©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The NH-terminal Half of the Tn10-specified Tetracycline Efflux Protein TetA Contains a Dimerization Domain (*)

(Received for publication, May 16, 1995; and in revised form, June 30, 1995)

Laura M. McMurry (§) Stuart B. Levy

From the Center for Adaptation Genetics and Drug Resistance and the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The 43.1-kDa tetracycline-cation/proton antiporter TetA from Tn10 comprises two equal-sized domains, alpha and beta (amino-terminal and carboxyl-terminal halves, respectively). An inactivating mutation in the alpha domain can complement a mutation on a second polypeptide in the beta domain to restore partial tetracycline resistance in bacterial cells, suggesting that intermolecular interactions permit this transport protein to act as a multimer. In the present studies, multimer formation was examined in mixtures of dodecylmaltoside extracts of membranes from Escherichia coli cells containing different TetA derivatives. TetA, TetA, and TetA were each fused genetically to a six-histidine carboxyl-terminal tail. The ability of these fusions, immobilized on a nickel affinity column, to bind wild type TetA or other Tet fusions was determined. An interaction between alpha domains on different polypeptides which resulted in multimerization was seen. The binding was specific for Tet protein and did not occur with other membrane proteins or another polyhistidine fusion protein. No alpha-beta interactions were detected by this method, although they are postulated to occur in the intact cell based on the alpha-beta genetic complementations. A dimeric model for TetA having intermolecular alpha-alpha and alpha-beta interactions is presented.


INTRODUCTION

TetA(B), a cytoplasmic membrane protein encoded by Tn10, is a member of a family of related tetracycline efflux proteins in Gram-negative bacterial cells(1, 2) . It mediates resistance to tetracyclines by pumping a cation-tetracycline complex across the membrane outwardly in an electroneutral exchange for an inwardly moving proton(3, 4, 5, 6) . Experiments with a collection of point mutations had shown that inactivating mutations in the first half of the protein complemented those in the second half in cells containing both polypeptides(7, 8) . Complementation also occurred with protein fragments(9) . However, each half of TetA did not have a unique function completely independent of that of the other half, since full or even half-resistance was rarely restored in complementations, even in cases where the presence of both polypeptides was confirmed. These results suggested that synergistic physical interaction between the two halves of the protein was required for resistance, and that such interaction could occur intermolecularly in a dimeric or higher multimeric state.

Further evidence for the required interaction between the two halves and for dimerization came from Tet protein chimeras. The sequences of the related tetA genes from the family of tetracycline resistance determinants predicts that each TetA protein has two sets of six putative membrane-spanning alpha-helices separated by a putative large cytoplasmic loop(2, 9, 10, 11, 12) . TetA proteins from classes A and C are more closely related (78%) than either are to the class B (Tn10) protein (45%)(1) . An ``A/C'' chimera, containing the first (alpha) half from class A and the second (beta) half from class C, was active in expressing tetracycline resistance, whereas a B/C or C/B chimera was not(13) . Evidently the alpha and beta halves functioned together only if they were related closely enough. The B/C and C/B chimeras together in the same cell, however, showed about 20% complementation of tetracycline resistance, indicating multimer formation(13) . alpha-beta interaction was also suggested by the ability of the cloned alpha half to stabilize the cloned beta half when both were present on separate polypeptides in the same cell(14) . Complementation occurred in this case also.

The present work was designed to determine whether TetA extracted from the cell existed as a multimer. We genetically fused six histidines to the carboxyl terminus of TetA, TetA, and TetA of class B. The ability of such a ``6H'' fusion to bind different Tet protein molecules was measured using Ni affinity chromatography.


EXPERIMENTAL PROCEDURES

Construction and Description of Plasmids

See Table 1for summary of plasmids and Fig. 1for diagrams of protein constructs.




