(Received for publication, March 4, 1997, and in revised form, May 2, 1997)
From the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
In the preceding two papers (Hall, J. A.,
Gehring, K., and Nikaido, H. (1997) J. Biol. Chem.
272, 17605-17609; Hall, J. A., Thorgeirson, T. E., Liu, J.,
Shin, Y.-E., and Nikaido, H. (1997) J. Biol. Chem.
272, 17610-17614), we showed that ligands that bind to the
Escherichia coli maltose-binding protein (MBP) without producing the closure of its two lobes are not transported into the
cytoplasm. Here, we examine various combinations of ligands, MBPs, and
membrane-associated transporters, by utilizing reconstituted proteoliposomes, right side-out membrane vesicles, and intact cells.
Closed forms of wild type MBP, complexed with maltose or maltodextrins,
interacted with wild type transporter complex to stimulate the
hydrolysis of ATP by MalK ATPase located on the other side of the
membrane, as shown earlier for the maltose-MBP complex (Davidson, A. L., Shuman, H. A., and Nikaido, H. (1992) Proc. Natl. Acad. Sci.
U. S. A. 89, 2360-2364). In contrast, open forms of liganded
MBPs, such as the complex containing wild type MBP and reduced,
oxidized, or cyclic maltodextrins or the complex containing the mutant
MBP MalE254 and unmodified maltodextrins, did not stimulate ATP
hydrolysis, suggesting that the proper interaction between the
ligand-MBP complex and the external surface of the transporter requires
the former to be in the closed conformation. However, when a mutant
transporter containing MalG511 was used, the already significant basal
level of ATP hydrolysis was further stimulated not only by ligand MBPs
in the closed form but also by those in the open form (except that
containing -cyclodextrin), data suggesting that the mutant
transporter does not always require the closed MBP complex presumably
because of its exceptionally strong affinity to MBP, described earlier
(Dean, D. A., Hor, L.-I., Shuman, H. A., and Nikaido, H. (1992)
Mol. Microbiol. 6, 2033-2040). Furthermore, this mutant
transporter was able to transport reduced maltodextrin, and cells
expressing the transporter were able to grow by using reduced
maltodextrin, if the periplasmic concentrations of MBP were kept low so
as not to inhibit the transport process.
Maltose and maltodextrins are transported in Escherichia coli by an ATP-dependent process, which requires the interaction between soluble, liganded maltose-binding protein (MBP)1 and a membrane-associated transporter (MalFGK2) composed of one copy each of MalF and MalG channel proteins and two copies of MalK ATPase (1, 2). The participation of MBP is absolutely needed for this transport process, and one of the reasons may be that the liganded MBP, by binding to the external surface of the transporter, sends a transmembrane signal so that the MalK ATPase can become activated on the opposite, internal surface of the membrane (3). The requirement for MBP, however, can be circumvented in "MBP-independent" transporter mutants, such as the one containing mutant MalG511 (MalFG511K2) (4), which constitutively hydrolyze ATP even in the absence of the liganded MBP (3).
We have shown in the preceding papers (5, 6) that MBP can bind its
ligands in two different ways. Maltose and linear maltodextrins bind to
MBP in a way that produces a slight red shift of the intrinsic
fluorescence emission spectrum of the protein (called R mode (for
red shift)) (5, 7) and a characteristic hypochromatic trend
in the <265-nm region of UV absorbance spectrum (5, 8). An earlier
study showed that the binding of maltose and -anomers of
maltodextrins produced a large upfield shift of the NMR resonance of
3H on the anomeric carbon of the reducing glucose residue
("end-on" mode) (9), and the R and end-on modes appear to refer to
the same manner of ligand binding. In contrast, when the MBP binds
-cyclodextrin, or reduced or oxidized derivatives of maltodextrins, the fluorescence emission spectrum is blue-shifted (B mode (for blue shift)), and the UV absorbance differential spectra
show no hypochromatic trend in the <265-nm region (5, 8). Since
-cyclodextrin and the modified dextrins just mentioned do not contain a reducing sugar residue with its anomeric carbon, we hypothesize that this mode corresponds to the binding mode that does
not involve the tight interaction of the hydrogen on the anomeric
carbon with the binding site of MBP, i.e. the middle mode,
earlier observed by 3H NMR for
-anomers of maltodextrins
(9). Interestingly, a mutant MalE254 MBP, which allows the transport of
maltose but not of maltodextrins (10), binds unaltered maltodextrins
exclusively via the B mode (5). By using representative ligands, we
showed further that the two lobes of MBP become closed when the R mode binding occurs, whereas there is little or no closing of the lobes when
the binding occurs through the B mode (6).
