INTRODUCTION

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The Na⁺/proline transporter of Escherichia coli (PutP) is an integral membrane protein that catalyzes the coupled translocation of Na⁺ ions and proline (1-3). PutP belongs to a family of homologous Na⁺-dependent membrane transport proteins (Na⁺/solute cotransporter family, SCF)¹ that comprises more than 35 members of bacterial, yeast, insect, nematode, and mammalian origin (4, 5). Based on the hydropathy profile of the amino acid sequence of PutP, a secondary structure model has been proposed according to which the transporter consists of a short N-terminal tail, 12 transmembrane domains (TMs) in -helical conformation that traverse the membrane in zig-zag fashion connected by hydrophilic loops, and a hydrophilic C-terminal tail (6) (Fig. 1). Experimental evidence for the cytoplasmic location of the C terminus comes from immunological studies (7). However, recent N-glycosylation scanning mutagenesis of the human Na⁺/glucose transporter (SGLT1), another member of the SCF, suggests a 14-helix motif for this transporter (8). Because PutP lacks a C-terminal extension, 13 TMs are predicted for this and other prokaryotic members of the SCF (5).

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Secondary structure model of PutP of E. coli. The model is based on the hydropathy plot of the primary amino acid sequence of the transporter (6). The cytoplasmic location of the C terminus was shown by immunological analysis (7). Transmembrane domains are represented as rectangles and numbered with Roman numerals. Arabic numerals correspond to hydrophilic cytoplasmic and periplasmic loops starting with the N terminus. Functional important amino acid residues are shown enlarged. In addition, sites of AspN cleavage before (filled arrows) and after equilibration (open arrows) of the protease with the proteoliposome interior are indicated.

In this study, we have tested the different topological predictions for PutP. In addition, our attention is focused on the topological location of the recently identified functional important amino acid residues, Asp-55 and Ser-57, which are proposed to be involved in ligand binding (9, 10). To obtain experimental evidence on the topology of PutP, we have generated and characterized a series of putP-phoA and putP-lacZ fusions. In addition, information on the arrangement of the PutP polypeptide has been gained from a Cys accessibility analysis and site-specific proteolysis. Periplasmic alkaline phosphatase (PhoA) and cytosolic -galactosidase (LacZ) have already been used successfully as reporter proteins to determine the topological arrangement of a variety of membrane proteins in the bacterial cytoplasmic membrane (11-13). Also, site-directed labeling and specific proteolysis of membrane proteins are established methods for topology analysis (14-16). The results obtained in the course of this study demonstrate that the N terminus of PutP is located on the periplasmic side of the membrane whereas the C terminus faces the cytoplasm. Furthermore, amino acids of former periplasmic loop (pL) 2 are proposed to form an additional TM (now TM II) whereas the boundaries of former TM II (now TM III) are shifted by eight amino acids toward the C terminus of PutP. The resulting 13-helix motif is discussed as a common structural feature of members of the SCF.

	EXPERIMENTAL PROCEDURES

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Materials-- L-[¹⁴C]proline (261 µCi/µmol), sheep anti-(mouse-IgG)-horseradish peroxidase and streptavidin-horseradish peroxidase conjugate were purchased from Amersham Buchler, Braunschweig, Germany. Mouse anti-FLAG-M2-antibody was from Integra Biosciences, Fernwald, Germany. Ni-NTA spin columns were from Qiagen GmbH, Hilden, Germany. The endoproteinase AspN was purchased from Sigma, Deisenhofen, Germany.