Figure 1: Tet protein constructs. Drawings are approximately to scale, except for an enlargement of the T7tag and the 6H tail. [Xa] indicates a protease factor Xa cleavage site.



pACT7 (encoding T7 RNA polymerase regulated by the lacUV5 promoter) (Kan,^R p15A origin). This plasmid (16) was used in trans with pET21b-Tet6, pLY17, and pLY22.

pET21b-Tet6 (encoding Tet-6H, 45.6 kDa). This plasmid, derived from pET21b (Novagen), provides a T7 promoter and lac operator regulating Tet-6H. It also provides lacI, the gene for the lac repressor, and was used in conjunction with pACT7. Its construction has been described. (^1)Tet-6H comprises an initial methionine followed by (in order) an 11 residue ``T7 tag,'' TetA (minus the initial methionine), leucine, glutamate, and then the six histidines (6H).

pLY17 (encoding Tetalpha-6H, 24 kDa). A 0.6-kilobase EcoRI-XhoI fragment representing the beta half of TetA was deleted from pET21b-Tet6. The 5` ends were filled in with Klenow DNA polymerase prior to ligation. TetA was thereby in frame with the polyhistidine tail encoded 3` to the XhoI site. Loss of the 0.6-kilobase fragment was confirmed by loss of the ScaI site within it, and by the 6.0 kilobase size of the resulting plasmid. pLY17 was used in combination with pACT7.

pLY22 (encoding Tet beta-6H, 24 kDa). The same tetA PCR product used to make pET21b-Tet6 was restricted with EcoRI (in the central loop of TetA) and XhoI (at the end of TetA) and cloned into identically restricted pET21b. This put the TetA domain in-frame with both the upstream ``T7 tag'' and the downstream polyhistidine tail encoded by pET21b. pLY22 was used in combination with pACT7.

pMalc-Tet1 (encoding MalE-Tet, 86 kDa). A tetA PCR product having BamHI sites on each end was restricted with BamHI and cloned into BamHI-restricted pMAL-C2 (New England BioLabs). This created an in-frame fusion between maltose-binding protein MalE (missing its signal sequence) and the (cytoplasmic) amino terminus of the intact TetA, with an intervening 28-amino acid linker containing a factor Xa cleavage site. The fusion protein was regulated by P together with the lacI^Q gene on the plasmid. Transformants were selected on 20 µg/ml tetracycline without IPTG. (^2)The strain synthesized several species of fusion protein, the largest and most abundant migrating at 70 kDa. The largest species was probably the intact fusion protein since it reacted with antiMalE, it bound to an amylose column by the MalE domain, and it reacted with antiCt to the carboxyl terminus of TetA.^3 The fusion protein was cleavable between MalE and TetA by factor Xa, as expected. (^3)

pQEGH12 (encoding 6H-IICB). This plasmid (17) was used in strain ZSC112L(17) , which has a glucose transporter ptsG mutation. The fusion protein is regulated by the ptsG promoter and is expressed constitutively in ZSC112L.

pRAR1020 (encoding wild type TetA of class B) and pRAR1027 (encoding C/B chimera of TetA). Both have the tet promoter regulated by TetR(13) . They were used in strain BC32(13) .

pRKH21 (encoding Tet279-LacZ, approximately 144 kDa). This plasmid (15) in strain RV200 (15) is regulated by P(L). It is accompanied by pcI857, a compatible Kan^R plasmid encoding the temperature-sensitive cI857 repressor(15) . The fusion protein was induced by a shift in temperature from 30 to 42 °C. pRKH21 had resulted from a spontaneous fusion between TetA and LacZ (15) . Junction sequencing has now been performed (DNA Sequencing Center, Division of Endocrinology, New England Medical Center); the junction is at base pair 836 of TetA, fusing leucine 279 (at the amino terminus of the putative ninth transmembrane helix) of TetA to proline 8 of LacZ.

R222 (encoding wild type TetA of class B). This large, naturally occurring, very low copy number plasmid bears Tn10, which carries the complete class B tet determinant including the tet repressor(1, 18) . It is compatible with both ori pMB1 and ori p15A plasmids. Expression of TetA was induced by tetracycline.