There are some hints that the B mode binding may not lead to the successful transport of ligands through the MalFGK2 complex. For example, maltodextrin derivatives that are reduced, oxidized, or substituted at their reducing glucose units bind to wild type MBP exclusively via the B mode as described above, and are not transported by the wild type E. coli (11). The mutant MalE254 MBP, which binds unmodified maltodextrins exclusively via the B mode (see above), does not allow the transport of these ligands (10). The failure to be transported can result either because the B mode complex fails to stimulate the ATPase activity of the transporter or because the B mode complex cannot deliver the ligand into the transport channel. The present paper examines this question and shows that the stimulation of ATP hydrolytic functions of the wild type MalFGK2 transporter is not induced by B mode ligand-MBP complex. We also show that ligands bound via the B mode to MBP are transported nevertheless through the mutant MalFG511K2 complex and that these mutant cells are indeed able to grow by using reduced maltodextrins as their carbon and energy source.
These are shown in Table
I. Strains HN889, HN892, HN924, and HN930 were
constructed by transferring the F factor carrying the
lacIq mutation and Tn5 (kanamycinr)
from HN596 to HN933, NT411 containing pLH16, NT229 containing pLH16,
and HN931, respectively, using standard mating technique (17). High
affinity lac repressor in these strains was necessary to
decrease the level of transcription of malE alleles under
control of the lacUV5 promoter (18) to a minimum when
uninduced. Antibiotic concentrations added to media were as follows:
streptomycin, 25 µg/ml; ampicillin, 100 µg/ml; and kanamycin, 50 µg/ml.
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Nonradioactive sugars have been described (5). [14C]Maltose was purchased from either Amersham Corp. or ICN Biomedicals, Inc. [3H]Maltotetraose was prepared at the National Tritium Labeling Facility from maltotetraose by exchange with tritium gas in the presence of palladium on BaSO4 (9). The specific activity of [3H]maltotetraose was estimated by assuming complete recovery of the sugar from the labeling reaction as well as by NMR. [3H]Maltotetraitol was made by reducing [3H]maltotetraose with sodium borohydride as described (5).
Preparation of MBPWild type MBP and malE254 MBP were prepared as described (5), except that malE254 MBP was exclusively from strain HS2019 containing pEH1. Bound maltose was removed from malE254 MBP by extensive dialysis as described by Silhavy et al. (19), and from wild type MBP by denaturation in 6 M guanidine HCl followed by renaturation through dialysis. Fluorescence emission spectroscopy was used to show that both the mutant and wild type MBPs were free of maltose and active (5).
Preparation of the Membrane Transporter ComplexThe
transporter complex (MalFGK2 or MalFG511K2) was
prepared from strain HN741 containing pMR11 and either pFG23 or pLH33. Cells were grown, induced, and harvested as described (3). Total
membrane fractions were prepared as described (2). Membrane proteins
were solubilized by keeping vesicles on ice for 30 min in 20 mM KPO4, pH 6.2, 20% glycerol, 5 mM MgCl2, 1 mM dithiothreitol, 1.1% octyl--D-glucopyranoside (membrane
protein:octyl-
-D-glucopyranoside ratio was about 3:1).
This mixture was centrifuged at 160,000 × g for 1 h at 4 °C, and the supernatant was aliquoted and stored at
70 °C until use. Membrane vesicles containing the MalG511 transporter that were destined for use in the proteoliposome substrate uptake assay were solubilized as above except that sonicated E. coli acetone/ether-washed total lipids (Avanti Polar Lipids, Inc.) were added to a final concentration of 8-9 mg/ml. This mixture was
then processed as above, and the supernatant fraction was used
immediately.
