From the Department of Physiology, Johns Hopkins University Medical School, Baltimore, Maryland 21205
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ABSTRACT |
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Site-directed and second site suppressor
mutagenesis identify an intrahelical salt bridge in the eleventh
transmembrane segment of UhpT, the sugar phosphate carrier of
Escherichia coli. Glucose 6-phosphate (G6P) transport by
UhpT is inactivated if cysteine replaces either Asp388 or
Lys391 but not if both are replaced. This suggests that
Asp388 and Lys391 are involved in an
intrahelical salt bridge and that neither is required for normal UhpT
function. This interpretation is strengthened by the finding that
mutations at Lys391 (K391N, K391Q, and K391T) are recovered
as revertants of the inactive D388C variant. Further work shows that
although the D388C variant is null for G6P transport, movement of
32Pi by homologous
Pi/Pi exchange is unaffected. This raises the possibility that this derivative may have latent function, a
possibility confirmed by showing that D388C is a gain-of-function
mutation in which phosphoenolpyruvate (PEP) is the preferred substrate. Added study of the Pi/Pi exchange shows that in
wild type UhpT this partial reaction is readily blocked by G6P but not
PEP. By contrast, in the D388C variant, Pi/Pi
exchange is unaffected by G6P but is inhibited by both PEP and
3-phosphoglycerate. These latter substrates are used by PgtP, a related
Pi-linked antiporter, which lacks the
Asp388-Lys391 salt bridge but has instead an
uncompensated arginine at position 391. For this reason, we conclude
that in both UhpT and PgtP position 391 can serve as a determinant of
substrate selectivity by acting as a receptor for the anionic carboxyl
brought into the translocation pathway by PEP.
In Escherichia coli, transport of hexose phosphates is
mediated by the Pi-linked antiport carrier, UhpT (1-3).
This well characterized transporter is one of a class of secondary
transport systems that together form the Major Facilitator Superfamily
(4-6), the largest known collection of related secondary transporters. Although members of the Major Facilitator Superfamily show great diversity in their substrate specificity and kinetic mechanism, they
all share a common structural theme, one characterized by the presence
of approximately 12 transmembrane segments thought to transverse the
membrane in an One approach to the analysis of helix packing in membrane proteins
involves identification of interacting charged residues in
transmembrane helices. Ordinarily, one expects the presence of an
uncompensated electric charge to be highly destabilizing to such
structures, due to the low dielectric of the hydrophobic environment.
However, this circumstance can be mitigated if oppositely charged amino
acids are brought together to form an ion pair or salt bridge (10, 11).
The idea that such a salt bridge may stabilize secondary structure has
both theoretical and experimental support (11-13). Moreover, intra-
and interhelical salt bridges have been identified in both globular and
in intrinsic membrane proteins (9, 13-18).
In work described here, we used site-directed mutagenesis and second
site suppressor analysis to search for salt bridges in UhpT. This
approach led to identification of an intrahelical ion pairing involving
Asp388 and Lys391 in TM11.1
Further study indicated that this salt
bridge is essential for proper UhpT function and that at least one
member of this pair, Lys391, can play a direct role in
determining substrate specificity.
Strains and Plasmids--
Strain XL1-Blue (recA1 endA1
gyrA96 thi1 hsdR17 supE44 relA1 lac (F' proAB
lacIqZ Mutagenesis--
Site-directed mutagenesis was performed by
using either sequential polymerase chain reaction (22) or by the method
of Kunkel (23). Mutant uhpT alleles were sequenced using the
Sequenase (v. 2.0) reaction (Amersham Pharmacia Biotech) to confirm the desired mutation and rule out the presence of other changes.
Isolation of Second Site Suppressor Mutants--
Strain RK5000
carrying a pTrc(HisC6S) derivative encoding the D388C
mutation was grown overnight at 37 °C in LB broth containing F6P
(0.15%) and ampicillin (100 µg/ml). Overnight cultures were washed
and resuspended in an equal volume of M63 minimal medium salts (24),
and 0.1-ml aliquots were plated on M63 minimal plates containing
thiamine (2 µg/ml), required amino acids (each 50 µg/ml), ampicillin (100 µg/ml), and F6P (0.15%) as a carbon source. Plates were incubated at 37 °C for 4-5 days, after which colonies arising from the background lawn were picked and restreaked on minimal plates
to isolate single colonies. To ensure that growth was due to suppressor
mutation(s) in uhpT, candidate plasmids were isolated, and
uhpT was excised at its flanking restriction sites by
digestion with NcoI and NgoMI. The uhpT gene was
reinserted into a parental vector, which was then used to transform
strain RK5000 to confirm the original phenotype. In most cases, the
phenotype traveled with uhpT, and in these instances the
gene was completely sequenced to identify the responsible mutation(s).
