; C6, pJAlOl; P5, pJA105; C5, pJA1OG; P4, pJAlO7; C4, pJA123; P3, pJAlO9; C3, pJAllO; P2, pJAlll; C2, pJA112; PI , pJA113. The position of a PhoA-sized degradation prod- uct is indicated on the right, and the positions of molecular weight markers (M, X are indicated on the left. Panel B, derivatives of TG1 containing pBST324 and the indicated tetA-phA fusion plasmid were induced for 90 min. Equal amounts of solubilized cells were electrophoresed in an SDS-polyacrylamide gel and TetA-PhoA fusion proteins were visualized by immunoblotting using anti-PhoA serum as described under “Experimental Procedures.” Lane designations are the same as in panel A .

samples pulse labeled for only 1 min (data not shown). The C1 fusion (to TetA amino acid 6) is unique among the fusions that we constructed in that the PhoA moiety should presumably be free in the cytoplasm (not membrane-bound). Al- though cytoplasmically localized PhoA is often less stable than periplasmically localized PhoA, this difference is not usually so great as to preclude detection in a 1-min pulse- labeling experiment (Boyd et al., 198713; San Millan et al., 1989; Calamia and Manoil, 1990). We suspect, therefore, that the C1 fusion protein is synthesized at a very much lower rate than other TetA-PhoA fusion proteins. The loss of sequences downstream from TetA amino acid 6 may reduce the efficiency of TetA translation. However, loss of downstream sequences is not the only factor, since we can detect synthesis of a protein with LacZ fused to TetA amino acid 6 (data not shown). In this regard, we note that the transcript for the C1 TetA-PhoA fusion protein has the potential to form a local RNA secondary structure involving the tetA Shine-Dalgarno sequence and the IS50 sequence near the tetA-phoA fusion junction.

There are significant differences in methionine content between the shortest and longest TetA-PhoA fusion proteins

seen in Fig. 3A. The P1 fusion protein contains 8 methionines, whereas the C7 fusion protein contains 23 methionines. Tak- ing these differences into account and taking care to scan appropriately exposed films, we determined the relative rates of fusion protein synthesis (Table 111). For most of the fusions (C2, C4, P4, C5, P5, C6, P6, and C7), the differences in rates of synthesis are less than 2-fold. However, there are several exceptions; the P1 fusion is synthesized at a 3-4-fold higher rate, and the P2, C3 and P3 fusions are synthesized at 2-3- fold lower rates relative to the other fusion proteins. The basis for these apparent differences in “initial” rates of synthesis is not obvious. There are no obvious correlations between fusion protein length, activity or toxicity, and the rate of synthesis. Although differences in fusion protein toxicity lead to significant growth differences 30-60 min after induction, they do not affect incorporation of [“S]methionine into total cell protein 10 min after induction (data not shown). One possibility that is hard to rule out is differential detection owing to differences in fusion protein solubility or aggrega- tion. Regardless of the explanation for the apparent differences in initial rates of fusion protein synthesis, these differences are not sufficient to account for either the high PhoA activity of the C6 fusion or the low PhoA activity of the P1, P2, and P5 fusions.

To correct for differences in the rates of synthesis of different leader peptidase-PhoA fusion proteins, San Millan et al. (1989) calculated PhoA “specific activities” (PhoA activity/ rate of synthesis). In the end, we chose not to calculate PhoA- specific activities based on the synthesis data in Table 111. As noted by San Millan et al. (1989), the validity of this calcu- lation is complicated by the fact that it relates the activity of the total amount of fusion protein that accumulates during the induction period to the rate of synthesis at a specific time. Rates of synthesis determined early in the induction period (as in our study) or at the end of the induction period (as in the San Millan et al. study) very likely overestimate or un- derestimate, respectively, the total synthesis of toxic fusion proteins. However, we want to emphasize that there are no obvious correlations between either initial rates of synthesis or toxicity and the PhoA activities of the anomalous TetA- PhoA fusions (C6, P1, P2, and P5).

We also determined the effects of the Asp+Ala mutations on the synthesis of the P1, P2, and P5 fusion proteins (Fig. 4A and Table IV). The D17A and D287A mutations have little effect on the rate of synthesis of the P1 and P5 fusions, respectively. In contrast, the D86A mutation increases synthesis of the P2 fusion, but the 2.3-fold increase in P2 synthesis clearly does not fully account for the 13.6-fold increase in PhoA activity that is observed. The D86A mutation also increases the mobility of the P2 fusion protein in SDS- polyacrylamide gels (Fig. 4A). We were initially concerned about this mobility change and went to some effort to verify the structure of the plasmid and the DNA sequence of its tetA region. In fact, there are many precedents for single amino acid substitutions changing protein mobilities in SDS gels, especially when the mutations introduce charge differences (e.g. Noel et al., 1979).