The affinities were determined from the concentration dependence of the quenching of the intrinsic protein fluorescence (7), measured on a Perkin-Elmer MPF-44B spectrofluorometer. The MBPs (0.2 µM) were dissolved in 10 mM KPO4, pH 7.0. Excitation was at 280 nm with a bandwidth of 7 nm, and emission was recorded at 348 and 346 nm for wild type and malE254 MBPs, respectively, with a bandwidth of 7 nm.
ATP Hydrolysis in ProteoliposomesSolubilized membrane
proteins were reconstituted into liposomes via a detergent dilution
method (13). In all experiments 4.5 mg of sonicated E. coli
acetone/ether-washed total lipids (Avanti Polar Lipids, Inc.) in 20 mM KPO4, pH 6.2, 2 mM
-mercaptoethanol was mixed with 90 µg of solubilized proteins, and
octyl-
-D-glucopyranoside was added to a final
concentration of 1.1%. This mixture (0.54 ml) was then incubated on
ice for 30 min, diluted into 14 ml of 20 mM
KPO4, pH 6.2, 1 mM dithiothreitol, and
centrifuged at 160,000 × g for 1 h at 4 °C to
isolate the proteoliposomes. MBP was added prior to dilution to give a
final concentration of 1.5 µM (for assays using wild type
MBP) or 0.15 µM (for assays using malE254 MBP)
after dilution. In these experiments, the weight ratio of E. coli phospholipids, MBP, and solubilized protein was 50:8:1 when
wild type MBP was used and 50:1:1 when MalE254 MBP was used. Also,
transport substrate was added prior to dilution to give a final
concentration after dilution such that >90% of the MBP would be in
the liganded form.
ATP hydrolysis by the MalFGK2 transporter was measured as
described (3). Proteoliposomes were resuspended in 20 mM
KPO4, pH 6.2, 3 mM MgCl2, 10 µM sugar substrate (if added when preparing proteoliposomes) to a final concentration of 0.45 µg of protein/ml and incubated with 100 µM [-32P]ATP (50 mCi/mmol) at room temperature, and released Pi was
determined at various time points.
Strains HS3368 and NT411 were grown at 37 °C overnight in medium 63 containing 0.4% glycerol and l µg/ml thiamine. The culture was diluted 1:20 into 200 ml of the same medium and grown to a cell density of 2 × 109 cells. Membrane vesicles were prepared by a modification (20) of the Kaback procedure (21), except that vesicles were separated from unlysed spheroplasts and whole cells by two centrifugations at 2000 × g for 10 min and then collected by centrifugation at 35,000 × g for 15 min. Vesicles were resuspended in 20 mM KPO4, pH 6.2, 3 mM MgCl2 to a final concentration of 1.5-2.0 mg of protein/ml, electron donors (10 mM ascorbate, 100 µM phenazine methosulfate) were then added as indicated, and the preparation was used immediately. [14C]Maltose (150 µCi/µmol), [3H]maltotetraose (150 µCi/µmol), or [3H]maltotetraitol (120 µCi/µmol) was added to a final concentration of 10 µM, and 25-µl aliquots were removed at specified time points, diluted 1:10 with cold 20 mM KPO4, pH 6.2, 3 mM MgCl2, and passed through a 0.22-µm Millipore GSTF filter. Filters were then washed with 5 ml of cold 50 mM LiCl, dried, and counted by liquid scintillation using Ecolume (ICN) as scintillant.
Substrate Uptake into ProteoliposomesThe accumulation of maltose and maltotetraose inside proteoliposomes was measured as described (13). Proteoliposomes were prepared as above (see "ATP Hydrolysis in Proteoliposomes") except that neither MBP nor substrate was added. Instead, ATP was added prior to dilution to give a final concentration of 5 mM after dilution. Proteoliposomes were resuspended to a final concentration of 0.45 µg of protein/ml in 20 mM KPO4, pH 6.2, 3 mM MgCl2 with or without 1 µM MBP and incubated with 10 µM [14C]maltose (150 µCi/µmol), 10 µM [3H]maltotetraose (150 µCi/µmol), or 10 µM [3H]maltotetraitol (120 µCi/µmol) at room temperature. At specified times, 25-40 µl of the reaction mixture was diluted 1:10 with 20 mM KPO4, pH 6.2, 3 mM MgCl2, filtered through a Millipore filter (0.22-µm GTSF), and washed with 5 ml of 50 mM LiCl. Filters were dried and counted as described above.
