From the Department of Genetics and Cell Biology and the Institute for Advanced Studies in Biological Process Technology, University of Minnesota, St. Paul, Minnesota 55108
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ABSTRACT |
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In this study, we have examined the transport characteristics of the wild-type lactose permease, single mutants in which Lys-319 was changed to asparagine or alanine or Glu-325 was changed to glutamine or alanine, and the corresponding double mutant strains. The wild-type and Asn-319 mutant showed high levels of lactose uptake, with Km values of 0.42 and 1.30 mM and Vmax values of 102.6 and 48.3 nmol of lactose/min/mg of protein, respectively. The Asn-319/Gln-325 strain had a normal Km of 0.36 mM and a moderate Vmax of 18.5 nmol of lactose/min/mg of protein. By comparison, the single E325Q strain had a normal Km of 0.27 mM but a very defective Vmax of 1.3 nmol of lactose/min/mg of protein. A similar trend was observed among the alanine substitutions at these positions, although the Vmax values were lower for the Ala-319 mutations. When comparing the Vmax values between the single position 325 mutants with those of the double mutants, these results indicate that neutral 319 mutations substantially alleviate a defect in Vmax caused by neutral 325 mutations.
With regard to H+/lactose coupling, the wild-type permease
is normally coupled and can transport lactose against a gradient. The
position 325 single mutants showed no evidence of H+
transport with lactose or thiodigalactoside (TDG) and were unable to
facilitate uphill lactose transport. The single Asn-319 mutant and
double Asn-319/Gln-325 mutant were able to transport H+
upon the addition of lactose or TDG. In addition, both of these strains
catalyzed a sugar-dependent H+ leak that
inhibited cell growth in the presence of TDG. These two strains were
also defective in uphill transport, which may be related to their
sugar-dependent leak pathway. Based on these and other
results in the literature, a model is presented that describes how the
interactions among several ionizable residues within the lactose
permease act in a concerted manner to control H+/lactose
coupling. In this model, Lys-319 and Glu-325 play a central role in
governing the ability of the lactose permease to couple the transport
of H+ and lactose.
A central pathway for the uptake of many solutes by living cells
involves membrane proteins known as cation/substrate cotransporters or
symporters. Symporters are able to couple the inwardly directed cation
electrochemical gradient to the transport of solute so that secondary
active transport can be achieved with regard to the solute (1, 2). The
lactose permease of Escherichia coli has provided a model
system in which to investigate secondary active transport (3). This
protein is found in the E. coli cytoplasmic membrane and
couples the transport of H+ and lactose with a
stoichiometry of 1:1 (4). From the cloning and nucleotide sequence of
the lacY gene, the lactose permease contains 417 amino acids
with a molecular mass of 46,504 Da (5, 6). Hydropathicity plots, as
well as genetic studies, are consistent with a secondary structural
model in which the protein contains 12 hydrophobic segments that
traverse the membrane in an The analysis of sequences among a variety of transporters has led to
the conclusion that a large and diverse group of solute transporters,
known as the major facilitator superfamily, are evolutionarily related
to one another (10-12). At the functional level, members encode a
diverse array of proteins that transport many different substrates such
as sugars, Krebs cycle intermediates, and antibiotics. For example, the
lactose permease of E. coli, the glucose transporter of red
blood cells, and bacterial tetracycline exporters are members.
Mechanistically, this group is rather variable and includes proteins
that catalyze symport, uniport, or antiport.