Stability of Fusion Proteins-Comparing the relative band intensities of pulse-labeled samples (Fig. 3A) to the band intensities of samples detected by immunoblotting (Fig. 3B) suggests that there are substantial differences in fusion protein stability. For example, the C5 and P5 fusions are under- represented on the immunoblot relative to the pulse-labeled samples. Calamia and Manoil (1990) noted a general tendency for less active Lacy-PhoA fusion proteins to be less stable. Overall, we see a similar pattern for TetA-PhoA fusion pro-

17816 Membrane Topology of thepBR322 Tetracycline Resistance Protein

B

FIG. 4. Effects of Asp+Ala mutations on synthesis and accumulation of TetA-PhoA fusion proteins. Derivatives of TG1 containing pBST324 and the indicated tetA-phoA fusion plasmid were induced and processed as in Fig. 3. Panel A , immunoprecipitation of TetA-PhoA fusion proteins. From left to right: PI, pJA113; P l -

pJA105; P5-D287A; pJA105-D287A. Panel B, immunoblot of same TetA-PhoA fusion strains as in panel A.

D17A, pJA113-Dl7A; P2, pJAll1; PZ-D86A, pJA111-D86A; P5,

teins. The correlation between activity and stability is particularly striking for TetA-PhoA fusions containing A s p A l a mutations. Each of the A s p A l a mutations increases both the PhoA activity and stability of the fusion to the following periplasmic segment, as judged by the intensities of the protein bands on immunoblots (Fig. 4B). This is most evident for D17A and D287A, which do not increase the rate of fusion protein synthesis, but do significantly increase the net accumulation of intact fusion protein over the 90-min induction period. To further examine these stability differences, we performed pulse-chase experiments on strains expressing the P5 fusion protein with and without the D287A mutation (Fig. 5). The active P5-D287A fusion protein decays with a half- life of about 55 min. In contrast, the 9.2-fold less active P5 fusion protein shows biphasic decay kinetics; the major component decays with a half-life of about 12 min, and the minor component decays with a half-life of about 50 min. The more stable component presumably corresponds to the small frac- tion of the P5 fusion protein in which the PhoA moiety is periplasmically localized. Calamia and Manoil (1990) observed similar biphasic decay kinetics for partially active Lacy-PhoA fusion proteins. The effects of the A s p A l a mutations on fusion protein stability are also consistent with

P5-D287A P5

' 0 5 10 20 30 60 90' ' 0 5 10 20 30 60 90 '

FIG. 5. Effect of the D287A AspAla mutation on the stability of the P5 TetA-PhoA fusion protein. Derivatives of TG1 containing pBST324 were induced and pulse-labeled with [%]methionine as in Fig. 3, and then incubated for 0, 5, 10, 20, 30, 60, and 90 min in the presence of excess unlabeled methionine. Samples were immunoprecipitated and processed as in Fig. 3. P5-D287A, pJA105- D287A, P5, pJA105.

TABLE VI Effect of TetA A s p A l u mutations on tetracycline resistance

Minimum inhibitory concentrations of tetracycline are for TGI derivatives containine the indicated dasmid. ~

Plasmid t e a genotype Minimum inhibitory tetracycline

w?lml

pBR322 tetA + 80 pZ150 tetA+ 80 pJAlO2 tetA-phoA (C7-396) 45 pJA114 tetA-phoA (Cl-6) 1 pZ150-Dl7A tetA (Asp-l7+Ala) 2 pZ150-D86A tetA (Asp-8bAla) 7 pZ150-D287A tetA (Asp-287-Ala) 1

earlier observations that signal sequence mutations reduce the stability of wild-type PhoA (Michaelis et al., 1983; Boyd et al., 1987b). Finally, since the reduced stability of cyto- alasmic PhoA is due, at least in part, to its failure to assume a native, enzymatically active conformation (Akiyama and Ito, 1989; Derman and Beckwith, 1991), it is clearly not useful to calculate PhoA-specific activities based on steady-state levels of fusion proteins (e.g. immunoblot band intensities).