Bacterial Strains and Plasmids-- E. coli JM109 (endA1 recA1 gyrA96 thi hsdR17 supE44 relA1 (lac-proAB) (F' traD36 proAB⁺ lacI^q ZM15)) (17) served as carrier for the plasmids described. E. coli WG170 (F trp lacZ rpsL thi (putPA)101 proP219) (18) harboring given plasmids was used for overexpression of the putP gene and transport assays. Plasmid-encoded putP-lacZ and putP-phoA fusions were maintained in E. coli CC181 (F128 lacI^q (ara, leu)7679 lacX74 (phoA)20 galE galK thi rpsE rpoB, argE(am) lacY328 (am) recA1) (13). Plasmid pT7-5/putP (9) a derivative of plasmid pT7-5 (19) containing the lac promoter/operator region as well as the putP gene was used for genetic manipulations and expression of the putP gene and putP-phoA and putP-lacZ fusions. Plasmid pTrc99a (20) was applied for overexpression of the putP gene. PutP gene fusions were overexpressed using plasmid pBAD24 (21).

Site-directed Mutagenesis-- To facilitate genetic manipulations, a cassette version of the putP gene was generated by site-directed mutagenesis using plasmid pT7-5/putP (9) and mutagenic oligonucleotides. The resulting pT7-5/putP(cassette) contained unique restriction sites of the following enzymes (cutting sites are shown in parentheses): BamHI (292), NcoI (296), ApaI (428), XbaI (661), BssHII (740), NheI (868), SalI (976), PstI (1080), AflII (1320), SpeI (1486), MluI (1573), Eco47III (1776), and HindIII (1805) (only sites located within or at the ends of putP were listed). The nucleotide substitutions introduced into the putP gene did not alter the amino acid sequence of PutP.

Proline Transport in Cells-- Active transport was measured in E. coli WG170 producing PutP or its variants with 5 µM L-[¹⁴C]proline in the presence of 20 mM D-lactate and 50 mM NaCl at 25 °C using the rapid filtration method as described (10).

Labeling of PutP(FH) in Cells-- E. coli WG170 harboring plasmid pTrc99a/putP(FH) encoding Cys-free or single Cys PutP(FH) was grown aerobically in LB medium (23) containing 100 µg/ml ampicillin at 37 °C, and expression was initiated by addition of 0.3 mM isopropyl-1-thio--D-galactopyranoside (IPTG) at the middle of the exponential growth phase. After further cultivation for 25 min (or 45 min if indicated), cells were harvested by centrifugation, washed with 100 mM KP_i, pH 7.5, containing 0.5 mM phenylmethylsulfonyl fluoride (buffer A) and resuspended in the same buffer to give a final protein concentration of 14 mg/ml. For blocking of periplasmic thiol groups, an aliquot of the cell suspension (1.5 ml) was preincubated with 400 µM (or 1 mM if indicated) of the membrane impermeant thiol reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonate (SM) at room temperature. After 30 min, cells were washed three times with buffer A and resuspended in the same buffer as described above. Aliquots (1.5 ml) of blocked and untreated cells were reacted with 400 µM 3-(N-maleimidylpropionyl)biocytin (BM) at room temperature for 30 min. Subsequently, labeled cells were washed three times with buffer A and disrupted by sonication followed by low speed centrifugation at 12,000 × g for 30 min at 4 °C to remove unbroken cells. Membranes were collected by centrifugation at 230,000 × g for 90 min at 4 °C, washed with 100 mM KP_i, pH 8.0, and resuspended in the same buffer. For solubilization of labeled Cys-free or single Cys PutP(FH), the membrane suspension was supplemented with 2 mM -mercaptoethanol and 10% glycerol. -D-Dodecylmaltoside was added dropwise to yield a final concentration of 1.5% (w/v) while stirring on ice. After additional stirring for 30 min, the sample was centrifuged at 230,000 × g for 20 min. The supernatant was supplemented with 10 mM imidazole and 300 mM NaCl and loaded onto a Ni²⁺-NTA spin column pre-equilibrated with 100 mM KP_i, pH 8.0, 300 mM NaCl, 10 mM imidazole, 10% glycerol (v/v), 0.04% -D-dodecylmaltoside (w/v) (buffer E). Unbound protein was removed by washing the Ni²⁺-NTA spin column with 0.6 ml of buffer E and 1.8 ml of buffer E containing 30 mM imidazole. The transporter was eluted from the spin column with 200 µl of a 200 mM imidazole solution in buffer E. Aliquots of the purified protein were subjected to SDS polyacrylamide gel electrophoresis (PAGE) (10% acrylamide) (43). The amount of protein was judged after Coomassie Blue staining. Reaction of single Cys PutP(FH) with biotin maleimide was determined by Western blot analysis using streptavidin-horseradish peroxidase.