Immunoblot Analysis--
SDS-polyacrylamide gel electrophoresis
was performed using cell extracts without preheating in sample buffer
(25). Protein was transferred to nitrocellulose and probed with a
peptide-directed rabbit polyclonal antibody reactive to a UhpT
C-terminal epitope (26, 27). Western blots were developed using
chemiluminescence (Amersham Pharmacia Biotech), and after scanning the
films, UhpT expression was quantitated by densitometry of digitized
images, as described (27, 28).
Transport Assays--
Cells grown overnight in LB broth plus
antibiotic were diluted 200-fold in the same medium and grown at
37 °C to a density of 2-5 × 108 cells/ml. Cells
were harvested by centrifugation, washed twice, and then resuspended in
Buffer A (50 mM MOPS/K, 100 mM potassium sulfate, 1 mM magnesium sulfate, pH 7.0) to achieve an
OD660 of 1.4, equivalent to about 2 × 109
cells/ml. After equilibration at room temperature, G6P or PEP transport
was initiated by adding a one-twentieth volume of labeled substrate
(final concentration, 50 µM), after which aliquots were removed for filtration on Millipore filters (0.45-µm pore size) and
washing (twice) with 5 ml of Buffer A lacking magnesium sulfate. Transport of Pi was monitored in the same way, using cells
pregrown in M63 minimal medium (as above) with 0.2% glucose as carbon
source. The high Pi content of M63 (24) ensured maximal
repression of other Pi transporters (Pst and Pit), so that
most Pi transport occurred via UhpT.
To assay transport (and metabolism) of F6P or PEP in vivo,
overnight broth cultures were diluted 1000-fold into M63 salts (above)
containing 0.15% F6P or PEP as carbon source. Cultures were placed at
37 °C with continuous shaking, and growth was monitored by changes
in optical density at 660 nm.
Chemicals--
Unlabeled substrates (G6P, F6P, 3-PGA, PEP,
2DG6P, and glycerol 3-phosphate), from Sigma, contained 0.5-1.0%
inorganic phosphate (30), which was ignored in calculation of kinetic
constants. [14C]G6P (56.6 µCi/µmol) and
[32P]KPi (1 Ci/mmol) were obtained from NEN
Life Science Products. [14C]PEP (38.6 µCi/µmol) was
from Amersham Pharmacia Biotech.
Identification of an Intrahelical Salt Bridge--
Our main
objective was to identify possible inter- or intrahelical salt bridges
that might exist among or within UhpT transmembrane helices. The
topological map of this transporter (Fig.
1), as well as results from earlier
studies (28), led us to choose four cationic residues
(Lys82, Arg275, Lys391, and
Lys404) and four anionic residues (Asp279,
Glu305, Asp388, and Asp400) as
candidate participants in salt bridges. Our experimental approach to
this question then proceeded in two phases. Initially, we replaced each
target with cysteine to determine whether normal function was
disrupted. For those cases in which a significant deficit was recorded,
we then asked whether function was preserved if a residue of like
charge was present (Table I). In this
screen, only the K404C variant retained partial function, indicating
that most of our target residues were essential for G6P transport by UhpT. In most of these latter cases, it appeared that this requirement was specific, because only for position 279 and 388 was it possible to
substitute a residue of like charge and recover significant function
(Table I).
The second step in our analysis was based on the fact that three pairs
of these residues were in a registration (i, i+3)
or (i, i+4) consistent with their participation
in a salt bridge in an Isolation of Second Site Suppressor Mutants--
To further test
the idea of an interaction between Asp388 and
Lys391, we isolated revertants of the inactive D388C
mutation by screening for growth on F6P as a carbon source. This
genetic screen was initiated with the expectation that if
Asp388 and Lys391 interact so as to eliminate
an uncompensated charge, at least some revertants of D388C might map to
position 391. We performed this screen in an otherwise cysteine-less
background rather than with the wild type protein, because the
partially reduced activity of the cysteine-less protein (compare Tables
I and III) allowed for a denser
background lawn on minimal plates,2 increasing the
frequency of revertants. In addition, this cysteine-less parental
protein had an N-terminal polyhistidine tag (20) to facilitate later
biochemical work.