Strains, Medium, Chemicals, and beta-Galactosidase Assays

Unless otherwise specified, the host strain of Escherichia coli was DH5alpha (Life Technologies, Inc.; relevant loci recA, hsdR) and cells were grown in LB (per liter: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl) at 37 °C. Antibiotics were purchased from Sigma, except that 5a,6-anhydrotetracycline was prepared by Mark Nelson of this laboratory. beta-Galactosidase (LacZ) assays were performed as described (19) . The LacZ assay was used to calculate picomoles of Tet279-LacZ (as monomers) assuming 1 pmol of LacZ hydrolyzes 34.9 pmol of ONPG/min at 28 °C (see (19) ). The specific LacZ activity of Tet279-LacZ was within a factor of two of that expected for LacZ itself,^3 showing that the Tet moiety was not detrimental to the tetramerization of LacZ required for activity. A plasmid bearing the gene for a 6H-LacZ fusion protein cloned into pET14b was provided by Novagen in host BL21(DE3) (see below); this strain was used to prepare a membrane-free cell lysate containing 6H-LacZ.

Complementation Assays

The host strain was BL21(DE3) (Novagen), which bears a chromosomal T7 RNA polymerase gene regulated by the lac repressor. This strain was transformed with pLY22 (Ap^R, bearing the Tetbeta-6H gene and having a pMB1 origin of replication). A second mutant plasmid known to encode an active class B TetA domain and which had the compatible p15A origin of replication and encoded Cm^R was also introduced; the second plasmid was either pLR1097 (bearing wild type tetA with a deletion in the beta-domain(9) ) or pRAR1032 (bearing the B/C chimeric gene(13) ). In these second plasmids the mutant tetA gene was regulated by the tet repressor TetR; non-inhibitory autoclaved chlorotetracycline (10 µg/ml) was used for induction(7) . Resistance to tetracycline was measured by gradient plates (7) containing the autoclaved chlorotetracycline and 20 µM IPTG.

Preparation of Membrane Extracts, Use of Ni-NTA Columns, and Subsequent Analyses

Growing cells bearing the appropriate plasmids were induced (at A = 0.8) for 1.5-3 h with the appropriate agents (100 µM IPTG for pET21b-Tet6, pLY17, pLY22, pMalc-Tet1; 42 °C for pRKH21; tetracycline at 2 µg/ml for R222; 0.02 µg/ml anhydrotetracycline (another gratuitous tet inducer(20) ) for pRAR plasmids. Cells were harvested and used immediately or stored at -80 °C. Cells were lysed by sonication at A = 100 in 50 mM sodium phosphate, 2 mM MgCl(2), 100 µg/ml lysozyme, pH 7.4. Membranes were sedimented (105,000 times g, 35 min, TLA 100.3 rotor (Beckman)) and resuspended in 10 mM sodium phosphate, pH 7.2 (0.3 ml/original 100 A units). n-Dodecyl-beta-D-maltoside (Anatrace, 8% alpha) was added to 1.5%. After 30 min of occasional mixing (4 °C), unsolubilized membranes were sedimented as before and discarded. The detergent extracts were stored at -80 °C.

Two different dodecylmaltoside extracts (usually 10-50 µl of each) were combined if desired to allow ``mixed multimers'' to form. After 30 min of occasional mixing at 4 °C, 1/7 volume of 8-fold concentrated column buffer was added (column buffer final concentration was 50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 5 mM imidazole, 0.03% dodecylmaltoside). Small (0.1) ml columns of Ni-NTA (Qiagen) (Ni bound to nitrilotriacetate immobilized on Sepharose CL-6B), were prepared in Pasteur pipettes and washed in column buffer. The samples were loaded onto the columns (50 µl every 5-7 min) and washed with column buffer (0.2 ml, 2 min, times6). When desired, an elution in column buffer at 40 mM imidazole was then performed at the same rate. Finally, an elution in column buffer at 1 M imidazole (pH adjusted to 8) was done (0.08 ml, 5 min, times3). In some cases eluates were used for dot-blots or assayed for LacZ. Otherwise they were precipitated with trichloroacetic acid (10% trichloroacetic acid, 15-30 min at 4 °C, centrifuged 15,000 times g, 10 min), dissolved in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (21) , and processed by SDS-PAGE (9 or 10%) 0.75-mm thick minigels. Some gels were then electroblotted onto Immobilon P (Millipore) and probed with antiCt or antiTet antiserum, followed by I-Protein A, as described.^1 AntiCt was specific for the carboxyl-terminal 14 amino acids of TetA(22) ; its reaction with Tet-6H was less than 2% of that with wild type TetA (determined by Molecular Dynamics Computing Densitometer evaluation of x-ray film exposed to immunoblots of SDS-PAGE gels), presumably due to the altered carboxyl terminus of Tet-6H. AntiTet reacted with an epitope between residues 127 and 201 of TetA and reacted equally well with Tet-6H.^1 Some gels were simply stained with Coomassie Brilliant Blue R-250 and dried in a Tut's Tomb frame between two sheets of Ultraclear Cellophane (both from Idea Scientific Co.). Protein bands on stained SDS-PAGE gels were quantitated using the Computing Densitometer; glyceraldehyde-6-phosphate dehydrogenase or ovalbumin served as standards. The total amount of a 6H fusion in extracts was defined as the sum of the amounts in the 40 mM and 1 M imidazole eluates from Ni-NTA.