Secondary structural models for the lactose permease and other members
of the superfamily usually depict a membrane protein with 12 transmembrane segments traversing the membrane in an Many studies have centered on the role of ionizable amino acid residues
in the mechanism of H+/lactose symport and active lactose
transport. Six ionizable residues within the lactose permease have been
implicated to play a central role, or an auxiliary role, in the
mechanism of H+/lactose coupling (15-25). These residues
are Asp-240, Glu-269, Arg-302, Lys-319, His-322, and Glu-325. In the
current study, we have focused our efforts on understanding the roles
of Lys-319 and Glu-325 because mutations at these sites cause very
striking effects on H+/lactose coupling, whereas mutations
at the other four sites tend to dramatically decrease the rate of
transport without affecting coupling. In previous studies, we showed
that nonionizable substitutions at Lys-319 uncouple
H+/lactose transport and change the stoichiometry to 0.3 (H+/lactose, Ref. 16). Furthermore, mutations at codon 319 cause a slow sugar-independent H+ leak and a fast
sugar-dependent H+ leak. In addition, mutations
at codon 319 alter sugar specificity (15). Other studies have shown
that nonionizable substitutions of Glu-325 cause a different phenotype
(17, 18). These mutations can catalyze a normal exchange of lactose but
are almost completely defective at unidirectional transport. Due to the
very low rate of net transport, these previous studies have not
determined if 325 substitutions can catalyze H+ transport.
However, the exchange reaction becomes pH-insensitive, suggesting that
325 mutations are unable to recognize H+ ions (18). With
these known effects of 319 and 325 substitutions, we decided to explore
the effects of double mutations, Asn-319/Gln-325 and Ala-319/Ala-325,
in which nonionizable residues were substituted at both positions, and
compare these results to those of the corresponding single mutant strains.
Reagents--
Lactose
(O- Bacterial Strains and Methods--
For downhill lactose
transport, E. coli strain
HS4006/F'IQZ+Y
Plasmid DNA was isolated by the NaOH-SDS method (29) and introduced
into the appropriate bacterial strain by the CaCl2
transformation procedure of Mandel and Higa (30).
Stock cultures of cells were grown in YT medium (31) supplemented with
tetracycline (0.01 mg/ml). For transport assays, cells were grown to
midlog phase in YT medium containing tetracycline (0.005 mg/ml), and
0.25 mM
isopropyl-1-thio- Sugar Transport Assays--
For the experiments shown in Figs. 1
and 3, midlog cells were washed in phosphate buffer, pH 7.0, containing
60 mM K2HPO4 and 40 mM
KH2PO4 and resuspended in the same buffer to a
density of approximately 0.5 mg of protein per ml. Cells were then
equilibrated at 30 °C, and radioactive sugar was added at the
designated concentrations. At appropriate time intervals, 0.2-ml
aliquots were withdrawn and filtered over a membrane filter (pore size,
0.45 µm). The external medium was then washed away with 5-10 ml of
ice-cold phosphate buffer, pH 7.0, by rapid filtration. As a control,
the lacY Calculations--
The Km and
Vmax values for lactose transport were
determined by plotting 1/v versus
1/[S] in a Lineweaver-Burke double reciprocal plot
(32).
H+ Transport--
For the H+ transport
experiments shown in Fig. 4, cells were grown to midlog phase and
washed twice with 120 mM KCl. The cells were then suspended
in 120 mM KCl and 30 mM potassium thiocyanate to a density of approximately 5 mg of protein per ml. 2.5 ml of cells
were placed in a closed vessel with a lid containing tight-fitting openings for the insertion of a pH electrode, the introduction of
nitrogen, and the insertion of (gas-impermeable) Hamilton syringes. Cells were made anaerobic under a continuous stream of nitrogen for at
least 30 min. To initiate sugar-induced H+ transport, an
anaerobic solution containing lactose or thiodigalactoside (TDG)1 was added to a final
concentration of 2 mM. The change in external pH was
measured with a Radiometer pH meter (PHM82) and electrode (GK2401C).
Changes in pH were continuously recorded on a Radiometer chart recorder
that had been modified to expand the scale of pH changes to a range
where a 0.1-unit pH change caused a 10-cm deflection in the chart recording.
