From the Department of Physiology, Johns Hopkins University Medical School, Baltimore, Maryland 21205
Received for publication, March 6, 2001, and in revised form, May 2, 2001
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ABSTRACT |
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In Escherichia coli, transport of
hexose 6-phosphates is mediated by the Pi-linked antiport
carrier, UhpT, a member of the major facilitator superfamily. We
showed earlier that Lys391, a member of an intrahelical
salt bridge (Asp388/Lys391) in the
eleventh transmembrane segment (TM11) of this transporter, can function
as a determinant of substrate selectivity (Hall, J. A., Fann,
M.-C., and Maloney, P. C. (1999) J. Biol. Chem.
274, 6148-6153). Here, we examine in detail the role of TM11 in
setting substrate preference. Derivatives having an uncompensated
cationic charge at either position 388 or 391 (the D388C, D388V, or
D388K/K391C variants) are gain-of-function mutants in which
phosphoenolpyruvate, not sugar 6-phosphate, is the preferred organic
substrate. By contrast, when an uncompensated anionic charge is placed
at position 388 (K391C), we observed behavior consistent with an
increased preference for monovalent rather than divalent sugar
6-phosphate. Because positions 388 and 391 lie deep within the UhpT
hydrophobic sector, these findings suggested that an extended length of
TM11 may be accessible to external substrates and probes. To explore this issue, we used a panel of TM11 single cysteine variants to examine
the transport of glucose 6-phosphate in the presence and absence of the
membrane-impermeant, thiol-reactive agent
p-chloromercuribenzosulfonate (PCMBS). Accessibility to
PCMBS, together with the pattern of substrate protection against PCMBS
inhibition, leads us to conclude that TM11 spans the membrane as an
Secondary transport systems use the chemiosmotic energy generated
by the movement of ions down their electrochemical gradients to
facilitate the accumulation of small solutes (1-3). The best-studied secondary transporters of this sort belong to the
MFS1 (4, 5), an
evolutionarily related collection that accounts for a large fraction of
known solute transporters (6). This taxonomic group is comprised of
single-polypeptide carriers that show great diversity in both substrate
specificity and kinetic mechanism. Despite this heterogeneity, most
members of the MFS share a common structural theme, one characterized
by the presence of 12 transmembrane segments believed to transverse the
membrane in In the absence of detailed information concerning the structure of such
transport proteins, helix relationships and helix function in members
of the MFS have been analyzed by less direct genetic approaches, such
as second-site suppressor, site-directed, and cysteine-scanning
mutagenesis. Application of these techniques has clarified structural
features for several important model systems. For example, second-site
suppressor and site-directed mutagenesis has been used to identify
intra- and interhelical salt bridges in the H+/lactose
cotransporter LacY, the Pi:sugar 6-phosphate antiporter UhpT, and the H+:metal-tetracycline exchanger TetA(B), as
well as in the unrelated Na+/melibiose carrier MelB
(11-18). Cysteine-scanning mutagenesis, along with agents that exploit
sulfhydral chemistry, has provided a way to probe the functional and
structural significance of specific amino acid residues in LacY, UhpT,
and the oxalate:formate exchanger OxlT (12, 19-22). Such approaches
have also given insight concerning residues likely to interact with
substrate and helices that line the substrate translocation pathway
(19, 20, 22). Taken together, this information has led to formulation
of rational models for helix packing in the MFS (12, 14, 23, 24).
In work described here, we examined UhpT, the Pi-linked
hexose phosphate antiport carrier of Escherichia coli
(25-27), with an emphasis on the study of TM11. We focused on this
target because earlier work showed that one of its residues,
Lys391, can play a direct role in determining substrate
selectivity (15), suggesting that TM11 lines the substrate
translocation pathway. In the present analysis, we used both
cysteine-scanning and site-directed mutagenesis to broadly probe the
role(s) of TM11 and to ask whether other residues in this segment might
also influence substrate selectivity. Our analysis implicates positions 388 and 391 in TM11 as crucial determinants of substrate selectivity and suggests that TM11 is an 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 the sequential polymerase chain reaction (29). To confirm the
desired mutation and to rule out the presence of other changes, mutant
alleles were sequenced either in the laboratory, using the Sequenase
(version 2.0) reaction (Amersham Pharmacia Biotech), or at the
Biosynthesis and Sequencing Facility of Johns Hopkins Medical School.