Site-specific Proteolysis-- PutP(FH) was purified and reconstituted into proteoliposomes in an inside-out orientation as described (42). Proteoliposomes containing PutP(FH) were diluted in 10 mM Tris/HCl, pH 8.0, to yield a final protein concentration of 0.25 mg/ml. The endoproteinase AspN was added at a PutP(FH):AspN ratio of 200:1 (w/w), and proteolysis was carried out at 37 °C. The reaction was stopped by addition of 25 mM EDTA after 0.5 or 17 h of incubation. Subsequently, the protein was solubilized in 1% SDS, subjected to SDS-PAGE (10% acrylamide) according to Schägger and Jagow (24), and stained with silver (25). N-terminal sequencing was performed as described (26).

Generation of putP-phoA and putP-lacZ Gene Fusions-- Initially, a unique NheI site was introduced at the 3' end of a cassette version of the putP gene in plasmid pT7-5 lacking the NheI site at position 868 by oligonucleotide-directed site-specific mutagenesis. For fusion of putP with phoA, the resulting plasmid pT7-5/putPNheI was digested with NheI and BanI. The fragment (4,064 base pairs) containing full-length putP and part of plasmid pT7-5 was ligated with a 1,527-base pair fragment obtained from plasmid pT7-5/lacY-phoA (27) digested with the same two restriction enzymes. The latter fragment carried the phoA gene and the remaining part of plasmid pT7-5. The procedure yielded plasmid pT7-5/putP(S502)phoA,² which contained the full-length putP gene followed at its 3' by a unique NheI site and the phoA gene. For fusion of putP with lacZ, the latter gene was amplified by PCR, thereby introducing restriction sites for NheI and HindIII at the 5' and 3' end, respectively, of lacZ. The PCR fragment was cloned into plasmid pT7-5/putPNheI using the NheI and HindIII restriction sites yielding plasmid pT7-5/putP(S502)lacZ. Further putP-phoA and putP-lacZ fusions were generated using PCR with antisense primers that were complementary to the 3' termini of the desired putP fragments. In addition, the primers created an NheI site at the 3' termini of the different putP fragments. The sense primer was complementary to an appropriate sequence upstream of the newly generated NheI site. The amplified fragments were digested with NheI and an appropriate enzyme upstream of the NheI site and then cloned into plasmids pT7-5/putP(S502)phoA and pT7-5/putP(S502)lacZ digested with the same two restriction enzymes. For expression, the gene fusions were cloned into plasmid pBAD24. Desired gene fusions were identified by restriction analysis and finally sequencing of plasmid DNA (22).

Assay of Alkaline Phosphatase and -Galactosidase Activities-- E. coli CC181 harboring the desired putP-phoA or putP-lacZ fusion in plasmid pT7-5 (or pBAD24) was cultivated in LB medium at 37 °C. In the exponential growth phase, the cells were induced with 0.5 mM IPTG (or 0.2% arabinose (w/v) in case of pBAD24) for 2 h. Alkaline phosphatase and -galactosidase activities were assayed by measuring the rate of hydrolysis of p-nitrophenyl phosphate and o-nitrophenyl--D-galactoside, respectively, by permeabilized cells (13, 28). Assays were performed in triplicate.