Determination of Periplasmic MBP ConcentrationCells were
grown in M63 medium containing 1 µg/ml thiamine, 0.4% glycerol, any
necessary antibiotics, and 250 µM IPTG (if required), at
37 °C to a density between 2 × 108 and 5 × 108 cells/ml. Cells were then harvested and osmotically
shocked as described above. The shock fluid containing periplasmic
proteins was cleared of cells and cellular debris by passage through
Whatman 1 filter paper. Proteins were then separated via
SDS-polyacrylamide gel electrophoresis (2) and either visualized by
Coomassie Blue staining or transferred to a nitrocellulose membrane for immunoblot analysis. The latter procedure was carried out by using an
anti-MBP rabbit antiserum and alkaline phosphatase-conjugated anti-rabbit IgG antibody (Sigma) (22). The total amount of MBP loaded
in each lane was quantified by comparison with serial dilutions of a
pure MBP standard. Periplasmic MBP concentrations were then estimated,
based on the total dry weight per cell of 2.7 × 1012 g (23) and the periplasmic volume, assumed to be
30% of the total cell volume (24).
Strains to be used in all experiments were grown overnight at 37 °C in M63 medium (17) containing 1 µg/ml thiamine, 0.4% glycerol, and any necessary antibiotics. Overnight cultures were diluted 1:20 into fresh medium of the same composition, except for the addition of 250 µM IPTG (if required), and grown at 37 °C to a density of approximately 5 × 108 cells/ml. Cells were washed twice with M63 and then resuspended in the same medium at a density of 2 × 109 cells/ml. The assay was initiated by the addition of labeled substrate ([14C]maltose (75-150 µCi/µmol), [3H]maltotetraose (200 µCi/µmol), or [3H]maltotetraitol (200 µCi/µmol)) to a final concentration of 10 µM. Portions of 50 µl each were removed, diluted 1:10 with cold M63, and filtered through a 0.45-µm HAWP Millipore filter. Filters were then washed with 5 ml of 50 mM LiCl, dried, and counted as described above. All values were corrected for background counts on filters.
To assay the ability of strains to transport and metabolize various substrates, overnight cultures were diluted 1:200 into 2.0 ml of fresh M63 media containing 1 µg/µl thiamine, any necessary antibiotics, 250 µM IPTG (if required), and maltose, maltotetraose, or maltotetraitol at the indicated concentration. Cultures were grown at 37 °C with continuous shaking, and growth was scored at various times.
Protein DeterminationEither the BCA protein assay (Pierce) or the method of Brown et al. (25) was used.
The wild type complex, MalFGK2, required the presence of both maltose and MBP inside the vesicles to hydrolyze, rapidly, ATP added from the outside, as noted earlier (3) (data not shown). When MBP and various ligands were added inside proteoliposomes at concentrations that would make MBP more than 95% liganded, only those ligands that bound to MBP via the R mode stimulated ATP hydrolysis by the MalFGK2 complex (Table II). Interestingly, maltotriose and maltotetraose both stimulated the transporter to a slightly higher degree than did maltose and maltohexaose. In contrast, ligands that bound to MBP exclusively via the B mode, such as reduced, oxidized, or cyclic maltodextrin derivatives, caused little stimulation (Table II). A marginal stimulation seen with maltotriitol and maltotetraitol could have been caused by traces of unmodified maltodextrins remaining in these preparations.
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Transport by
the wild type MalFGK2 transporter was examined in various
systems. Fig. 1A shows the accumulation of
various substrates into proteoliposomes. In these experiments, ATP was trapped inside the proteoliposomes, and wild type MBP and substrate were added to the outside. Clearly, both maltose and maltotetraose (binding to MBP via the R mode) were transported quite well, but maltotetraitol (binding via the B mode) was not accumulated to a
significant level.