Immunoblot Analysis--
SDS-polyacrylamide gel electrophoresis
was performed using cell extracts without preheating in sample buffer,
as described (30). Protein was transferred to nitrocellulose and probed
with a peptide-directed rabbit antibody reactive to a UhpT C-terminal epitope (31, 32). Western blots were developed using chemiluminescence (Amersham Pharmacia Biotech), and expression of UhpT and its
derivatives was quantitated by densitometry of digitized images (32,
33). Expression levels for each mutant were determined in at least three independent trials; mean values were used to normalize G6P transport for calculation of specific activity.
Transport Assays--
Unless otherwise indicated, overnight
cultures were diluted 200-fold into LB broth plus antibiotics (10 µg/ml streptomycin, 100 µg/ml ampicillin), grown at 37 °C to a
density of 2-5 × 108 cells/ml, and harvested by
centrifugation. Cells were washed twice and resuspended in buffer A (50 mM MOPS/K+, 100 mM
K2SO4, 1 mM MgSO4, pH
7) at OD660 = 1.4, equivalent to about 2 × 109 cells/ml. Cell suspensions were allowed to equilibrate
at room temperature, and tests of G6P, F6P, or PEP transport were
initiated by adding a one-twentieth volume of labeled substrate to a
final concentration of 50 µM. At the indicated times,
aliquots were removed for filtration on Millipore filters (0.45-µm
pore size), followed by two washes with 5 ml of buffer A lacking
MgSO4. To monitor PCMBS inhibition of G6P and PEP
transport, cells were first incubated for 10 min with PCMBS (0.05-200
µM) at room temperature. Aliquots were then filtered to
remove the inhibitor, and after two washes with 5 ml of buffer A, tests
of transport were initiated by overlaying cells (on the filter) with
buffer A containing 50 µM labeled substrate. To assess
substrate protection, the same procedure was used except that cells
were preincubated with either 1 mM unlabeled substrate or 1 mM unlabeled substrate plus PCMBS.
For assays of UhpT-mediated Pi transport, cells were grown
in M63 minimal medium (34) (pH 7) containing thiamine (2 µg/ml), required amino acids (50 µg/ml), necessary antibiotics, and 0.2% (w/v) glucose as carbon source. The high Pi content of M63
ensured maximal repression of other Pi transporters (Pst
and Pit) present in broth-grown cells, so that most Pi
transport occurred via UhpT (15, 35). (Broth- and M63-grown cells had
comparable behavior with regard to the organophosphates transported by
UhpT.)
The maximal velocity (Vmax) and Michaelis
constant (Km) of Pi transport were
estimated using the method of Hofstee (36). The inhibition constants
(Ki) for 2-dG6P, F6P, or PEP as inhibitors of
Pi transport were determined using linear Dixon plots (37),
with the assumption of competitive inhibition between Pi
and the test substrate. In these latter assays, the unlabeled test
substrates (G6P, 2-dG6P, F6P, PEP) contained 0.5-1.0% Pi
(38), and because this was ignored in calculation of kinetic constants,
the derived Ki values should be considered minimal estimates.
To monitor in vivo function of UhpT and its derivatives,
cells from an overnight broth culture were diluted 1000-fold into M63
containing 0.15% F6P or 0.15% 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--
[14C]G6P (52.8 µCi/µmol) and
[32P]KPi (1 Ci/mmol) were obtained from
PerkinElmer Life Sciences. [14C]F6P (300 µCi/µmol) and [14C]PEP (29 µCi/µmol) were from
American Radiolabeled Chemicals, Inc. and Amersham Pharmacia Biotech,
respectively. Unlabeled organophosphate substrates (G6P, 2-dG6P, F6P,
PEP) were obtained from Sigma.
