(Received for publication, May 16, 1995; and in revised form, June 30, 1995)
From the
The 43.1-kDa tetracycline-cation/proton antiporter TetA from
Tn10 comprises two equal-sized domains, and
(amino-terminal and carboxyl-terminal halves, respectively). An
inactivating mutation in the
domain can complement a mutation on
a second polypeptide in the
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
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
-
interactions were detected by this
method, although they are postulated to occur in the intact cell based
on the
-
genetic complementations. A dimeric model for TetA
having intermolecular
-
and
-
interactions is
presented.
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 -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 (
) half from class
A and the second (
) half from class C, was active in expressing
tetracycline resistance, whereas a B/C or C/B chimera was
not(13) . Evidently the
and
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) .
-
interaction was also suggested by
the ability of the cloned
half to stabilize the cloned
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.
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, 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. ()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 Tet-6H, 24 kDa). A 0.6-kilobase EcoRI-XhoI fragment representing the
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 -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
gene on the plasmid.
Transformants were selected on 20 µg/ml tetracycline without IPTG. (
)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.
The fusion protein
was cleavable between MalE and TetA by factor Xa, as expected. (
)
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
. It is accompanied by
pcI857, a compatible Kan
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.
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,
6). 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,
3). 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
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.
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.
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.
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.
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.
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
domains
of the two different polypeptides, or between an
and a
domain, but possibly not between the
domains, since Tet279-LacZ
had the entire
domain but only the first two helices and
associated loops of the
region.
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 Tet
-6H (Fig. 3, lanes 3). These results
extended the previous finding with Tet279-LacZ, showing that an
domain of TetA was sufficient for binding to full-length Tet.
Therefore,
-
interactions were not necessary.
Figure 3:
Association of MalE-Tet with Ni-NTA-bound
Tet-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, Tet
-6H.
Molecular mass standards (kDa) are in lane S. The molar ratio
of MalE-Tet
Tet
-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.
Figure 4:
Association of B/B but not C/B with
Ni-NTA-bound Tet-6H. The method of Fig. 3was employed
except that immunoblots are shown. An extract representing 9 A
units of the Tet
-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
(Tet
-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
Tet
-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 Tet
-6H.
The Tet-6H protein was identified on
Coomassie-stained SDS-PAGE gels of Ni-NTA-bound protein as a band
migrating slightly more slowly than Tet
-6H and not present in
fusionless host cells (data not shown). Quantification of these bands
indicated that cells containing pLY22, encoding Tet
-6H, produced
only about 2% as much fusion protein as did cells bearing pLY17
(encoding Tet
-6H).
The functionality of Tet-6H encoded by
pLY22 was assayed in vivo by the ability to complement TetA
having a mutated
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 Tet
-6H domain was
functional, at least in the intact cell expressing a complementing Tet
protein.
Biochemical studies were then performed. Extracts of cells
containing Tet-6H or Tet
-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
Tet
-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 Tet
-6H but only 0.002 for
Tet
-6H. Tet
-6H also bound no MalE-Tet observable on SDS-PAGE
even though the MalE-Tet
Tet
-6H molar ratio applied to
Ni-NTA was about 7 (data not shown). MalE-Tet was bound to Tet
-6H
in the presence of Tet
-6H extract, as expected, although the
required large volume of Tet
-6H extract increased background bands
on SDS-PAGE, making quantification difficult (data not shown). These
results showed that Tet
-6H was neither able to bind Tet containing
both
and
domains (in MalE-Tet), nor able to bind the
domain in Tet279-LacZ. Our earlier failure to see
-
interaction in extracts was therefore confirmed, as was the absence of
-
interaction. The results also suggested that
-
interactions between Tet279-LacZ and Tet
-6H were responsible for
Tet multimerization in this assay.
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
and
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.
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.
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) domains, rather than between an
and a
domain. However, in intact cells, besides the genetic data there are
also physical indications of
-
interaction. The amount of a
polypeptide comprising the
half of TetA in whole cells was
increased 1.5-fold or more by the presence of the
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
; a simple explanation
for those results could be that the C/B protein formed a multimer with
the B/C protein via same-class
-
interactions and stabilized
it, although other explanations are possible. The fact that in the
present work we did not see
-
interactions after the Tet
protein had been extracted may mean that the
domain for the C/B
and Tet
-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
and
domains in that polypeptide do have approximately
the expected
-helical content.
A TetA dimer may be
held together both by -
interactions (seen in the present
study for proteins extracted from membranes by dodecylmaltoside) and by
-
interactions (not apparent using extracts, but inferred
from genetic and biochemical studies in whole cells). A model in which
both
-
and
-
interactions occur within a TetA dimer
is shown in Fig. 5. During complementation of B/C with C/B in vivo, the
-
interactions would presumably not
occur, but the
-
ones would. Two active sites/wild type
dimer, or one/ complementing dimer, would be expected. Our model might
explain why Tet
-6H was found in cells at high concentrations
similar to those of the full-length fusion Tet-6H, while the amount of
Tet
-6H was 50-fold lower, since the model allows
to bind to
(or to
), and such associations may prevent degradation.
Absence of self-association for
, as modeled, would lead to
degradation of
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 . A ribbon
representing the large cytoplasmic loop connects the
and
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 -
interactions and the altered topology of the
central loop (Fig. 5). A monomer of TetA has both the domains
(
and
) required for activity, and we cannot discount the
possibility that a complex consisting of only one
and one
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.