Similar results were obtained when right side-out membrane vesicles from HS3368 (containing the wild type transporter) were used; the addition of maltose, maltotetraose, and maltotetraitol (in addition to MBP) resulted in the uptake of 0.38, 0.18, and 0.02 nmol of substrate/mg of protein/min, respectively. Without MBP, the addition of these sugars produced a residual uptake value of 0.02-0.03 nmol/mg of protein/min. There were some differences between the membrane vesicles and proteoliposomes. First, the uptake rates of maltose and maltotetraose were much higher in proteoliposomes; this is most likely due to the partial purification of the transporter via selective solubilization. Second, maltotetraose was transported into proteoliposomes better than maltose, whereas in membrane vesicles the opposite was true. A possible explanation is that many of the inner membrane vesicles were surrounded by residual outer membranes, which could have hindered the access of maltotetraose.
We also assayed substrate accumulation in whole cells of strain HS3368 (Fig. 1B). Interestingly, maltose was transported approximately 25 times more rapidly than maltotetraose. Maltodextrin transport in whole cells is most likely limited by diffusion through the LamB channel (26, 27). Maltotetraitol accumulation was close to the base-line level.
ATP Hydrolysis by the Wild Type Transporter in the Presence of MalE254 MBPThe malE254 allele was isolated
by Wandersman et al. (10) and is a member of a
class of "maltodextrin-negative" malE mutants, which are
unable to utilize maltodextrins although their MBPs have good affinity
for these substrates. The MalE254 MBP, unlike wild type MBP, binds both
maltodextrins and their nonreducing derivatives exclusively by the B or
open mode (5). However, it binds maltose (at high concentrations) by
the R mode (5). The MalE254 MBP was trapped inside proteoliposomes
containing the wild type MalFGK2 complex together with
various ligands (at concentrations that would assure at least 90%
saturation of MalE254 MBP), and ATP hydrolysis was measured. We found
that this mutant MBP could stimulate ATP hydrolysis only with maltose
(Fig. 2). The maltotetraose-MalE254 complex showed no
stimulation of ATP hydrolysis (Fig. 2), in contrast to the strong
stimulatory activity of maltotetraose-wild type MBP complex (Table
II).
ATP Hydrolysis by the MalG511 Transporter
The
malG511 allele is a member of a class of mutant
malF and malG alleles that allow a cell to
transport maltose in the absence of MBP (4). The MalFG511K2
transporter, when reconstituted into proteoliposomes, hydrolyzed ATP in
the absence of MBP and transport substrates (not shown), reproducing
earlier results (3). Its ATP hydrolytic activity was not stimulated
significantly by the incorporation of either MBP alone or substrate
alone into the intravesicular space. However, ATP hydrolysis was
stimulated when proteoliposomes were reconstituted so that they
contained any substrates (except -cyclodextrin) in addition to the
wild type MBP (Table II). In other words, even those ligands that
appeared to bind exclusively via the B mode, such as maltotetraitol and maltohexaitol, showed enhancement almost as strong as the unmodified parent compounds.
In the early experiments with proteoliposomes, the mutant transporter was found to be unstable, when extracted, and showed no apparent uptake activity. However, when solubilized in the presence of E. coli total lipids (see "Experimental Procedures"), it showed good transport activity upon reconstitution. (The presence of total lipid extract during MalG511 transporter solubilization also resulted in higher ATP hydrolysis rates (not shown)). Proteoliposomes containing the MalFG511K2 complex accumulated maltose, maltotetraose (both R mode binders), and maltotetraitol (an exclusive B mode binder), at a rate of 0.06, 0.12, and 0.14 nmol/mg of protein/5 min, respectively. In the absence of MBP there was little uptake of these substrates (<0.01 nmol/mg of protein/5 min).
The same pattern of transport was seen with NT411 right side-out membrane vesicles containing the same mutant transporter, with rates of 0.51, 0.12, and 0.13 nmol/mg of protein/5 min for maltose, maltotetraose, and maltotetraitol, respectively. Thus, these vesicles transported maltotetraitol quite well in the presence of MBP. We also note that maltose was transported better than maltotetraose by these membrane vesicles, whereas in the proteoliposomes the opposite was true.
Substrate Transport by Intact Cells in the Presence of Wild Type MBP and the MalG511 TransporterMaltotetraitol that bound via the B mode was transported into proteoliposomes and membrane vesicles containing MalG511 transporter. Since active transport of modified maltodextrins has not been seen in E. coli (see Ref. 11), we examined whether the transport occurred in intact cells. However, the MalG511 mutant transporter, as well as other "MBP-independent" mutant transporters, cannot transport any ligands when wild type MBP is present at high concentrations that are usually found in induced cells, presumably because transport is inhibited by the excessively tight binding of MBP-ligand complex to the mutant transporters (3, 28). We therefore constructed strains that expressed MBP to different levels and studied their capacity to transport and metabolize various substrates.