TM11 as a Determinant of Substrate Specificity--
In previous
work, we had identified Asp388 and Lys391 of
UhpT TM11 as participants in an intrahelical salt bridge (15). Further study of this ion pair showed that if the anionic partner is replaced by cysteine, the derivative protein has a substrate selectivity biased
toward compounds, such as PEP, that carry an additional anionic charge
(15). Because PgtP, the related PEP transporter of Salmonella
typhimurium, has arginine at this position, we argued that the
gain-of-function phenotype reflects the presence of an uncompensated
positive charge (lysine) at position 391 and not the presence of
cysteine, a proton donor, at position 388. In the present study, this
supposition is confirmed by the finding of an equivalent result after
replacement of Asp388 with Val, the amino acid occupying
the corresponding position in PgtP. Thus, although phosphate transport
by both the D388C and D388V variants shows a kinetic profile resembling
the parent protein (Table I), each mutant
has a 20-25-fold reduced affinity for hexose 6-phosphate, coupled with
a 10-fold increase in the affinity for PEP (Table I). Tests of both
growth and transport confirmed that each mutant, but not their parent,
could use PEP as sole carbon source for growth (Fig.
1).
Such findings confirm that an uncompensated positive charge at position
391 alters selectivity so as to favor substrates with an increased
anionic character. With this in mind, we next asked whether the
location of such charge is also a critical determinant of specificity.
To do this, we constructed the D388C/K391C variant to remove both
partners in the natural salt bridge. This variant, like its parent, is
able to transport sugar 6-phosphates but not PEP (Fig. 1). We then
scanned this derivative with lysine placed at positions in a
registration (i, i ± 3 or 4) that would
span four turns of an
The finding that UhpT selectivity is biased in favor of more anionic
substrates upon placement of a lone cationic residue at position 388 or
391 led us to ask if a change in selectivity would also arise on
introduction of a negative charge at either of these positions. The
K391D/D388C variant, in which aspartate is moved to position 391, was
too poorly expressed for such study, so that only the K391C variant was
examined. We considered two possible models. On the one hand, an
uncompensated anionic residue at position 388 might bias selectivity to
favor substrates carrying an extra cationic group. Equivalently,
altered selectivity might favor substrates with one less negative
charge. That the K391C variant had near parental activity, with only a
2-5-fold decrease in affinity for both G6P and F6P (Tables I and
II; Figs. 1A, 2C, and
3B) did not favor the
first model. In the same way, tests (data not shown) using either
glucosamine 6-phosphate and O-phosphorylethanolamine as
inhibitors of Pi transport failed to support the idea.
Our next tests addressed the idea that the uncompensated negative
charge at position 388 biased selectivity toward substrates carrying
one negative charge rather than two (i.e.
HG6P1 UhpT TM11 Lies on the Substrate Transport Pathway--
Because the
topology of UhpT shows the Lys391-Asp388 salt
bridge to be deep within the hydrophobic sector (15, 19, 39, 40), it
seemed plausible that a water-filled pathway would extend inward from
the periplasm to at least position 388. If so, we reasoned that one
might identify the part of TM11 bordering this pathway by use of a
suitable probe. Accordingly, we next analyzed a library of
single-cysteine variants encompassing the whole of TM11 (residues 383-404), so that individual positions along TM11 could be probed by
PCMBS, an impermeant, thiol-directed agent with the same molecular volume and charge as the normal substrates of UhpT (20).
An initial screen of TM11 single-cysteine variants showed that most
were expressed at a level sufficient for functional analysis (usually
30%) (Table II). In the preceding work (Table I, Fig. 1; Ref.
15), we found that gain-of-function variants in which Lys391 acts as a selectivity determinant retained
UhpT-mediated Pi transport despite a defect in transport of
G6P. By contrast, of the six TM11 single-cysteine derivatives with
substantially reduced G6P transport (~10% or less) (Table II), all
but one had comparable reductions in Pi transport (data not
shown); the exceptional case was the D388C variant, which retained
normal levels of Pi transport, as described in the earlier
study (15). Thus, generation of a gain-of-function phenotype by
cysteine substitution may be a rare event.