Immunological Analysis-- E. coli CC181 harboring plasmid pBAD24 with the desired putP-phoA or putP-lacZ fusion was cultivated and induced as described above. Cells from a 1-ml aliquot of the cultures were harvested by centrifugation and resuspended in SDS lysis buffer (60 mM Tris/HCl, pH 6.8, 2 mM -mercaptoethanol, 10% glycerol (v/v), 2% SDS (w/v), 0.005% bromphenol blue (w/v)). Ten micrograms of total cells protein from each culture were subjected to SDS-PAGE using 7.5% (PutP-LacZ) or 11% (PutP-PhoA) polyacrylamide gels. Proteins were then transferred to a nitrocellulose membrane (0.45 µm pore size), and hybrid proteins were probed with mouse antiserum raised against alkaline phosphatase or -galactosidase followed by incubation with horseradish-peroxidase linked sheep-anti-mouse-IgG antibody.

Protein Determination-- Protein determination was performed using a modification of the method of Lowry (29) with bovine serum albumin as standard.

	RESULTS

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putP-phoA and putP-lacZ Fusion Analysis-- For generation of gene fusions, a unique NheI site was introduced at the 3' end of full-length putP followed by the phoA or lacZ gene. Based on these constructs, further putP-phoA and putP-lacZ fusions were generated by shifting the NheI site (junction site) from the 3' end of putP toward the 5' end by oligonucleotide-directed, site-specific mutagenesis. The gene fusions were expressed in E. coli CC181 from the lac promoter in plasmid pT7-5, and the activity of the reporter proteins was determined in permeabilized cells (Table I). Hybrid proteins with junction sites after Glu-446, Pro-393, Leu-302, Glu-217, and Gly-157 of PutP showed high PhoA (>100 units) and low LacZ (<5 units) activities, indicating a location of these sites at or close to the periplasm. A reverse activity pattern (PhoA < 20, LacZ > 120 units) was observed when the junction site was placed after Ser-502, Thr-426, His-365, Met-259, Gln-190, Lys-121, Gly-116, Glu-102, and Arg-96 of the transporter. The latter results indicate that the C-terminal amino acids of the corresponding PutP fragments are located at or close to the cytosolic side of the membrane. Further analysis of PutP-PhoA hybrid proteins revealed a 5- to 10-fold increase in alkaline phosphatase activity upon shifting the junction site from Arg-96 to Trp-90 in PutP. Hybrid proteins with PutP-PhoA junction sites in the loop between putative TMs I and II showed intermediate alkaline phosphatase activities. The corresponding PutP-LacZ hybrids exhibited relatively high -galactosidase activities. Because the latter results were not conclusive, the topology of the N-terminal part of PutP could not be further defined by the gene fusion method.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Immunological detection of PutP-PhoA hybrid proteins in E. coli CC181. Cells were cultivated as described under "Experimental Procedures." Ten micrograms of total cells protein from each culture were separated by SDS-PAGE using 11% polyacrylamide gels. Proteins were then transferred to a nitrocellulose membrane (0.45 µm pore size), and hybrid proteins were probed with mouse antiserum raised against alkaline phosphatase followed by incubation with horseradish-peroxidase-linked sheep-anti-mouse-IgG antibody.

View larger version (39K): [in this window] [in a new window]   Fig. 3.   Immunological detection of PutP-LacZ hybrid proteins in E. coli CC181. The experimental procedure was as described in Fig. 2 except for 7.5% polyacrylamide gels used for SDS-PAGE and anti--galactosidase antibodies used to probe -galactosidase.

Generation and Properties of Cys-free PutP-- As a prerequisite for site-directed labeling of PutP, all five native Cys residues of the transporter were replaced simultaneously with Ala (Cys-12, -141, -281) or Ser (Cys-344, -349). Cells of E. coli WG170, producing the resulting transporter, catalyzed Na⁺-coupled proline uptake with 50% of the V_max and up to 100% of the steady-state level of proline accumulation of cells containing wild-type PutP. Further kinetic analysis of Cys-free PutP revealed an apparent K_m for proline of 5 ± 0.4 µM compared with 2 ± 0.2 µM wild-type. In contrast to the effect on wild-type PutP, N-ethylmaleimide did not inhibit transport catalyzed by the Cys-free protein. Furthermore, immunological analysis did not reveal significant differences between the amounts of wild-type and Cys-free PutP in the membrane. These results indicate that none of the Cys residues was essential for activity and/or insertion of the protein into the membrane.