As noted under "Experimental Procedures," we were able to eliminate
revertants arising from mutations outside the UhpT structural gene.
Those that remained contained the original D388C mutation along with a
second site suppressor at position 391 in which Lys391 was
replaced by one of three uncharged residues, threonine, glutamine, or asparagine.
We characterized these revertants in several ways. For example,
phenotypic growth tests in liquid medium confirmed that each of these
double mutants utilized F6P (Fig. 2).
Indeed, they grew slightly more rapidly than did the parental,
cysteine-less protein (Fig. 2). By contrast, the host strain, RK5000
( Altered Substrate Specificity of the D388C Mutant--
UhpT and
its relatives comprise a coherent family of transporters and receptors
(4, 6), each having high specificity for some organic phosphate ester
and low affinity toward inorganic phosphate (1). This family has twelve
bacterial members, seven of which have been functionally characterized
(28). A multiple alignment of the amino acid sequences within this
group (28) suggests that some residues within TM11 are highly
conserved. This is not true, however, for residues assigned to the
intrahelical salt bridge in UhpT, Asp388 and
Lys391 (Fig. 3). Instead,
most family members have either an aliphatic (Ala or Val) or polar
(Ser, Thr, or Asn) residue at these positions. However, in one case,
the phosphoenolpyruvate transporter of Salmonella typhimurium, we noted the presence of Val388 and
Arg391. This was of interest for two reasons. First, the
presence of an apparently uncompensated positive charge in the PgtP
TM11 contrasted sharply with evidence that this was detrimental to
UhpT. Second, because of their acidic carboxyl group, substrates of
PgtP (PEP and PGAs) (35, 36) carry one more negative charge than do substrates of UhpT. For this reason, we considered it feasible that
Arg391 in PgtP might serve as an internal receptor for the
anionic carboxyl on PEP and PGA. And, if so, we reasoned that the D388C
variant in UhpT, which no longer transports F6P or G6P, might
show an unexpected bias toward transport of PgtP substrates due to the remaining Lys391.
To test this idea, we exploited the fact that UhpT and PgtP (and other
members of this family) each display a Pi self-exchange reaction (3, 37, 38). Thus, if the UhpT D388C derivative retained this
partial reaction, one might justify an extended search for substrates
other than G6P. When we examined Pi transport in both the
D388C mutant and its parental protein, it became clear that despite its
null phenotype for G6P transport (Tables I and III), the mutant
displayed considerable Pi self-exchange (Fig. 4). For example, the reactions had
comparable Michaelis constants (Km values of 740 and 550 µM, respectively, for parent and mutant),
and nearly equivalent maximal velocities (105 and 59 nmol/min/mg
protein, respectively). Given that the D388C variant is expressed at
only 60% of the parental level (Table III), we concluded that D388C
UhpT displays an essentially normal Pi self-exchange reaction.
Having found that Pi self-exchange is normal in the D388C
mutant, we used this reaction as a vehicle to compare substrate preference for parental and mutant proteins. This was essential because
UhpT itself does not transport three-carbon organophosphates (29), and
the mutant we wished to analyze does not respond to sugar 6-phosphates
(Table III). Accordingly, for both wild type and mutant proteins, we
ranked putative substrates with respect to their capacity to inhibit
Pi self-exchange. As expected from earlier work (29), this
reaction was strongly inhibited by G6P or the nonmetabolizable 2DG6P in
wild type UhpT, whereas PEP and 3-PGA were ineffective inhibitors (Fig.
5A). These findings contrasted sharply with results obtained with the D388C variant, which was strongly inhibited by PEP and 3-PGA but not by G6P and 2DG6P (Fig. 5B). Neither protein responded to glycerol 3-phosphate, a
substrate of the related GlpT antiporter (37, 38). To quantitate these observations, we calculated for each substrate an inhibition constant (Ki) from linear Dixon plots (40) (Fig.
5C and Table IV). This showed
that the D388C mutation causes a 50-100-fold decrease in the
Ki value for normal UhpT substrates while at the
same time increasing by 20-fold the apparent affinity for PEP or
3-PGA.
These findings strongly suggested that D388C is a gain-of-function
mutation in UhpT, and to test this directly we examined this protein
with respect to both growth on and transport of PEP. The first test
clearly documented that the D388C variant was able to use PEP as sole
carbon source, in contrast to its revertants or to the wild type
protein (Fig. 6A). Similarly,
only the mutant, not its revertants, was able to transport PEP (Fig.