RESULTS

Formation of Mixed Multimers between Wild Type TetA and Tet-6H

DH5alpha cells containing pACT7 plus (i) the vector containing the tet-6H gene (pET21b-Tet6), (ii) the vector (pET21b) alone plus the wild type tetA gene on Tn10 (on naturally occurring plasmid R222), or (iii) pET21b-Tet6 plus R222, were induced with IPTG. The wild type gene in the strains with Tn10 was also induced with tetracycline. Dodecylmaltoside detergent extracts of membranes from all cells were prepared. In one case, extracts from i and ii were mixed in equal volumes for 5 min. The extracts were passed through a Ni-NTA column to bind Tet-6H and co-associated proteins. Bound proteins were eluted with 1 M imidazole, and both the original extracts and the bound proteins were examined for wild type TetA and Tet-6H by immunodot blot using antisera and I-Protein A.

The original extracts loaded onto the Ni-NTA columns were analyzed first. Probing with antiCt antiserum, which reacts with TetA but not with Tet-6H, revealed that the amount of TetA in the two strains bearing Tn10 was similar (Fig. 2, column A, rows 1 and 3). As expected, the extract from the strain with only Tet-6H (without Tn10) showed a low, probably host cell background, reaction with antiCt (Fig. 2, column A, row 2). Use of antiTet antiserum, which reacts similarly with both TetA and Tet-6H, showed that cells synthesized less wild type TetA than overproduced Tet-6H (Fig. 2, column B, row 1versusrow 2), as shown before.^1


Figure 2: Detection of wild type TetA and Tet-6H before and after Ni-NTA chromatography of dodecylmaltoside membrane extracts (dot-blot). Rows 1-3, extracts applied to Ni-NTA; (from 0.1 A units of cells): wild type TetA (1), Tet-6H (2), or both (3) from the same cell. Rows 4-7, 1 M imidazole eluates from Ni-NTA (from 0.5 A units of cells): wild type TetA (4), wild type TetA applied after Tet-6H (5), wild type TetA and Tet-6H from the same cell (6), or wild type TetA plus Tet-6H premixed before application (7). Blots were probed with antiCt (A) or antiTet antiserum (B).



The samples which bound to the Ni-NTA columns were then analyzed. Use of antiCt demonstrated that little wild type TetA bound to Ni-NTA in the absence of Tet-6H (Fig. 2, column A, row 4). On the other hand, in the presence of Tet-6H bound to the resin, TetA binding was clearly detectable. This was true whether the two versions of Tet had been synthesized in the same cell (Fig. 2, column A, row 6), or came from separate cells but were mixed together prior to loading onto Ni-NTA (Fig. 2, column A, row 7). If Tet-6H was loaded first, followed by TetA, TetA was still bound (Fig. 2, column A, row 5). Use of antiTet confirmed that similar amounts of Tet-6H were bound to Ni-NTA in all cases (Fig. 2, column B, rows 5-7). From Coomassie-stained gels this was estimated to be about 5 µg (110 pmol). To evaluate the contribution of the -6H region of Tet-6H to this binding, we mixed a membrane-free cell lysate containing 6H-LacZ with a membrane extract containing TetA. There was excellent binding of 6H-LacZ to Ni-NTA (about 100 µg, or 950 pmol of monomers) but no binding of TetA (data not shown). Therefore, the binding of TetA to Tet-6H was not via the polyhistidine region.