Influence of Amino Acid Substitutions on the Activity of Cys-free PutP-- To provide a chemical reactive group at a defined position in the primary structure of the transporter, different amino acids were replaced individually with Cys in Cys-free PutP. Analysis of Na⁺-coupled proline uptake revealed that the substitution of Ile-3, Thr-5, Ala-12, Ser-28, Leu-37, Phe-45, Ser-54, Ser-71, Glu-75, Ile-80, or Lys-91 by Cys has no or only little effect on the initial rates of proline transport (>70% of the Cys-free PutP value) and the steady-state levels of proline accumulation (>75% of the Cys-free PutP value) (Fig. 4). PutP-G39C,³ PutP-R40C, and PutP-M62C exhibited intermediate transport activities with transport parameters corresponding to 30-70% of the Cys-free PutP values. Proline uptake by PutP with Cys in place of Ser-50 or Ala-53 caused a dramatic decrease of the initial rate of proline uptake (<10% of the Cys-free PutP value) (Fig. 4). However, all PutP molecules showed a significant activity indicating that none of the substituted residues is essential for active transport.

View larger version (55K): [in this window] [in a new window]   Fig. 4.   Influence of the replacement of different amino acids in the N-terminal part of Cys-free PutP on Na+-coupled proline uptake. Cells of E. coli WG170 were grown and treated as described under "Experimental Procedures." Transport of L-[U-14C]proline (5 µM final concentration) was assayed in the presence of 50 mM NaCl and 20 mM D-lactate (Na+ salt) as the electron donor at 25 °C under aerobic conditions using a rapid filtration method (10). Initial rates of proline uptake (black columns) and steady-state levels of proline accumulation (gray columns) are expressed as percentage of the corresponding Cys-free PutP value. Cys-free PutP catalyzed proline uptake with an initial rate of 6.5 nmol/min × mg of cell protein to a steady-state level of proline accumulation of 13 nmol/mg of cell protein.

Location of the N-terminus of PutP-- Information on the location of the N terminus of PutP in intact cells was gained by monitoring the accessibility of the sulfhydryl group in single Cys PutP-I3C and PutP-T5C to membrane permeant (BM) and impermeant (SM) sulfhydryl reagents. Since the C terminus of PutP was shown to be located on the cytoplasmic side of the membrane by immunological studies (7) and gene fusion analysis (this study) single Cys PutP-S502C was used for comparison. Cells of E. coli WG170 producing the desired PutP variant were preincubated with SM to block periplasmic Cys residues. Blocked and untreated cells were then incubated with BM and labeled PutP was isolated as described under "Experimental Procedures." Reaction with BM was assessed by Western blotting using streptavidin-horseradish peroxidase. Applying this procedure, Cys at the position of Ile-3, Thr-5, or Ser-502 was shown to react with BM in untreated E. coli WG170 cells (Fig. 5). Furthermore, reaction of BM with PutP-S502C was blocked by preincubation with the membrane impermeant SM only in disrupted cells but not in intact cells. These results confirm a cytoplasmic location of the C terminus. In contrast to these experiments, the thiol groups at positions 3 and 5 of PutP were blocked almost completely already in intact cells by pretreatment with the membrane impermeant SM (Fig. 5). These results indicate a periplasmic location of the N terminus and suggest an uneven number of TMs.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Accessibility of Cys residues in PutP in intact and disrupted cells of E. coli WG170. Cells producing PutP with a single Cys at a given position were incubated with 400 µM BM for 30 min at room temperature. Where indicated (SM +), cells were preincubated with SM for 30 min. Subsequently, cells were disrupted and the transporter was purified as described under "Experimental Procedures." After subjection of the purified transporter to SDS-PAGE (10% polyacrylamide) reaction with BM and the amount of protein was estimated by Western blot analysis using streptavidin-horseradish peroxidase (panel BM) and Coomassie staining (panel P), respectively. A, expression of putP in E. coli WG170 was induced with 0.5 mM IPTG for 45 min, and 1 mM SM was used for labeling. B, expression of putP was induced with 0.5 mM IPTG for 25 min, and SM was used at a concentration of 0.4 mM.