Construction of Plasmids Carrying Mutant Permeases--
In
previous studies, we have described the isolation of single E325Q and
E325A mutants (18) and a double A177V/K319N mutant (15). For the
current study, we isolated a single K319A mutant by polymerase chain
reaction mutagenesis using a primer that annealed across a unique
NdeI site found at codon 322. To construct the single K319N
strain, the plasmid carrying the double A177V/K319N mutation was cut
with AflIII and BstXI yielding a 1.0-kbp fragment containing the coding sequence of the lacY gene that
contains the K319N mutation but not the A177V mutation. This fragment
was ligated into the plasmid, pLac184, carrying the wild-type
lacY gene in which the AflIII/BstXI
1.0-kbp fragment had been removed.
To construct the double mutants (K319N/E325Q and K319A/E325A), the
plasmids carrying the single mutations were digested with SalI (which has a unique restriction site within the
plasmid) and NdeI. A unique NdeI restriction site
is found between codons 319 and 325 in the lacY gene. This
digestion yields a 3.8-kbp fragment carrying codon 319 and a 2.8-kbp
fragment carrying codon 325. To construct the K319N/E325Q and
K319A/E325A strains, the 3.8-kbp fragment from the pK319N and pK319A
plasmids was ligated to the 2.8-kbp fragment from the pE325Q and pE325A
plasmids, respectively.
DNA Sequencing--
The presence of the mutations within the
plasmids was confirmed by the sequencing of double-stranded plasmid DNA
isolated using MagicTM Minicolumns from Promega and
sequenced according to Kraft et al. (33).
Membrane Isolation and Immunoblot Analysis--
For Western blot
analysis, T184 cells containing the appropriate plasmid were grown as
described above for the sugar transport assays. 40 ml of late log cells
were pelleted by low speed centrifugation and resuspended in 10 ml 50 mM Tris-HCl, pH 7.5, with protease inhibitors
phenylmethylsulfonyl fluoride (25 mg/ml) and
L-1-tosylamido-2-phenylethyl chloromethyl ketone (0.1 mg/ml). Cells were then French pressed at 18,000 p.s.i. The cell debris
was removed by two repeated centrifugation steps (10 min at 11,000 × g). The membrane vesicles were collected by high speed
centrifugation (45 min at 150,000 × g) and resuspended in the Tris buffer with protease inhibitors. 50 µg of total membrane protein were subjected to sodium dodecyl sulfate polyacrylamide gel
electrophoresis. Proteins were electroblotted to nitrocellulose and
then probed using an antibody that recognizes the carboxyl terminus of
the lactose permease. The amount of lactose permease was then
determined by laser densitometry. All of the mutant strains described
in this study had levels of permease that were very similar to
wild-type. Normalized to wild-type (at 100%), they were as follows:
K319N, 105%; E325Q, 103%; pK319N/E325Q, 101%; K319A, 107%; E325A,
108%; and K319A/E325A, 104%.
Sugar Transport--
As described under "Materials and
Methods," we constructed single mutations at codons 319 and 325 in
which polar mutations (i.e. K319N and E325Q) and nonpolar
mutations (i.e. K319A and E325A) were introduced into the
lacY gene. To examine the transport characteristics of the
parent and mutant strains, their phenotype on MacConkey plates was used
as a qualitative measure of transport activity. As shown in Table
I, the wild-type, K319N, and K319A mutant
strains could effectively transport lactose (a
In earlier studies, we have shown that YT plates, with and without
As expected, the growth of the strain carrying the wild-type lactose
permease was not affected by the presence of TDG because this strain is
normally coupled (Table I). The E325Q, E325A, K319A, and K319A/E325A
mutants also showed no evidence for a sugar-dependent H+ leak. However, the K319N and K319N/E325Q strains both
catalyze a robust TDG-dependent H+ leak that
completely inhibits growth. These results indicate that both of these
strains catalyze H+/TDG influx and uncoupled TDG efflux at
a substantial rate.