Formation of Mixed Multimers of Tet279-LacZ with Tet-6H, but Not with 6H-IICB

We also tested Tet279-LacZ, encoded by pRKH21. This fusion contained the alpha portion of TetA plus the first two putative transmembrane helices and associated extramembrane loops of the beta domain. In this case the Tet moiety could be quantitated by LacZ activity. An extract containing 480 pmol of Tet279-LacZ was used alone or mixed with one containing 240 pmol of Tet-6H and passed over a Ni-NTA column. In the absence of Tet-6H, 8.7 pmol of Tet279-LacZ was bound to the column, while 37 pmol was bound in the presence of Tet-6H. These results indicated a possible association between Tet279-LacZ and Tet-6H.

In a second experiment we included an extract containing 6H-IICB as a control to see if Tet would stick nonspecifically to another membrane protein immobilized on the Ni-NTA column. 6H-IICB is an E. coli membrane protein of 8 putative transmembrane segments which transports glucose as part of the phosphotransferase system and which has a polyhistidine head at the amino terminus(17) . As a final control to measure background binding of Tet279-LacZ, we also included an extract from cells containing no polyhistidine fusion protein. We found that the background binding of Tet-279-LacZ (Table 2) was 1.5% of that applied, similar to that in the first experiment (1.8%). Even though three times as much 6H-IICB as Tet-6H was applied and bound to the Ni-NTA column, only one-tenth as much net Tet279-LacZ was bound to it as to Tet-6H (Table 2). These results showed that the binding of Tet279-LacZ was specific for Tet-6H and did not occur with an unrelated integral membrane protein. The results also showed that the Tet-Tet interaction might be between the alpha domains of the two different polypeptides, or between an alpha and a beta domain, but possibly not between the beta domains, since Tet279-LacZ had the entire alpha domain but only the first two helices and associated loops of the beta region.



beta-beta Interactions Were Not Required for Multimer Formation

In Tetalpha-6H the beta domain is completely absent. If indeed beta-beta interaction was not required for Tet-Tet binding, Tetalpha-6H should be able to bind a Tet protein containing both alpha and beta domains. As its prospective partner, we used the MalE-Tet fusion in which the entire TetA protein was fused genetically via a linking region to the carboxyl terminus of MalE. The MalE-Tet fusion could be detected by its large size (migrating at 70 kDa) on Coomassie-stained SDS-PAGE. We again included 6H-IICB as a negative control. Dodecylmaltoside extracts containing MalE-Tet were mixed with extracts containing no fusion, IICB, or Tetalpha-6H and passed over a Ni-NTA column. Sequential elutions were done at 40 mM and 1 M imidazole.

The MalE-Tet polypeptide was not bound in absence of polyhistidine fusion (Fig. 3, lanes 1) nor to 6H-IICB (Fig. 3, lanes 2). MalE-Tet was, however, bound to Tetalpha-6H (Fig. 3, lanes 3). These results extended the previous finding with Tet279-LacZ, showing that an alpha domain of TetA was sufficient for binding to full-length Tet. Therefore, beta-beta interactions were not necessary.


Figure 3: Association of MalE-Tet with Ni-NTA-bound Tetalpha-6H or 6H-IICB. Dodecylmaltoside extracts of membranes from 6 A units of cells containing MalE-Tet were mixed with the same amount of a second dodecylmaltoside extract (see below) and loaded onto Ni-NTA columns. Proteins which bound and eluted at 40 mM or 1 M imidazole are shown by SDS-PAGE (1.4 A unit/lane). The second extract contained: lanes 1, no fusion (host extract); lanes 2, 6H-IICB; lanes 3, Tetalpha-6H. Molecular mass standards (kDa) are in lane S. The molar ratio of MalE-Tet Tetalpha-6H was about 0.9 for the material loaded, 0.5 for the 40 mM eluate, and 0.09 for the 1 M eluate. The molar ratio of MalE-Tet 6H-IICB loaded was 2.0.