To measure transport, NT411 was used as a parent strain because it is
unable to metabolize maltose and maltodextrin due to the absence of
amylomaltase and maltodextrin phosphorylase. Detectable amounts of
maltose were not transported into NT411 cells (Fig. 3A). This result may seem unexpected, since
strain NT411 expresses the "MBP-independent" MalG511 transporter.
However, since the transport assays were done using a concentration of
maltose (10 µM) far below the Km of
this mutant transporter (~2.0 mM), any significant
transport is not expected. As has been shown (4), maltose transport was
readily detected if maltose was present at 500 µM (not
shown). When MBP was present at a very low concentration in the
periplasm (HN889, uninduced), maltose transport into the cytoplasm was
enhanced greatly in comparison with NT411 (Fig. 3A). This
stimulatory effect of MBP continued up to MBP concentrations of at
least 250 µM (HN889 and HN933, both induced). As the
periplasmic concentration of MBP approached that found in the periplasm
of induced cells (~1.0 mM; HN934), MBP showed an
inhibitory effect instead (Fig. 3A). This is in stark
contrast to the system containing the wild type MalFGK2 transporter (HS3368, Fig. 3A), in which the fully induced
level of MBP supported maltose transport at a nearly maximal rate.
The transport of maltotetraose followed a somewhat similar pattern (Fig. 3B). As shown earlier (4), strain NT411 could not transport maltotetraose in the absence of MBP. Maltotetraose transport in uninduced HN889 cells was strongly stimulated by the presence of a low level of MBP (Fig. 3B). At MBP concentrations around 250 µM, however, maltotetraose transport became inhibited (Fig. 3B) in contrast to the strong stimulation seen for maltose transport under similar conditions (Fig. 3A), suggesting that the MalFG511K2 transporter became inhibited more strongly by the maltotetraose-MBP complex than by the maltose-MBP complex.
Maltotetraitol, although it binds to MBP via the normally inactive B binding mode, was transported (Fig. 3C), generally much more rapidly than maltotetraose (compare with Fig. 3B), when MBP was present up to concentrations of 250 µM. As periplasmic MBP concentrations approached 1.0 mM, maltotetraitol transport also became somewhat inhibited. It was not transported by either HS3368 (producing wild type transporter) or NT411 (not producing any MBP) (Fig. 3C).
The growth on various substrates was also tested (Table III). Strain NT229 (malP+ malQ+) was used as the parent strain producing the mutant MalFG511K2 transporter as well as varying levels of MBP. All such strains were capable of growing upon media containing 6 mM maltose, although HN932, with a high level of MBP, could not grow in 1, 2, or 4 mM maltose, confirming the result of Treptow and Shuman (4). At lower maltose concentrations, those strains that produced lower levels of MBP (uninduced HN930 and IPTG-induced HN931 and HN930) grew more rapidly than those that had high periplasmic MBP concentrations (HN932) or produced no MBP at all (NT229) (Table III and results not shown). These results agree well with the active transport data and again indicate that high levels of MBP inhibit transport through the MalG511 transport complex.
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Maltotetraose was not utilized by strain NT229 (Table III), confirming the earlier report (4). On the other hand, strains producing MBP at noninhibitory levels were capable of growing not only on maltotetraose but also on the alcohol maltotetraitol (see uninduced and induced HN930 as well as HN931 in Table III).
Substrate Transport in Intact Cells in the Presence of MalE632 MBP and the MalG511 TransporterThe malE632 allele was isolated due to its ability to allow a malG511 mutant strain to grow on maltodextrin as a sole carbon source in the presence of fully induced levels of MBP (29). We studied the interaction between the MalE632 suppressor MBP and the MalG511 transporter to see whether the substrate specificity of this MalFG511K2 system, which allowed the transport of ligands binding via the B mode, was restored to the more restrictive pattern found in the wild type system.