To obtain a quantitative description of the transport process, in
vitro transport experiments were also conducted. Fig.
1 shows the results of a "downhill"
lactose transport assay that was carried out on the wild-type and
mutant strains. For this experiment, plasmids containing the wild-type
or mutant lacY genes were transformed into a
lacZ+ E. coli strain
(HS4006/F'Z+Y
Consistent with the results of the MacConkey plating, the wild-type,
K319N, and K319A mutants were able to transport lactose at substantial
rates. Likewise, the K319N/E325Q and K319A/E325A double mutants could
transport lactose at a rate that was substantial, although
significantly less than the wild-type strain. The E325Q and E325A
strains, however, were very defective in their rates of downhill
lactose transport.
To examine the effects of these mutations on lactose transport in
greater detail, a kinetic analysis was conducted in which the
Km and Vmax values for
transport were measured in the wild-type and mutant strains. As shown
in Table II, the wild-type permease
catalyzed downhill transport with a Km of 0.42 mM and a Vmax value of 102.6 nmol of
lactose/min/mg of protein. With regard to the polar substitutions, the
K319N mutant exhibited a 3-fold higher Km and a
2-fold lower Vmax. The other single mutant,
E325Q, had a relatively normal Km but a
Vmax value that was only 1% of the value of the
wild-type strain. The double mutant had a Km that
was also normal, and a Vmax value that was
defective, but only moderately so. The addition of the K319N mutation
greatly alleviated the defect in Vmax seen in
the E325 single mutant. As shown in Table II, the K319N/E325Q double
mutant had a Vmax value that was 14-fold higher
than the E325Q single mutant.
A similar trend was seen with the nonpolar mutants. The Ala-319 single
mutant had a slightly elevated Km for lactose, and a
Vmax value that was about 4-fold lower than
wild-type. The Ala-325 mutant had a Km that
was about 2-fold lower than wild-type, but a greatly reduced
Vmax value. The double Ala-319/Ala-325 mutant
had an intermediate Vmax value, again suggesting
that a neutral substitution at position 319 partially relieves the
defect imposed by a neutral substitution at position 325.
Fig. 2 also shows the results of downhill
transport assays conducted at a variety of external pH values. The
wild-type permease exhibited a pH optimum for downhill transport that
was approximately 6.0. The pH optima of the single and double mutant
strains carrying polar mutations were also similar to this value.
The wild-type lactose permease obligatorily couples the transport of
H+ and lactose with a 1:1 stoichiometry. This coupling
enables the bacterium to actively accumulate lactose against a
concentration gradient. Therefore, this "uphill" accumulation of
lactose provides a way to examine the coupling between H+
and lactose transport. To conduct an uphill transport assay, plasmid
DNA was transformed into a bacterial strain that is
H+/Sugar Symport--
Another way to evaluate
H+/sugar coupling is to directly measure the rate of
H+ uptake upon the addition of sugar by using a pH
electrode. These results are shown in Fig.
4. As expected, when lactose was added, the wild-type strain exhibited a rapid alkalinization of the medium due
to the cotransport of H+ and sugar into the bacterial
cytoplasm. After a short period of time, this alkalinization was
followed by an acidification due to the metabolism of lactose and the
excretion of acidic products. The addition of the nonmetabolizable
sugar TDG also promoted a rapid alkalinization.
Likewise, readily detectable alkalinizations also were observed for the
K319N and K319N/E325Q mutant strains. The data seen with the
K319N/E325Q strain are the first demonstration that a nonionizable
mutation at this position can still facilitate H+/sugar
cotransport. The only exception was a H322D/E325S double mutant that
had an acidic residue at position 322, which is on the same face of
helix 10 (25). Otherwise, previous studies have consistently suggested
that an acidic residue is required at position 325 (or 322) to
facilitate H+ transport via the lactose permease.