6B). Coupled with the observation that UhpT transports G6P
only when the Asp388/Lys391 salt bridge remains
intact or when both its elements are removed, these findings lead us to
conclude that in both UhpT and PgtP position 391 can serve as a
determinant of substrate selectivity.
The study of membrane transport proteins reveals several instances
in which a functional or structural role can be assigned to one or more
charged residues in a transmembrane helix. The former category is well
represented by several cases in E. coli, including two
charged residues (Glu126 and Arg144) thought
necessary for substrate binding in the lactose permease, LacY (8), a
pair of cationic residues (Arg46 and Arg275)
that may function as substrate recognition elements in UhpT (28), and
acidic residues (Asp51 and Asp120) that help
set co-ion specificity for MelB, the Na+/melibiose
cotransporter (41). Such residues may also act as stabilizing factors
by virtue of their interaction in a salt bridge. Thus, in
bacteriorhodopsin, Arg227 and Asp96 form an
interhelical salt bridge, whereas Arg82 and
Asp85 are involved in an intrahelical ion pair (14).
Interhelical salt bridges in LacY have also been recognized (9, 17);
indeed, these have been indispensable to development of current models of helix packing in this symporter (9, 17). Similarly, it has been
proposed that Arg499 and Glu503 form an ion
pair in the Na+/glucose cotransporter, SGLT1 (42).
In the present study, we used site-directed and second site suppressor
mutagenesis as tools to ask if charged residues in the UhpT hydrophobic
sector might take part in salt bridges, basing our interpretation on
the original analysis of salt bridges in LacY (43). This approach
highlighted two residues in TM11, Asp388 and
Lys391, as interacting in an intrahelical ion pair. Thus,
sugar phosphate transport was not found after individual replacement of
either Asp388 or Lys391, but function was
retained when uncharged residues replaced both of them (Tables II and
III).
Initially, the null phenotype arising when Asp388 and
Lys391 were removed individually led us to believe that
UhpT function was disrupted due to inappropriate helix packing, but
this view did not easily explain why a related antiporter, PgtP (32%
identity), contained an apparently uncompensated arginine at position
391. We therefore considered the possibility that lack of function in
some of our derivatives was related to altered substrate specificity rather than a change in structure. This seemed more likely when we
found that Pi self-exchange was unaffected by the D388C
mutation (the K391C mutant has not been tested). Subsequently, by using this partial reaction as a screening tool, we showed that D388C is a
gain-of-function mutation that biases substrate preference toward PEP
and 3-PGA and away from G6P and F6P (Figs. 4-6 and Table IV).
Two questions are raised by our findings. 1) What is the molecular
basis of the new substrate specificity in the D388C variant? 2) Why is
there at the same time a discrimination against normal UhpT substrates?
No definitive answers can be offered to either question, but a single
perspective does address both issues. An early model (44) specified
that two electropositive centers in UhpT associate with a pair of
negative charges brought into the active site by the anionic
substrates, Pi and G6P; indeed, we recently identified two
required arginines (Arg46 and Arg275) that may
fulfill this function (28). If the configuration that accepts sugar
phosphate must achieve an electroneutrality, one might expect that
substrates such as PEP and PGA, which carry an additional negative
charge relative to G6P, might require the presence of a third positive
center before productive binding can occur. The second question is
answered in much the same way. That is, when the D388C variant is
presented with G6P, failure to achieve an electrostatic balance at the
sugar phosphate binding site would be associated with an abortive
carrier-substrate complex.
Although such arguments account for our findings related to transport
of PEP or G6P, this reasoning does not directly explain why the
Pi self-exchange is unimpaired in the D388C mutant. At the
least one must conclude that TM11 is not involved in binding of
Pi. This might be expected if the Pi
self-exchange exploits a different configuration of the protein (44),
possibly relying predominantly on factors related to either or both of
the two essential arginine residues, one of which lies in TM1
(Arg46), the other in TM7 (Arg275). The absence
of either of these inactivates G6P transport (28), but in light
of the work reported here, it could be instructive to examine the
Pi self-exchange reaction as these arginines are removed individually.
The finding of PEP transport by the D388C mutant also leads one to
question the stoichiometry and electrical character of the reaction.