Interestingly, the C4 and C7 TetA-PhoA fusions, which have very low PhoA activities, appear to be more stable than other low activity fusions. This difference could be related to the structural integrity of the TetA moiety in these two cases. Several lines of evidence indicate that the N- and C-terminal halves of TetA constitute two structural and perhaps functional domains (Curiale et al., 1984; Rubin et al., 1990). In this regard, the C4 fusion contains the intact N-terminal domain, while the C7 fusion contains both domains.

Conserved Aspartates in TM1, TM3, and TM9 Are Critical for Tetracycline Resistance-The aspartates in TM1, TM3, and TM9 of pBR322 TetA are conserved in the related TetA proteins encoded by TnlO (Nguyen et al., 1983) and Tnl721 (Waters et al., 1983). Their conservation, together with their location in otherwise hydrophobic transmembrane segments, suggested that they might be important for TetA function. To test this, we introduced the D17A, D86A, and D287A mutations into the wild-type tetA gene on pZ150 and then determined the MIC of tetracycline for cells harboring the mutant plasmids (Table VI). All three of the A s p A l a mutations dramatically reduce tetracycline resistance. D287A has the most severe effect; the MIC for pZ150-D287A is the same as that for pJA114, the tetracycline-sensitive control.

We do not know whether the A s p A l a mutations affect the membrane insertion or stability of TetA, however, the same mutations increase both membrane insertion and stability of TetA-PhoA fusion proteins. Therefore, it seems


unlikely that the observed changes in tetracycline resistance are due primarily to reduced membrane insertion or stability. There are two interesting possible roles for these conserved aspartates: (i) involvement in the mechanism of proton translocation, as shown for AspR5 and Asps6 in bacteriorhodopsin (Henderson et al., 1990) and (ii) involvement in metal-tetracycline binding. Yamaguchi et al. (1991) have also suggested that AspR4 and AspzR5 in TnlO TetA may be involved in proton translocation or metal-tetracycline binding.

The C-terminal TetA-PhoA fusion protein confers a significant level of tetracycline resistance; the MIC for pJAlO2 is over 50% that for pZ150, the wild-type TetA control. This result suggests that the TetA moiety of the fusion protein is inserted into the inner membrane in an essentially wild-type conformation. The MICs in Table VI are for strains that express wild-type or mutant TetA proteins constitutively. Because the presence of the tetR repressor plasmid pBST324 reduces MICs 50-75%, comparable MICs for toxic fusion plasmids such as pJA103 could not be determined. However, TG1 harboring pBST324 and pJA103 fails to grow on LB- Ap-Km plates containing 10 pg/ml tetracycline, whereas TG1 harboring pBST324 and pJAlO2 grows well on these plates. This result is consistent with previous reports that the C- terminal 14-17 amino acids of TnlO TetA are important for wild-type function (Jorgensen and Reznikoff, 1979; Nguyen et al., 1983; Hickman and Levy, 1988).

DISCUSSION

We have used the alkaline phosphatase (PhoA) gene fusion strategy (Manoil and Beckwith, 1986) to analyze the topology of the pBR322 tetracycline resistance protein (TetA) in the inner membrane of E. coli. We first developed an explicit working model based on the 12 transmembrane segments predicted by the hydrophobicity scale and criteria of Eisen- berg et al. (1984). Eckert and Beck (1989) and Henderson and Maiden (1990) have also proposed similar 12-transmembrane- segment models for the membrane topology of TnlO TetA and pBR322 TetA, respectively.

Previous biochemical and immunological studies on TnlO TetA have provided experimental support for the cytoplasmic location of six of the seven predicted cytoplasmic segments shown in Fig. 1. (i) The N-terminal methionine is accessible to cyanate modification in inside-out membrane vesicles but not in spheroplasts (Eckert and Beck, 1989). (ii) Five of the predicted cytoplasmic segments are accessible to protease cleavage in inside-out membrane vesicles but not in spheroplasts (Eckert and Beck, 1989; Yamaguchi et al., 1990a). More specifically, trypsin cleaves C2, C4, and C7; proteinase K cleaves C3 and C4; and endoproteinase LysC cleaves C6. (iii) Finally, antibodies raised against a synthetic peptide corre- sponding to the C-terminal14 amino acids of TnlO TetA bind preferentially to inside-out membrane vesicles (Yamaguchi et al., 1990a). However, there is no biochemical evidence for the periplasmic location of any of the predicted periplasmic segments of TetA; both pBR322 and TnlO TetA are resistant to protease cleavage from the periplasmic side of the membrane (Hengge and Boos, 1985; Eckert and Beck, 1989).