Ligand binding affinity of MalE632 was not grossly different from that
of wild type MBP, as assessed by the fluorescence quenching assay (data
not shown). Binding of maltose, maltotetraitol, or -cyclodextrin to
MalE632 produced the same shifts of fluorescence emission spectra as
seen with the wild type MBP (not shown). Maltotetraose, however, caused
a marked blue shift (5 nm) in MalE632, in contrast to the very slight
blue shift (0.7 nm) observed with the wild type MBP (5). This suggests
that maltotetraose uses mainly the B binding mode with the MalE632.
Transport data are shown in Fig. 4. IPTG-induced HN892,
which had a periplasmic MalE632 MBP concentration of approximately 300 µM, transported both maltose and maltotetraose at rates
considerably higher than that found in the malE strain
NT411. This strain also transported maltotetraitol, but the initial
rate (0.17 nmol/mg at 1 min) was much lower than in the strains
expressing the wild type MBP at similar levels (1.4 and 0.7 nmol/mg at
1 min for IPTG-induced HN933 and HN889, respectively, in Fig.
3C).
Strain NT229 grew only on maltose, while IPTG-induced strain HN924, producing MalE632, grew well on both maltose and maltotetraose (Table III). Interestingly, it seemed to grow only marginally on maltotetraitol, much more slowly than the strain producing the wild type MBP (HN930). The transport and growth data are thus consistent with the hypothesis that MalE632 MBP restores, at least partially, the wild type substrate specificity to the MalG511-containing cells.
Previous studies showed that MBP may bind its ligands in one of
the two ways. Thus, the wild type MBP binds maltose and -anomers of
maltodextrins in the end-on or R mode, which is accompanied by the
closure of its two lobes, whereas it binds reduced, oxidized, or cyclic
maltodextrins exclusively in the middle or B mode, which leaves its two
lobes essentially open (5, 6). In contrast, the mutant MalE254 MBP
binds even unmodified maltodextrins entirely via the middle mode (5).
It is known that ligands that are now known to be bound in the open or
middle mode fail to be transported (e.g. reduced, oxidized,
or cyclic maltodextrins in wild type cells or unmodified linear
maltodextrins in malE254 cells (see Introduction)). We
examined why the middle binding does not lead to transport.
One of the essential functions of liganded MBP is to interact with the
periplasmic face of the MalFGK2 transporter and to send a
transmembrane signal so that ATP hydrolysis by the MalK ATPase, located
on the opposite, cytosolic face of the membrane, could be initiated
(3). The middle mode binding may not lead to transport because the
liganded MBP in the open form may fail to stimulate this ATPase
activity. Our results with the wild type MalFGK2 complex
showed clearly, both with the wild type MBP and with the mutant MalE254
MBP, that little ATP hydrolysis occurred when any ligand was bound
exclusively via the B or middle mode (Fig. 2 and Table II, columns
labeled "By MalFGK2"). In addition, MBP-ligand
complexes of this type did not cause the transport of ligands,
confirming the previously published data (10, 11). In contrast, all
ligands that bind to MBP via the R or end-on mode, which produces the
closure of the two lobes of MBP, stimulated ATP hydrolysis and
substrate uptake activities of the MalFGK2 transporter
(Fig. 2; Table II). The simplest hypothesis then is that the open form
of MBP cannot produce ligand transport simply because it cannot produce
activation of the ATPase. We note that this hypothesis is also
consistent with the conclusion, from genetic suppression studies, that
the N-terminal and C-terminal lobes of the MBP interact, respectively,
with the external surfaces of MalG and MalF (30, 31), since the closure
of MBP may then bring the two lobes to the correct distance for their
interaction with these subunits of the transporter complex (see Fig.
5).