Nevertheless, in the current study, the H+ transport seen
with the K319N and K319N/E325Q strains was expected because both of
these strains were shown in Table I to catalyze a
sugar-dependent H+ leak. In contrast, the
single E325Q mutant, which had very low levels of downhill lactose
transport, also exhibited negligible levels of sugar-induced
H+ transport.
The surprising result obtained from this study is that an acidic
residue at position 325 is not obligatorily required for H+
transport via the lactose permease. This outcome was unexpected because
previous analyses of single mutants at position 325 have always
exhibited a phenotype that was consistent with uncoupled lactose
transport (17, 18). All single mutations at position 325 can exchange
lactose at wild-type rates, but neutral substitutions cannot
catalyze a detectable level of sugar-induced H+
transport. In addition, the efflux reaction of counterflow experiments is insensitive to pH, suggesting that the H+ recognition
site within the lactose permease has been abolished (18). One caveat of
these experiments, however, is that the rate of unidirectional lactose
uptake and efflux of position 325 single mutants is extremely low.
Therefore, the interpretation of undetectable H+ transport
in position 325 single mutants may be related to the low rate of
unidirectional transport, rather than an inability to bind both
H+ and lactose as a fully loaded carrier.
In other studies, a Ser-325 mutant was used as a parent strain to
isolate second site mutations with an increased rate of transport (25).
Substitutions of His-322 (i.e. Asn-322 and Asp-322) partially alleviated the inhibition of net sugar transport seen in the
single Ser-325 strain. The Asn-322 substitution was found to increase
the rate of sugar transport without allowing H+ transport,
whereas an Asp-322 mutation permitted H+/sugar cotransport.
These results also suggested that a carboxylate in this specific region
of the lactose permease is a critical feature of H+/lactose cotransport.
Nevertheless, the results obtained with the Asn-319/Gln-325 double
mutant in our current study clearly refute the idea that a carboxylate
on transmembrane segment 10 within the lactose permease is an absolute
requirement for H+/lactose cotransport. With regard to the
H+ recognition site, our data suggest that this site is
more complicated than a single, critical, ionizable residue. Even
though all of the residues within the lactose permease have been
altered by site-directed mutagenesis, the only single mutations that
abolished H+ transport were nonionizable mutations at
position-325. However, because the K319N/E325Q double mutant can
facilitate H+/lactose cotransport, other residues within
the permease must also be capable of providing an H+
binding site when a carboxylate is absent from this region. Even so,
this observation does not preclude the possibility that Glu-325 plays a
central role in H+ binding when it is present in the
wild-type protein.
A second issue addressed in the current study is the role of Lys-319.
Our data are consistent with the hypothesis that Lys-319 controls the
coupling between H+ and lactose transport even though it
does not function directly as an H+ binding site. The
single Asn-319 mutant has a striking uncoupled phenotype that exhibits
a sugar-dependent H+ leak (see Table I and Ref.
16). The double Asn-319/Gln-325 mutant has this phenotype as well.
According to the kinetic mechanism shown in Fig.
5, this leak may occur by permitting
conformational changes associated with the partially loaded
carrier.
INTRODUCTION
Top
Abstract
Introduction
References
-helical manner (7-9).
-helical manner. This arrangement of 12 transmembrane segments may have arisen
by a gene duplication/fusion event of an ancestral gene encoding a
protein with 6 transmembrane segments (13). More recently, we proposed
a three-dimensional model based on the structural characteristics of
transmembrane domains, hydrophilic loops, putative helical interactions
in the lactose permease, and rotational symmetry between the two halves
of the protein (14).
MATERIALS AND METHODS
-D-galactopyranosyl-[1,4]-
-D-glucopyranose)
and melibiose
(O-
-D-galactopyranosyl-[1,6]-
-D-glucopyranose)
were purchased from Sigma. [14C]Lactose was purchased
from Amersham Pharmacia Biotech. Restriction enzymes were purchased
from New England Biolabs, Inc. The remaining reagents were analytical grade.
was used. It is
lacZ-positive but lacY-negative (26). For uphill lactose transport, E. coli strain T184 was used. It is
negative for both the lacZ and lacY genes (27).