The kinetic parameters of Pi self-exchange by D388C deviate
little from its parent (Fig. 4), implying that heterologous Pi/3-PGA or Pi/PEP antiport by this mutant
would retain the exchange stoichiometry shown by wild type UhpT during
transport of G6P. In this latter case, early work suggests that UhpT
selects monovalent Pi to use in an electroneutral exchange
with divalent G6P, with an overall 2-for-1 stoichiometry (44). A
similar 2-for-1 stoichiometry during heterologous Pi/PEP
antiport is therefore expected to be electrogenic, carrying negative
charge inward, because for our assay conditions (pH 7) PEP or 3-PGA
mainly exists as the trivalent anion. Because this reaction would occur
against the cellular membrane potential (inside negative), one might
also expect, as is found, a relatively low growth yield on PEP (Fig.
6A) and a relatively low rate of PEP transport (Fig.
6B). Although we have not yet studied the electrical
character of heterologous exchange in D388C, it is clear that this
single mutation (D388C) could have mechanistic impact on more than one
attribute of the antiport reaction.
That substrate specificity can be altered by change of a single amino
acid residue has been demonstrated for several transporters. In LacY,
for example, point mutations lead to a preference for maltose (45),
sucrose (46), or malto-oligosaccharides (47). Similarly, point mutants
in the melibiose carrier of E. coli can show altered cation
and/or sugar specificity (48, 49). Most recently efficient transport of
lactose was described for a mutant of the E. coli maltose
ABC transporter (50). Even among this group, however, our finding that
the D388C mutation alters the substrate selectivity of UhpT is unusual.
Given that the new preference for PEP or 3-PGA reflects interaction
between Lys391 and the substrate carboxyl, we infer that in
wild type UhpT the C1 position of G6P lies on TM11 in the neighborhood
of the Asp388/Lys391 ion pair. Of additional
interest is the possibility that the approaches described here might
let us assign specific functional roles to other charged residues. For
example, TM7 contains two charged residues, Arg275 and
Asp279 (Fig. 3), one of which (Arg275) is known
to lie on the translocation pathway (51) and is believed to aid in
substrate recognition (28). Arg275 is conserved among
relatives of UhpT (28), but only UhpT has a charged residue at position
279. This situation is reminiscent of that found in TM11, where one of
a pair of charges (Asp388) is unique to UhpT, whereas the
other (Lys391) is more broadly represented (Fig. 3).
Although our tests did not identify the
Arg275/Asp279 pair as a simple salt bridge, we
should not rule out the possibility that these residues associate such
that a more general function of Arg275 is moderated by
Asp279 to optimize the features specific to UhpT.
INTRODUCTION
Top
Abstract
Introduction
References
-helical conformation. Direct information as to the
arrangement of these helices is limited, although several potential
models have been formulated in specific cases (7-9).
EXPERIMENTAL PROCEDURES
M15 Tn10))
(Stratagene Cloning Systems) was used for all cloning steps. Strain
RK5000 (araD139
(ArgF-lac)U169 relA1
rpsL150 thi gyrA219 non metE780
(ilvB-uhpABCT')2056 recA) (19)
served as host for tests of expression and function of plasmid-encoded
UhpT and its derivatives. Plasmids pBS281 and
pTrc(HisC6S) encoded wild type UhpT and a histidine-tagged
cysteine-less UhpT, respectively (20).2
pTrc(HisC6S) was constructed by replacing the six cysteines
found in UhpT with serine residues (21).1
RESULTS
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Fig. 1.
UhpT topology. Shown is the topology of
UhpT as derived from the analysis of hydropathy (30), the results of
reporter gene fusions in UhpT and GlpT (31, 32), and the accessibility
of residues in UhpT to impermeant probes (27). Targets for
mutagenesis in this study are shown as either circles or
squares, indicating anionic or cationic residues,
respectively.
UhpT function in mutants of charged residues within the hydrophobic
sector
-helix. Members of the remaining pair,
Lys82 and Glu305, were at the center of their
respective helices, where they too might interact (7). Accordingly, we
generated double mutants in which cysteine residues replaced each
target pair (Table II). This test
provided clear positive evidence that Asp388 and
Lys391 might normally interact as an ion pair, because
sugar phosphate transport in the D388C/K391C double mutant was about
half that found in the wild type protein (Table II); when normalized to UhpT expression, the specific activity of this double mutant was about
75% that of the wild type.2 In no other case, however, did
the specific activity in the double mutants rise above about 1% of the
parental protein. Thus, this approach did not provide evidence of
interaction for our other targets.