alpha-beta Interactions Did Not Contribute to Multimer Formation

The genetic complementation which occurred between alpha and beta domains on different polypeptides (7, 8, 9, 13, 14) had suggested that these two domains could interact physically. We looked for such an alpha-beta interaction with Tetalpha-6H. We mixed extracts containing either B/B (that is, wild type TetA from the class B tetracycline resistance determinant) or C/B (a chimera containing the alpha domain from class C, the beta domain from class B) (see Fig. 1) with an extract containing Tetalpha-6H. A MalE-Tet/Tetalpha-6H mixture was included as a positive control. Since B/B and C/B were not made by cells in sufficient quantities to be detected by Coomassie-stained SDS-PAGE, all proteins bound to Tetalpha-6H were detected by immunoblot of SDS-PAGE gels. The results are shown in Fig. 4. Binding of Tetalpha-6H to Ni-NTA was verified using antiTet (Fig. 4, lane 1"), as was binding of MalE-Tet to Tetalpha-6H (data not shown). Using antiCt, binding of B/B to Tetalpha-6H was seen (Fig. 4, lane 2`), as expected from the earlier experiments. However, little binding of C/B was seen (Fig. 4, lane 3`), even though much more C/B than B/B had been applied to the Ni-NTA columns, (Fig. 4, lane 3versuslane 2). These results suggested that the alpha domains of classes B and C interacted poorly. They also unexpectedly implied that there was little interaction between the beta domain of class B (on C/B) with the alpha domain of class B (on Tetalpha-6H).


Figure 4: Association of B/B but not C/B with Ni-NTA-bound Tetalpha-6H. The method of Fig. 3was employed except that immunoblots are shown. An extract representing 9 A units of the Tetalpha-6H strain was mixed with an extract representing 24 A units of the other strains. Each SDS-PAGE lane contains 0.075 A units (loaded onto Ni-NTA) or 1.7 A units (Tetalpha-6H, bound to Ni-NTA). Lanes 1-3 are the loaded samples: 1, host with no plasmid; 2, B/B; 3, C/B. Lanes 1`-3` are the bound samples, all containing Tetalpha-6H plus: 1`, host with no plasmid; 2`, B/B; 3`, C/B. Blots were probed with antiCt to reveal B/B and C/B or with antiTet to reveal Tetalpha-6H.



Use of Tetbeta-6H to Confirm Lack of alpha-beta and beta-beta Interactions: Observation of alpha-alpha Interactions

The fact that C/B did not bind to Tetalpha-6H suggested that the alpha and beta domains of class B did not interact, despite genetic data to the contrary. It was possible that the C/B protein was for some reason in a nonbinding conformation after extraction. Therefore, we constructed a polyhistidine fusion having only the beta domain of class B for binding studies. This fusion was designated Tetbeta-6H.

The Tetbeta-6H protein was identified on Coomassie-stained SDS-PAGE gels of Ni-NTA-bound protein as a band migrating slightly more slowly than Tetalpha-6H and not present in fusionless host cells (data not shown). Quantification of these bands indicated that cells containing pLY22, encoding Tetbeta-6H, produced only about 2% as much fusion protein as did cells bearing pLY17 (encoding Tetalpha-6H).

The functionality of Tetbeta-6H encoded by pLY22 was assayed in vivo by the ability to complement TetA having a mutated beta domain encoded on a compatible plasmid. Two different compatible mutant plasmids were tested in trans with pLY22 (see ``Experimental Procedures''). No plasmid offered tetracycline resistance alone (minimal inhibitory concentration of tetracycline <0.2 µg/ml). pLY22 complemented both mutant plasmids to give tetracycline resistance (minimal inhibitory concentration >10 µg/ml). Therefore, the Tetbeta-6H domain was functional, at least in the intact cell expressing a complementing Tet protein.