Accessibility of Cys Residues in pL2 and TM II of PutP-- To further investigate the topology of the N-terminal part of PutP, the accessibility of single Cys residues in pL2 and the adjoining putative TMs I and II to sulfhydryl reagents was analyzed. The studies revealed that Cys at the position of Ala-12 in TM I; Ser-28, Gly-39, Arg-40, Phe45, Ser-50, Ala-53, and Ser-54 in pL2; Met-62, and Ile-80 in TM II; or Lys-91 in putative cL3 showed only a low reactivity toward BM or did not react at all (data not shown). Incubation of PutP containing a single Cys at the position of Leu-37 in pL2, or Ser-71 or Glu-75 in TM II, with 200 µM BM resulted in significant labeling of the transporter in intact cells. In addition, reaction of PutP-S71C and PutP-E75C with BM was blocked by preincubation of the cells with membrane impermeant SM. However, the latter compound had relatively little effect on the BM labeling of PutP-L37C in intact cells although SM inhibited reaction of the Cys with BM after cells disruption similar to that observed for PutP-S502C (Fig. 5). The results are consistent with the idea that residues of pL2 form an additional TM (now TM II) thereby placing Leu-37 onto the cytosolic side of the membrane. Furthermore, the accessibility of the thiol groups in PutP-S71C and PutP-E75C to the membrane-impermeant SM indicate a location of these residues in a periplasmic loop region (now pL3). The latter modification requires a shift of the boundaries of TM II (now TM III) by at least eight amino acids toward the C terminus (Fig. 7).

Proteolysis-- The modification of the topological arrangement of PutP proposed by the Cys accessibility analysis was further tested by site-specific proteolysis using the endoproteinase AspN. This enzyme is known to cleave polypeptides before Asp residues (33, 34). For AspN treatment, PutP-wild type was purified and reconstituted into proteoliposomes in an inside-out orientation as described (42). In agreement with the 12-helix motif, AspN treatment of the proteoliposomes yielded peptide fragments within 30 min of incubation that contained Asp-112 (in cL3), Asp-262 (in cL7), or Asp-356 (in cL9) at the N terminus (Figs. 1 and 6 and Table II). In addition, PutP was cleaved before Asp-33 in pL2 while hydrolysis of peptide bonds at potential AspN sites in other periplasmic loops was not observed within 30 min. Only after an extended period of incubation of the proteoliposomes with AspN (6-17 h at 37 °C) and equilibration of the protease with the proteoliposome interior PutP was cleaved before Asp-228 in pL 6 (Figs. 1 and 6, and Table II). In addition, an unspecific cleavage occurred before Leu-302 in pL8. These findings are consistent with the results of the Cys accessibility analysis and support the idea that an additional TM is formed by amino acids of former pL2, thereby moving amino acids around Asp-33 and Leu-37, from the periplasmic to the cytosolic side of the membrane.

View larger version (51K): [in this window] [in a new window]   Fig. 6.   AspN proteolysis pattern of PutP reconstituted into proteoliposomes. PutP was purified and reconstituted into proteoliposomes in an inside-out orientation as described (42). The resulting proteoliposomes were incubated with the endoproteinase AspN at a PutP(FH):AspN ratio of 200:1 (w/w) at 37 °C for the times indicated. Subsequently, the protein was solubilized and subjected to SDS-PAGE (10% polyacrylamide) according to Schägger and Jagow (24) and stained with silver. Protein fragments are marked with a letter corresponding to that used in Table II.