To test the topology model in Fig. 1, we used oligonucleotide-directed deletion mutagenesis (Boyd et al., 1987a) to construct PhoA fusions to the C-terminal edges of the 13 predicted periplasmic and cytoplasmic segments of TetA. This in vitro approach is particularly attractive for topologically complex membrane proteins with multiple short periplasmic and cytoplasmic segments. Even when large numbers of fusions are isolated by TnphoA transposition, the existence of transposition hot spots, together with the small target size

presented by short periplasmic and cytoplasmic segments, often necessitates constructing some fusions in vitro (Manoil and Beckwith, 1986; Boyd et al., 1987a; San Millan et al., 1989; Calamia and Manoil, 1990). In addition, low activity fusions to cytoplasmic segments are inherently more difficult to obtain by TnphoA transposition.

The properties of many of the TetA-PhoA fusions that we constructed support the 12-transmembrane-segment model. More specifically, fusions to six of the seven predicted cytoplasmic segments have low PhoA activities and fusions to three of the six predicted periplasmic segments have high PhoA activities. On the other hand, two fusions to one of the predicted cytoplasmic segments (C6) have high PhoA activities. The protease accessibility of the C6 region in TnlO TetA (Eckert and Beck, 1989) is particularly important here because it provides independent evidence that C6 is cytoplasmically located in wild-type TetA. Boyd et al. (1987a) and San Millan et al. (1989) described active PhoA fusions to predicted cytoplasmic segments in MalF and leader peptidase, respectively. However, in both of these studies the anomalous fusions were near the beginning of the cytoplasmic segments, and their high activity could be attributed to the loss of topogenic determinants in the disrupted cytoplasmic segments; fusions near the end of the same cytoplasmic segments showed low activity. In contrast, we obtained an active PhoA fusion to the end of the C6 segment in TetA. The properties of this fusion (C6-239) suggest that the C6 segment is not effectively anchored on the cytoplasmic side of the membrane in the absence of downstream TetA sequences, i.e. sequences that are missing in the C6 fusion protein.

von Heijne (1986) proposed that transmembrane segments orient in the inner membrane such that there are more basic amino acids near their cytoplasmic ends than near their periplasmic ends. This generalization, referred to as the “positive-inside rule,” is supported by statistical analyses of membrane proteins of known topology (von Heijne, 1986; von Heijne and Gavel, 1988) and by mutational studies with MalF (Boyd and Beckwith, 1989) and leader peptidase (von Heijne, 1989; Nilsson and von Heijne, 1990; for a review, see Boyd and Beckwith, 1990). In fact, TMlO is the only predicted transmembrane segment in pBR322 TetA that does not follow the positive-inside rule; there is a single arginine on either side of TM10. Since deletion of the three aspartates in C6 does not reduce the PhoA activity of the C6 fusion, we suspect that it is the equal number of positive charges at either end of TM10, rather than the net negative charge of C6, that accounts for the high PhoA activity of the C6 fusion. Inter- estingly, all 12 predicted transmembrane segments in TnlO TetA, including TM10, follow the positive-inside rule; the arginine in P5 of pBR322 TetA is replaced by glutamate in TnlO.

Fusions to three predicted periplasmic segments (Pl , P2, and P5) have either low or intermediate PhoA activity. The properties of these fusions suggest that TM1, TM3, and TM9 cannot function as efficient signals for PhoA export. In each case, there is an aspartate near the middle of the transmembrane segment (Asp” in TM1, Asps6 in TM3, and Asp’87 in TM9). Two lines of experimental evidence indicate that these aspartates act like signal sequence mutations. (i) A mutant allele of prlA (prlA402) that suppresses signal sequence mutations increases the PhoA activity of the PI, P2, and P5 fusions. (ii) Asp+Ala mutations in TM1, TM3, and TM9 increase the PhoA activity of these same fusions. In general, the topological effects of the Asp-Ala mutations are local; that is, they have their greatest effect on the PhoA activity of the fusion to the following periplasmic segment. Interest-


ingly, the A s p A l a mutation in TM9 (D287A) not only increases the low activity of the P5 fusion, but also reduces the high activity of the C6 fusion. A possible interpretation is that Aspzs7 blocks TM9 membrane insertion, allowing TMlO to insert backwards and thereby export the PhoA moiety of the C6 fusion protein. According to this scenario, proper membrane insertion of both TM9 and TMlO normally requires the presence of downstream TetA sequences that have been deleted in the C6 fusion, but the D287A mutation promotes TM9 membrane insertion and thereby reduces backward insertion of TMlO in the C6-D287A fusion.