Biochemical studies were then performed. Extracts of cells containing Tetbeta-6H or Tetalpha-6H were loaded onto Ni-NTA columns at volumes which contained approximately equal amounts of each fusion protein. A volume of a host extract identical to the volume used for Tetbeta-6H was also loaded onto a column as a control. Then an extract containing Tet279-LacZ (or MalE-Tet in one case) was passed over the columns. Binding of Tet279-LacZ to the host extract column was considered as background. The molar ratio of Tet279-LacZ to 6H fusion applied to the column was about 2. The net molar ratio eluting at 1 M imidazole was about 0.038 for Tetalpha-6H but only 0.002 for Tetbeta-6H. Tetbeta-6H also bound no MalE-Tet observable on SDS-PAGE even though the MalE-Tet Tetbeta-6H molar ratio applied to Ni-NTA was about 7 (data not shown). MalE-Tet was bound to Tetalpha-6H in the presence of Tetbeta-6H extract, as expected, although the required large volume of Tetbeta-6H extract increased background bands on SDS-PAGE, making quantification difficult (data not shown). These results showed that Tetbeta-6H was neither able to bind Tet containing both alpha and beta domains (in MalE-Tet), nor able to bind the alpha domain in Tet279-LacZ. Our earlier failure to see alpha-beta interaction in extracts was therefore confirmed, as was the absence of beta-beta interaction. The results also suggested that alpha-alpha interactions between Tet279-LacZ and Tetalpha-6H were responsible for Tet multimerization in this assay.

Comparison of alpha Binding to alpha with alpha Binding to alpha+beta in Multimer Formation

It appeared that binding between different Tet proteins in extracts could occur solely by alpha-alpha interactions. To see whether alpha-alpha interactions might be fortified by additional alpha-beta ones, we compared binding of Tet279-LacZ to Tetalpha-6H with that to Tet-6H. In this experiment the amount of Tetalpha-6H recovered from cells and applied to the Ni-NTA column was five times the amount of Tet-6H. 180 pmol of Tet279-LacZ was mixed with Tetalpha-6H or Tet-6H and loaded onto a Ni-NTA column. Summation of values for elutions at 40 mM and at 1 M imidazole gave 37 pmol of Tet279-LacZ co-eluting with the 870 pmol of Tetalpha-6H bound to the column, while 8.8 pmol of Tet279-LacZ co-eluted with the 170 pmol of Tet-6H bound. On a molar basis, Tet-6H bound about the same amount of Tet279-LacZ as did Tetalpha-6H. Therefore, the additional alpha-beta interactions in Tet-6H were not helpful to the association. We concluded that, except for a possible role for the bit of the beta domain in Tet279-LacZ, the in vitro binding of one Tet polypeptide to another seen using polyhistidine fusions and Ni-NTA columns must occur by interactions between two or more alpha domains.


DISCUSSION

We report here initial biochemical studies on the quaternary structure of the tetracycline-cation/proton antiporter TetA. From genetic data described earlier we had expected that TetA protein was capable of functioning in vivo as a dimer or other multimer. We had also imagined that the interaction would be between the alpha and beta domains. Earlier we had found that a small proportion of either the B/B protein or the C/B chimeric protein could be cross-linked into a immunoreactive band having the molecular weight of a dimer, but that little coimmunoprecipitation of one Tet polypeptide by antibody specific for another occurred, with or without cross-linking.^3

In the present work we explored another biochemical method to test the multimer hypothesis. Immobilized Ni can be used to bind proteins having a polyhistidine region(23) . By the use of TetA-polyhistidine fusion proteins, we were able to clearly show specific association between two distinguishable Tet protein molecules from cell membrane extracts. These heteromultimers between two Tet species formed simply upon mixing a dodecylmaltoside extract containing one Tet species with an extract containing the other. Apparently, in the mixtures the original homomultimers have readily dissociated (within minutes) into subunits, followed by rapid association with a heterologous subunit into a multimer which was stable enough to detect. Presumably, the rates of both association and dissociation are high, while the former exceeds the latter to account for multimer stability on Ni-NTA. Binding did not occur between TetA and another polyhistidine fusion of an integral membrane transport protein, 6H-IICB, nor did other cell membrane proteins associate with Tet-6H to any notable extent, as was evident by its purity following Ni-NTA chromatography.^1 Therefore, we believe the Tet-Tet interactions to be specific.