	DISCUSSION

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The 12-helix secondary structure model of PutP (6) is based mainly on hydropathy profile analysis of the amino acid sequence. We have obtained experimental evidence on the topological arrangement of the secondary transporter by applying a gene fusion approach together with a Cys accessibility analysis and site-specific proteolysis. The results of the investigation lead to a new secondary structure model according to which PutP consists of 13 TMs with the N terminus on the outside and the C terminus facing the cytoplasm (Fig. 7). An additional TM is formed by amino acid residues Ser-41 to Pro-65 of former pL2 and TM II.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   New topological arrangement of PutP. The model is based on the analysis of a series of putP-phoA and putP-lacZ fusions, a Cys accessibility analysis and site-specific proteolysis. The nomenclature corresponds to that in Fig. 1.

Analysis of PutP-PhoA and PutP-LacZ hybrid proteins with junction sites in one of the putative loops in the C-terminal part of PutP (former loops 3-13) yields a reciprocal activity pattern for the two reporter enzymes that is in agreement with the prediction based on the hydropathy profile (Table I). Assuming that at least half a TM is required to translocate PhoA across the membrane (13, 27), the jump in PhoA activity detected upon moving the PutP-PhoA junction site from Arg-96 to Trp-90 in former cL3 could be interpreted as a first hint at a location of Trp-90 within a TM. Indeed, a shift of Trp-90 from a cytoplasmic loop into a TM is proposed as a consequence of the results of the Cys accessibility analysis. Surprisingly, however, the jump in PhoA activity is accompanied by constantly high LacZ activities. This unclear activity pattern maybe explained by a loss of basic amino acid residue(s) (i.e. Arg-96) that determine a stable cytoplasmic location of this loop. Thus, deletion of basic amino acids at the N-terminal end of cytoplasmic loops in MalF leads to an increase in PhoA activity of MalF-PhoA hybrid proteins (11, 35). In addition, placement of junction sites into former pL2 in PutP (after Thr-29 or Ser-50) does not provide conclusive results (Table I). To explain these observations, it is speculated that, aside from the loss of topological determinants, the large C-terminal deletion (11 TM are missing) disrupts protein-protein interactions necessary to stabilize, i.e. the proposed new TM II comprising amino acids Ser-41 to Pro-65 with its polar residues (Ser-50, Asp-55, Ser-57) in the membrane.

Because the gene fusion approach does not provide conclusive information, the topological arrangement of the N-terminal part of PutP is further investigated by a Cys accessibility analysis. The generation of a functional Cys-free PutP as a prerequisite for site-directed labeling indicates that Cys residues are not essential for Na⁺-coupled proline uptake. Similarly, Cys residues are shown to be completely replaceable in other secondary transporters like, i.e. the lactose permease (LacY) or melibiose permease (MelB) of E. coli (36, 37). Furthermore, individual substitution of a variety of amino acids in the N-terminal part of Cys-free PutP by Cys had no or only little effect on transport activity, indicating also a structural integrity of the corresponding PutP molecules. The substitution of Arg-40, Ser-50, Ala-53, and Met-62 by Cys results in reduced proline uptake rates (Fig. 4). In case of Ser-50, Ala-53, and Met-62, the effect on transport activity might be because of a location close to Asp-55 and Ser-57, which are proposed to be involved in ligand binding (9, 10). Arg-40 is conserved within the members of the SCF (4). A preliminary substitution analysis indicates that this basic residue affects Na⁺-dependent proline binding.⁴