The plasmids used in this study are derivatives of the
lacY-carrying plasmid pLac184 (see Ref. 18).
-D-galactopyranoside to induce the
synthesis of the lactose permease.
strain,
HS4006/F'IQZ+Y
/pACYC184, was also
assayed for radiolabeled sugar uptake to obtain an accurate value for
nonspecific sugar uptake. The sugar transport assay was also conducted
in a similar manner for the experiment shown in Fig. 2, except that the
pH was adjusted by varying the relative amounts of
K2HPO4 and KH2PO4.
RESULTS
-galactoside) as
evidenced by their red or pink phenotype at both sugar concentrations tested. With regard to melibiose (an
-galactoside), however, the
Ala-319 mutants were markedly defective. By comparison, the white
phenotypes of the E325Q and E325A single mutants indicated that neutral
substitutions at position 325 inhibit transport of either sugar. The
double mutants, K319N/E325Q and K319A/E325A, had intermediate
phenotypes. At high lactose concentrations, the double mutants had a
red or pink phenotype, and at the higher melibiose concentration, the
Asn-319/Gln-325 double mutant had a dark pink phenotype. Overall, the
results of Table I indicate that the position 319 mutations are least
inhibitory, and the position 325 mutations are the most inhibitory for
sugar transport. Coupling a position 319 mutation with a position 325 mutation appears to alleviate some of the inhibition seen in the single 325 mutant strains.
Phenotype on plates
-D-TDG, can be used to determine whether lactose
permease mutants catalyze a sugar-dependent H+
leak. This sugar is a nonmetabolizable
-galactoside that is cotransported with H+ via the lactose permease. We have
shown previously that TDG can promote an H+ leak via the
leak B pathway in certain lactose permease mutants (16). In the
operation of the leak B pathway, H+ and TDG are
cotransported inward, and then TDG is exported without an
H+ ion. This results in a net inward movement of
H+ without net sugar transport. When lactose permease
mutants catalyzing the leak B pathway are grown on rich plates
containing TDG, the inward flux of H+ collapses the
H+ electrochemical gradient and dramatically inhibits
growth. In previous studies, this observation provided the basis for
the isolation of second site suppressor mutations that had a diminished leak B pathway (34).
), which is
-galactosidase-positive. When lactose enters the cell, it is rapidly
metabolized so that the external lactose concentration is always higher
than the internal concentration (35). Therefore, this in
vitro assay measures lactose transport as it moves from a higher
to lower concentration, or downhill.
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Fig. 1.
Downhill lactose transport by wild-type and
mutant strains. The uptake of [14C]lactose was
measured as described under "Materials and Methods." Downhill
lactose uptake was performed at an external lactose concentration
of 1.0 mM using the designated plasmids transformed into
strain HS4006/F'IQZ+Y .
A, wild-type lacY plasmid (pLAC184) (
),
Asn-319 (pK319N) (
), Gln-325 (pE325Q) (
), or
Asn-319/Gln-325 (pK319N/E325Q) (
). B, Ala-319 (pK319A)
(
), Ala-325 (pE325A) (
), or Ala-319/Ala-325
(pK319A/E325A) (
). Note that the y axis is expanded
in B.
Table of apparent Km and Vmax values
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Fig. 2.
Downhill lactose transport at different
external pH values. The uptake of [14C]lactose was
measured as described under "Materials and Methods." Downhill
lactose uptake was carried out at an external lactose concentration of
1.0 mM in strain
HS4006/F'IQZ+Y containing the
wild-type lacY plasmid (pLAC184) (
), Asn-319
(pK319N) (
), Gln-325 (pE325Q) (
), or Asn-319/Gln-325
(pK319N/E325Q) (
) at the designated external pH values.