UhpT function in double mutants of selected pairs of charged residues
UhpT function and expression in second site suppressor mutants of D388C
UhpT
uhpT), and the D388C mutant showed greatly delayed
responses; eventual growth of these strains presumably reflects the
appearance of adaptive revertants arising after slow growth under
strong selective pressure (33, 34). We also examined the three
suppressor mutants with respect to their expression of UhpT and their
capacity to transport G6P (Table III). In the wild type protein, the
D388C mutation had reduced UhpT expression nearly 2-fold (not shown),
and a similarly reduced expression was found when the D388C mutation
was placed in the cysteine-less background (Table III). In both cases,
however, G6P transport was reduced to near background levels (Tables I and III). By contrast, in all suppressor mutations, both UhpT
expression and G6P transport were elevated to levels resembling that
found in the parental protein. It is evident, then, that neither
Asp388 nor Lys391 are required for normal UhpT
function. Equally clear, the presence of one without the other is
detrimental. This behavior strongly argues that Asp388 and
Lys391 form an ion pair in TM11.
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Fig. 2.
Growth of UhpT and its derivatives on
F6P. RK5000 carrying plasmids encoding the parental, cysteine-less
UhpT ( ), its D388C derivative (
), or one of three D388C
suppressors, D388C/K391T (
), D388C/K391N (
), and D388C/K391Q
(
), were grown in minimal medium M63 with F6P as carbon source.
Strain RK5000 (
) was also examined. Growth was monitored by changes
in optical density at 660 nm. The data, from a single experiment, are
representative of findings made in three independent trials.
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Fig. 3.
Conserved residues in TM11. Multiple
alignment of TM11 sequences for Pi-linked transporters and
receptors with ~30% identity to UhpT. Residues conserved in at least
five family members are highlighted with shading; charged
residues are shown in bold type. The alignment is modified
from Fann et al. (28) by addition of GlpT from
Rickettsia prowazekii (52).
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Fig. 4.
Kinetics of phosphate exchange.
A, rates of Pi self-exchange were estimated as
described under "Experimental Procedures" for strain RK5000 without
( ) and with plasmids encoding cysteine-less UhpT (
) or its D388C
derivative (
). B, the kinetics of Pi
self-exchange for cysteine-less UhpT (
) and its D388C derivative
(
), presented as recommended by Hofstee (39).
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Fig. 5.
Substrate specificity of UhpT and its D388C
derivative. Pi self-exchange was estimated for both
cysteine-less UhpT (A) and its D388C derivative
(B) in the presence of G6P ( ), 2DG6P(
), PEP (
), or
3-PGA (
) using 32Pi at 0.1 mM
along with the indicated concentrations of competing substrates. In the
absence of competing substrate, 32Pi transport
varied between 8.5-15.5 nmol/min/mg protein for cysteine-less UhpT,
and 6-11.7 nmol/min/mg protein for its D388C variant, and these values
were used to normalize values for the individual trials. C,
graphical presentation of the data using the method of Dixon (40):
cysteine-less UhpT with G6P (
) or PEP (
); the D388C variant with
G6P (
) or PEP (
). Ki values for these and
other test substrates are shown in Table IV.
Inhibition constants for UhpT substrates
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Fig. 6.
Growth on and transport of PEP.
Growth on (A) and transport of (B) PEP was
measured as described under "Experimental Procedures."
A, RK5000 without ( ) and with plasmids expressing
cysteine-less UhpT (
), its D388C derivative (
), or second site
suppressors, D388C/K391T (
), D388C/K391N (
), and D388C/K391Q
(
), were grown in M63 with PEP as a carbon source. Growth was
monitored as described in the legend of Fig. 2. B, transport
of PEP was examined for D388C UhpT (
) and various controls (
),
including RK5000, and RK5000 expressing cysteine-less UhpT and its
suppressors, D388C/K391T, D388C/K391N, and D388C/K391Q.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Wolfgang Epstein and Robert Brooker for helpful discussion and Robert Kadner for the gift of strain RK5000.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM24195.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.
Supported by National Research Service Award postdoctoral training
grant F32GM19421.
§ To whom correspondence should be addressed: Dept. of Physiology, Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205-2185. Tel.: 410-955-8325; Fax: 410-955-4438; E-mail: pmaloney{at}bs.jhmi.edu.
2 J. A. Hall, M.-C. Fann, and P. C. Maloney, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: TM, transmembrane segment; F6P, fructose 6-phosphate; G6P, glucose 6-phosphate; PEP, phosphoenolpyruvate; PGA, phosphoglyceric acid; 2DG6P, 2-deoxyglucose 6-phosphate; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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