In intact cells, Cys residues placed at or close to the N terminus (I3C, T5C) of PutP are readily accessible to membrane permeant (BM) and impermeant (SM) sulfhydryl reagents. Under these conditions, a Cys at the C terminus of the protein (S502C) reacts only with the membrane permeant BM but becomes accessible to the highly polar SM after cell disruption (Fig. 5). This accessibility pattern can only be explained by a location of the N and C terminus on the periplasmic and cytosolic side, respectively, of the membrane. This conclusion contradicts the 12-helix motif and implies the existence of an uneven number of TMs. Furthermore, the high accessibility of sulfhydryl groups placed at the position of Ser-71 or Glu-75 (former TM II) to BM and SM in intact cells suggests a location of these residues in a periplasmic loop. This modification requires a shift of the boundaries of former TM II by at least eight amino acids toward the C terminus of PutP. Contrary to the general high accessibility of sulfhydryl groups particularly at or close to the protein termini, Cys residues at most of the positions in former pL2 and adjacent TMs tested show little or no reaction with sulfhydryl reagents independent from the membrane orientation (Fig. 5). The low reactivity of the sulfhydryl group might be because of a hydrophobic environment (38) or a location that buries the sulfhydryl group within the protein. These results are consistent with the idea that residues of former pL2 are located in a transmembrane domain. This idea also explains the finding that antibodies raised against a synthetic peptide corresponding to the hydrophilic segment between TM I and II does not react with the transporter (3). Strong support for the formation of an additional TM in this region comes from the fact that the membrane impermeant SM blocks reaction of PutP-L37C with BM completely only after cell disruption but not in intact cells similar to that observed for PutP-S502C. These results suggest a shift of Leu-37 from the former pL2 into a cytosolic loop. The formation of an additional TM in this region of the protein is confirmed by the pattern of PutP fragments obtained after AspN proteolysis of transporter reconstituted into proteoliposomes in an inside-out orientation. Thus, the endoproteinase readily cleaves the polypeptide not only in the known cytoplasmic loops (cL3, cL7, and cL9 according to the 12-helix motif) as expected but also before Asp-33 in the former pL2. In conclusion, the data suggest a placement of amino acids Arg-27 to Arg-40 from former pL2 into a cytoplasmic loop (now cL2), whereas amino acids Ser-41 to Pro-65 form an additional TM (TM II).

The latter modification shifts amino acid residues proposed to be involved in ligand binding (Asp-55 and Ser-57) (9, 10) from former pL2 into the new TM II. This topological arrangement is in agreement with the fact that residues suggested to be involved, i.e. in binding of the coupling ion in transporters such as LacY and MelB, are located in TMs (31, 39). On the other hand, Glu-75 which is not important for PutP function (10), is shifted from the energetically unfavorable position in former hydrophobic TM II into pL3 (former pL2).

Although it is clearly not the aim of this study to investigate the mechanism of insertion of PutP into the cytoplasmic membrane, it is tempting to speculate how the transporter without a leader (signal) sequence translocates its N terminus across the membrane. Analysis of hybrid proteins generated from E. coli leader peptidase and the phage Pf3 coat protein reveals that short tails, which do not contain positively charged residues, are efficiently translocated independent of the Sec machinery (40). Depending on size and presence of acidic amino acid residues in the N-terminal tail, the translocation might be energized by a proton motive force (41). In the case of PutP, the short N-terminal tail and the adjoining first TM do not contain either positively or negatively charged residues. Instead, this part of the protein is highly hydrophobic. Therefore, it is possible that the energy required to translocate the short N-terminal tail of PutP across the membrane is gained from the transition of hydrophobic TM I from the aqueous phase into the apolar phase of the bilayer.

The new secondary structure model of PutP is in agreement with the recently proposed topological arrangement of TMs I to XIII of SGLT1, which is based on an N-glycosylation study (8). The results support the idea of a common topological motif for members of the SCF, according to which i.e. the bacterial transporters for proline (PutP) and pantothenate (PanF) and the mammalian Na⁺/I transporter are composed of 13 TMs (5). Transporters with a C-terminal extension (i.e. the human SGLT1 and myoinositol transporter (SMIT1)) are proposed to have an additional 14th TM (5, 8). In this context, it is interesting to note that MelB, which is not homologous to PutP and belongs to the family Na⁺/galactoside transporters (SGF) (4), is composed of 12 TMs as revealed by a melB-phoA fusion analysis (31). Thus, the 13-helix motif might be a characteristic feature of members of the SCF.