-galactosidase-negative. The assays were done at a low external
lactose concentration (0.1 mM) and the intracellular
accumulation of [14C]lactose was measured at various time
points by rapid filtration. The results of an uphill transport assay
are shown in Fig. 3. As seen here, the
wild-type permease catalyzed uphill transport to accumulation levels
that were over 30-fold higher than the external lactose concentration.
However, the single and double mutants were completely defective in
their ability to actively transport lactose. Among these mutants,
different reasons may account for the defects in lactose
accumulation. In the case of the E325Q strain, the inability to
actively transport lactose can be explained by its very poor rate of
net lactose transport (see Fig. 1 and Table II). In contrast, the K319N
and K319N/E325Q strains showed moderate downhill transport but were
still unable to actively transport lactose. The defect seen in these
two strains can be explained by their uncoupled phenotype. As shown in
Table II, both of these strains catalyze a sugar-dependent
H+ leak that inhibits cell growth. This phenotype is due to
H+/sugar influx in conjunction with uncoupled sugar efflux.
Therefore, the K319N and K319N/E325Q strains are not expected to
accumulate lactose against a concentration gradient because these
mutations permit the uncoupled efflux of lactose.
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Fig. 3.
Uphill lactose transport by wild-type and
mutant strains. The uptake of [14C]lactose was
measured as described under "Materials and Methods." Uphill lactose
uptake was performed at an external lactose concentration of 0.1 mM in strain T184 containing the wild-type lacY
plasmid or mutant plasmids as follows: pLAC184 ( ), K319N (
),
E325Q (
), K319N/E325Q (
), K319A (
), E325A (
), and
K319A/E325A (
).
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Fig. 4.
Sugar-induced H+ transport.
H+ transport was measured in the strain
HS4006/F'IQZ+Y containing the
designated plasmids as described under "Materials and Methods."
A, addition of lactose; B, addition of TDG.
DISCUSSION
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Fig. 5.
An unordered mechanism of
H+/lactose cotransport. C1 and C2 are outward-facing
and inward-facing conformations of the lactose permease, respectively.
The wild-type permease is only able to convert between C1 and C2 in the
unloaded and fully loaded states. Certain mutants can catalyze
uncoupled transport, as indicated by the open arrow.
Fig. 6 provides a working hypothesis to explain the mechanism of H+/lactose coupling via the lactose permease and suggests ways that Lys-319 and Glu-325 may exert their effects. This model is based on several types of observations. These include: 1) the general proximity of Asp-240, Glu-269, Arg-302, Lys-319, His-322, and Glu-325 inferred from spectroscopic and genetic studies (20-22); 2) the effects of single mutations at these positions on the mechanism of lactose transport (16-19); 3) the isolation of suppressor mutations that suggest particular interactions among these ionizable residues (23-25); and 4) the effects of the K319N/E325Q double mutant described in this study.
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The model suggests particular ionic interactions between residues of the lactose permease in the unloaded, partially loaded, and fully loaded carrier. It also proposes that H+ binding occurs at Glu-325, although our data suggest that the H+ binding site (or the hydronium ion binding site) also involves the coordinate interactions of other residues (which can form an H+ binding site when a carboxylate is absent from position 325). As shown in Fig. 6, B and C, we propose that specific ionic interactions in the partially loaded carrier prevent the C1/C2 interconversion described in Fig. 5. In contrast, ionic interactions in the unloaded carrier permit a slow interconversion of C1/C2, and other ionic interactions in the fully loaded carrier permit a faster interconversion. A faster interconversion of the fully loaded carrier is predicted from the rate of lactose exchange, which is much faster than the rate of unidirectional lactose transport. Overall, the model shown in Fig. 6 suggests a pattern of ionic interactions that explains how the wild-type lactose permease is able to catalyze coupled transport of H+ and lactose and prevent uncoupled transport.