Since neither the protease accessibility data for TnlO TetA (Eckert and Beck, 1989) nor the PhoA fusion data for pBR322 TetA provide direct support for the periplasmic location of the P2 and P5 segments, it is legitimate to ask whether the 12-transmembrane-segment model should be modified. For example, the TM3-P2-TM4 and/or TM9-P5-TM10 regions could be located in the cytoplasm. However, we continue to favor the 12-transmembrane-segment model for several rea- sons. (i) The mean hydrophobicities of TM3, TM4, TM9, and TMlO are well within the range seen for bona fide transmembrane segments in both the photosyntheic reaction center and bacteriorhodopsin (Engelman et al., 1986). (ii) The protease accessibility data for TnlO TetA (Eckert and Beck, 1989) do not support the placement of either TM3-P2-TM4 or TM9- P5-TM10 in the cytoplasm. (iii) Mutational studies suggest a mechanistic parallel between residues in TM8 and TM9 of TnlO TetA and TM9 and TMlO of Lacy (Yamaguchi et al., 1991). (iv) Finally, the similarities between the hydrophobicity profiles of TetA and a number of sugar transporters in bacteria and mammals, and the conservation of short sequence motifs among these proteins, suggest that TetA is a member of a large class of transport proteins that share a conserved 12-transmembrane-segment structural motif (Hen- derson and Maiden, 1990).

A number of authors have discussed general models for the organization of topogenic sequences in membrane proteins. Blobel and Friedlander (1980, 1985) proposed that the topology of inner membrane proteins could be accounted for by two classes of topogenic sequences. According to their model, the transmembrane segments that precede periplasmic domains constitute “signal sequences” (or “insertion sequences”), and those that follow periplasmic domains constitute “stop-transfer sequences.” They envisioned that membrane insertion is cotranslational and therefore proceeds sequentially from N terminus to C terminus. More recently, von Heijne and others have focused on the topogenic role of basic amino acids flanking transmembane segments (von Heijne, 1986; Nilsson and von Heijne, 1990; Boyd and Beck- with, 1990). Whether or not membrane insertion is cotranslational or proceeds sequentially from N terminus to C terminus, both of these models suggest that insertion and orientation of individual transmembrane segments is determined locally, i.e. by sequences within the apolar region of the transmembrane segment itself and the polar segments immediately adjacent to it. The two-stage model for membrane protein folding proposed by Popot and Engelman (1990) is even more explicit. These authors suggest that transmembrane a-helicies constitute “autonomous folding domains” that individually fold and insert into the membrane and only then interact to form functional structures. Indeed, the general success of the PhoA fusion strategy in analyzing membrane protein topology tends to support this same view (Man- oil and Beckwith, 1986; Manoil et al., 1990). However, the properties of several of the TetA-PhoA fusions described here suggest that some transmembrane segments behave differ-

ently. For four of the 12 transmembrane segments in TetA (TM1, TM3, TM9, and TMlO), proper membrane insertion appears to depend on TetA sequences C-terminal to both the transmembrane segment itself and the adjacent polar segments. That is, long range interactions may be more important in establishing TetA membrane topology than suggested by the general models discussed above. We note that it is the functional requirement for aspartates in TM1, TM3, and TM9 that compromises the ability of these TetA transmembrane segments to correctly fold and insert into the membrane on their own.

The helical hairpin hypothesis of Engelman and Steitz (1981) offers an alternative way to think about the organization of topogenic sequences in TetA. According to this model, pairs of adjacent transmembrane segments fold to form helical hairpins which then insert into the inner membrane. For example, the TM1-P1-TM2 segment of TetA may fold and insert into the membrane efficiently, whereas the TM1-P1 segment alone apparently cannot. Similarly, TM3-P2-TM4 may fold and insert as a unit. However, the anomalous behav- ior of the P5 and C6 fusions cannot be explained simply by supposing that TM9-P5-TM10 inserts as a unit. Perhaps the proper insertion of TM9-P5-TM10 depends on simultaneous or prior insertion of TM11-P6-TM12. We are currently test- ing some of these possibilities.

Acknowledgments-We thank Kathleen Postle and Linda Randall for providing plasmids and strains and for comments on the manu- script. We also thank Susan Johns of the Washington State Univer- sity VADMS computer facility for assistance.

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membrane topology of the pbr322 tetracycline … tetracycline resistance gene of pbr322 encodes a...

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