We also examined a mutant transporter complex, MalFG511K2,
which transports maltose in the absence of MBP, with an affinity 1000-fold less than the affinity shown by the wild type in the presence
of MBP (4, 30). Most significantly, such transporters hydrolyze ATP in
the absence of liganded MBP, i.e. in a partially constitutive manner (3). According to the hypothesis above, such
transporters should be able to transport reduced or oxidized maltodextrins, since they do not require closed MBP to start the ATP
hydrolytic cycle. However, as shown earlier by Treptow and Shuman (4),
cells of this mutant cannot transport even unmodified maltodextrins in
the absence of MBP. Proteoliposomes containing the mutant transporter
complex could not transport maltodextrins by themselves, confirming
these intact cell data (Fig. 3; see "Results"). Interestingly, the
MalFG511K2 complex could transport maltodextrins when MBP
was present (Fig. 3; see "Results"), suggesting that MBP must play
an additional role in the transport of maltodextrins, perhaps by
positioning the ligands correctly in the transport channel. It has been
shown earlier (3) that the addition of maltose-MBP stimulated further
the partially constitutive level of ATP hydrolysis by this mutant
transporter. Consistent with this finding, the closed maltodextrin-MBP
complex stimulated ATP hydrolysis by the MalFG511K2
transporter, but unexpectedly, the open, middle-bound complexes
containing reduced or oxidized maltodextrins also stimulated ATP
hydrolysis, although the -cyclodextrin-MBP complex was inactive
(Fig. 2; Table II, columns labeled "By
MalFG511K2").
These data suggest that our first hypothesis is too simplistic. It is
perhaps important that the MalFG511K2 transporter has an
unusually high affinity toward liganded MBPs (28). Possibly because of
this high affinity, even the open form MBP could be bound tightly in a
way to produce an additional stimulation of ATP hydrolysis. Since
-cyclodextrin does not cause this stimulation, it is tempting to
speculate that the open form MBP cannot be closed in this case because
of the strong steric hindrance of the bulky ligand (Fig.
5E), but with other ligands the MBP could become closed to
some extent (Fig. 5D).
The MalG511 transporter transports maltose in the absence of MBP, yet
the transport is inhibited when the fully induced levels of MBP (around
1 mM in the periplasm) are present, most probably because
of the exceptionally high affinity of the transporter complex to MBP
(4, 28). We have shown in this study, by regulating the expression of
MBP, that lower levels of MBP actually stimulated the transport of
various ligands in intact cells (Figs. 3 and 4), confirming the earlier
data obtained with membrane vesicles (28). One significant outcome of
these intact cell studies was the demonstration that malG511
mutant, in the presence of moderate levels of MBP, could transport
reduced and oxidized maltodextrins (Fig. 3C) and could
actually grow on these substrates (Table III). Such
"MBP-independent" mutants were previously shown to transport p-nitrophenyl--maltoside (32), although this occurred in
the absence of MBP.
The inhibition of maltose transport caused by the tight binding of MBP to MalFG511K2 transporter can be relieved by suppressor mutations in MBP (30). Such suppressor MBPs most likely act by decreasing the affinity of the liganded MBP complex for the MalG511 transporter (28). If the middle binders are transported by the mutant transporter because of the very high affinity of the transporter for MBP, it may be expected that a suppressor MBP will not allow the transport of middle mode binders such as maltotetraitol. We tested this possibility by using a suppressor MBP MalE632. The strain expressing both MalFG511K2 transporter and the MalE632 MBP indeed had difficulty in growing on maltotetraitol (Table III) and transported maltotetraitol more slowly than did the strains expressing the wild type MBP (compare Fig. 4 with Fig. 3C). However, rigorous comparison between the strains is difficult, because there were too many variables. Crystallization of wild type maltose transporter has been achieved (33), and the solution of its structure may eventually solve some of these uncertainties.
Finally, this study provided an interesting glimpse of the relationship between the length of a sugar substrate and its transport efficiency. The wild type transporter hydrolyzes ATP to a greater degree when interacting with MBP complexed with maltotriose and maltotetraose than when MBP is complexed with either maltose or maltohexaose (Table II). The same is true of the MalG511 transporter (Table II). This variation appears to be correlated with the subsequent transport of the sugar. Thus, in reconstituted proteoliposomes, maltotetraose (and maltotetraitol in MalFG511K2-containing proteoliposomes) is transported more rapidly than maltose (Fig. 1A; see "Results"). In contrast, in whole cells maltose is transported more efficiently than any of the maltodextrins (Fig. 1B). The flux of maltodextrins through the LamB channel, however, is far slower than that of maltose (26, 27), and it seems possible that the interaction between liganded MBP and the transport complex would favor transport of maltodextrins over maltose to counterbalance this effect of the outer membrane barrier.
We thank the staff of National Tritium Labeling Facility, Lawrence Berkeley Laboratory, for preparation of [3H]maltotetraose.