This model makes several functional predictions that are consistent with this and previous studies. First, a single mutation with a neutral residue at position 325 should be able to catalyze lactose exchange, but not unidirectional transport. When position 325 is neutral (as in a neutral mutation or when Glu-325 is protonated), the model proposes that Lys-319 interacts with Glu-269 and prevents the C1/C2 conformational change. Therefore, a neutral mutation at position 325 would be unable to catalyze unidirectional transport because the unloaded carrier could not interconvert. But a neutral substitution at position-325 should be able to exchange lactose because the binding of sugar disrupts the interaction between Lys-319 and Glu-269 when position 325 is neutral. Second, the single Lys-319 mutation should be able to catalyze lactose transport with or without H+. When Lys-319 is neutral, the partially loaded carrier is not prevented from making the C1/C2 interconversion. Therefore, it can translocate lactose when H+ is not bound, but it can also transport lactose when H+ is bound. Consistent with this prediction, the K319N mutation has a stoichiometry for H+/lactose transport that is less that 1:1 (i.e. 0.3 mol of H+/mol of lactose; Ref. 16) and it catalyzes a sugar-dependent H+ leak. A third and very striking prediction of our model is the phenotype of the K319N/E325Q and K319A/E325A double mutants. In the double mutants, the neutral residue at position 319 should circumvent the inability of the E325Q or E325A mutations from making the interconversion of the unloaded carrier. When positions 319 and 325 are both neutral, the unloaded carrier should be able to interconvert. Therefore, the double mutant should have a much higher rate of unidirectional transport compared with the single E325Q or E325A strains. This significant observation was made in the current study. Aside from these transport characteristics of mutations at positions 319 and 325, some additional observations are consistent with this model. Neutral mutations at Asp-240 and Glu-269 are rescued by neutral substitutions at Lys-319, suggesting an interaction between these residues (24). In addition, spontaneous mutations altering sugar specificity have involved substitutions at codon 319, suggesting a proximity of Lys-319 and the sugar binding site (15). In our model, the binding of sugar alters the ability of Lys-319 to interact with other ionizable residues in the permease.
For simplicity, we have not included Arg-302 and His-322 in this model. Mutations at these sites have significant effects on the rate of lactose transport, but they do not abolish H+ transport or alter the stoichiometry of H+/lactose cotransport (19, 36, 37). We speculate that either or both of these residues may play a role in transport by providing part of the H+ binding site and/or by interacting with other residues that may contribute to this site. They also could play an auxiliary role in influencing the pK values of other residues, such as Glu-325. Perhaps, when an ionizable side chain is eliminated at position 325, these residues may act as secondary H+ recognition sites.
Further research will be needed before we understand how the putative ionic interactions shown in Fig. 6 are able to control the ability of the lactose permease to make the C1/C2 conformational change. In other studies, we have proposed that this conformational change occurs at the interface between the two halves of the protein (38-40). Along these lines, it is interesting to note that Glu-269 is located on helix 8, which is at the interface between the two halves of the protein in our tertiary model of the permease (14). The topology of helix 8 may play a central role in governing the ability of the permease to make conformational changes.
Finally, there are several key differences between our model, shown in
Fig. 6, and an alternative model from another laboratory (28). First,
our model explicitly describes the states of the lactose permease that
permit the C1/C2 conformational change versus those that do
not. Second, our model proposes a key role for Lys-319 in promoting the
coupling between H+ and lactose cotransport. And lastly,
our model suggests that Glu-269 (a residue that we propose is at the
interface between the two halves of the protein) may control the
ability of the lactose permease to alternate between the C1 and C2 conformations.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM53259.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Bioprocess Technical
Institute, 240 Gortner Laboratories, 1479 Gortner Ave., St. Paul, MN 55108.
The abbreviations used are: TDG, thiodigalactoside; kbp, kilobase pair(s).
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REFERENCES |
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