Unexpectedly the crucial interaction in formation of Tet multimers in vitro appeared to be between two (or more) alpha domains, rather than between an alpha and a beta domain. However, in intact cells, besides the genetic data there are also physical indications of alpha-beta interaction. The amount of a polypeptide comprising the beta half of TetA in whole cells was increased 1.5-fold or more by the presence of the alpha half polypeptide, suggesting a physical interaction of the two (14) . We have observed that the amount of full-length B/C chimera in cells (normally very low) increased notably if the C/B chimera was present in the same cell^3; a simple explanation for those results could be that the C/B protein formed a multimer with the B/C protein via same-class alpha-beta interactions and stabilized it, although other explanations are possible. The fact that in the present work we did not see alpha-beta interactions after the Tet protein had been extracted may mean that the beta domain for the C/B and Tetbeta-6H constructs did not have native binding properties in our extracts or under our assay conditions. However, recent circular dichroism studies on purified full-length Tet-6H, at least, show that both alpha and beta domains in that polypeptide do have approximately the expected alpha-helical content.^3

A TetA dimer may be held together both by alpha-alpha interactions (seen in the present study for proteins extracted from membranes by dodecylmaltoside) and by alpha-beta interactions (not apparent using extracts, but inferred from genetic and biochemical studies in whole cells). A model in which both alpha-alpha and alpha-beta interactions occur within a TetA dimer is shown in Fig. 5. During complementation of B/C with C/B in vivo, the alpha-alpha interactions would presumably not occur, but the alpha-beta ones would. Two active sites/wild type dimer, or one/ complementing dimer, would be expected. Our model might explain why Tetalpha-6H was found in cells at high concentrations similar to those of the full-length fusion Tet-6H, while the amount of Tetbeta-6H was 50-fold lower, since the model allows alpha to bind to alpha (or to beta), and such associations may prevent degradation. Absence of self-association for beta, as modeled, would lead to degradation of beta when alone in a cell.


Figure 5: Model of possible Tet dimer and monomer. The plane of the page represents that of the membrane surface. Hypothetical active site is denoted by an times. A ribbon representing the large cytoplasmic loop connects the alpha and beta domains within a single polypeptide strain. This loop is located in the cytoplasm above the membrane surface.



The proposed structure of the dimer differs from that proposed for a monomer both because of the additional alpha-alpha interactions and the altered topology of the central loop (Fig. 5). A monomer of TetA has both the domains (alpha and beta) required for activity, and we cannot discount the possibility that a complex consisting of only one alpha and one beta domain is capable of functioning. On the other hand, even when these two domains are tethered together in a normal monomer, considerable interaction with other such monomers must be allowed in vivo, since intermolecular complementation can occur. Self-association of monomers into dimers might be favored in the two-dimensional membrane bilayer even more than the considerable degree seen here in detergent extracts.

Multimerization provides possibilities for scaffolding, interfaces, and allostery. Some other membrane transport proteins of the same superfamily (24) as TetA are known to occur as multimers, including the facilitated glucose transporter GLUT1(17, 25) , the erythrocyte anion exchanger Band 3(26) , and the Na/glucose cotransporter (27) . The relationship between these multimerizations and function is uncertain(25, 28) , and at least one example exists (the lactose permease, LacY) in which the transporter almost certainly functions as a monomer(29) . Our results strengthen the concept that the mechanism of action of TetA involves a multimeric state.


FOOTNOTES

*
This work was supported by Grant AI30646 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 617-636-4288; Fax: 617-636-0458.

(^1)
Aldema, M. L., McMurry, L. M., Walmsley, A. R., and Levy, S. B.(1995) Mol. Microbiol., in press.

(^2)
The abbreviations used are: IPTG, isopropyl-1-thio-beta-D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; ONPG, o-nitrophenyl-beta-D-galactopyranoside.

(^3)
L. M. McMurry and S. B. Levy, unpublished data.


ACKNOWLEDGEMENTS

We thank B. Erni for plasmid pQEGH12 and strain ZSC112L, A. Yamaguchi for antiCt, and Mark Nelson for anhydrotetracycline.


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