In other experiments, we examined each single-cysteine variant with
regard to its response to the impermeant thiol-reactive probe PCMBS.
The first trials monitored the effects of a 10-min exposure to 200 µM PCMBS. As judged by inhibition of G6P transport, we
concluded that eight positions were accessible to the probe (positions
391, 393, 395, 397, 398, 401, 402, and 404); study of PEP transport by
the D388C derivative showed that position 388 was also accessible to
PCMBS (Table III). For each of these PCMBS-responsive mutants, we then estimated the PCMBS concentration yielding 50% inhibition (K0.5) (Table III).
This effort suggested two classes of derivatives. Most mutants (seven
of nine) showed a relatively high sensitivity, with
K0.5 values in the range of 0.05-3
µM, whereas two variants showed relatively low
sensitivity (K0.5 of 20-40 µM)
(Table III). Finally, for each PCMBS-responsive protein, we evaluated
whether protection was afforded by coincubation with substrate (1 mM G6P or PEP), using a probe concentration near its
K0.5 value. Neither of the two variants of low
sensitivity to PCMBS showed evidence of substrate protection, whereas
six of the seven highly sensitive mutants benefited from the presence of substrate (Table III).
We conclude from these experiments that positions accessible to PCMBS
(Table III) may be found along nearly the full length of TM11
(positions 388-402 versus positions 383-404), suggesting that a hydrophilic pathway extends inward along a similar length. That
substrate protection is observed for many of these positions, especially those deep within the hydrophobic sector, indicates that
this pathway is closed by the structural changes that accompany substrate binding or transport.
For a number of membrane transport proteins, use of site-directed
and scanning mutagenesis has identified charged residues that
contribute to a substrate-binding domain. For example, within the MFS
two charged residues (Glu126 and Arg144)
coordinate substrate binding by LacY (24), several cationic residues
(Arg46, Arg275, and Lys391)
function as recognition elements in UhpT (15, 33), and a lysine
(Lys355) facilitates substrate binding by OxlT, the
oxalate:formate antiporter of Oxalobacter formigenes (22,
41). The MFS also contains examples in which one or more transmembrane
helices have been associated with the permeation pathway. Thus, TM5 of
the GLUT1 glucose uniporter and TM7 of UhpT are known to line the
sugar/sugar phosphate transport pathway (20, 42), as do residues on
TM11 of OxlT (41). Similarly, several helices in LacY have been
shown to line the pathway taken by lactose (12).
In the present study, we exploited site-directed and cysteine-scanning
mutagenesis, together with the use of thiol-directed probes, as tools
to analyze TM11 of UhpT. This focus was based on early work showing
that Lys391, normally part of an intrahelical TM11 salt
bridge, acts as a determinant of substrate specificity when present
without its normal partner, Asp388 (15). This finding
showed that at least one residue on TM11 must intersect with the UhpT
transport pathway. New evidence presented here both reinforces this
idea and extends the argument to implicate a substantial portion of
TM11 as lining the pathway. Thus, it is now clear that positive charge
placed (as Lys) at either position 388 or 391 can influence substrate
selectivity (see also below), as does a lone negative center at
position 388, strengthening the argument that residues on TM11 have
strategic value. Moreover, study of a single-cysteine panel now shows
that the impermeant and anionic probe PCMBS has access to a large tract
along TM11, encompassing nearly two-thirds of its length, from position
404 to position 388 (Table III; Fig. 4).
The periodicity of PCMBS accessibility (Fig. 4) suggests that TM11 is
an -helix, with approximately two-thirds of its surface lining a
substrate translocation pathway. We suggest that this feature is a
general property of carrier proteins in the major facilitator
superfamily and that for this reason residues in TM11 will serve to
carry determinants of substrate selectivity.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-helical conformation. In select cases, a high
preponderance of
-helix has been confirmed by circular dichroism or
electron spin resonance spectroscopies (7-9). Similar tests suggest
that an unrelated transporter, the Na+/H+
antiporter, NhaA, also has 12 transmembrane helices, and in this case
two-dimensional crystallography has confirmed the inference (10). In no
case, however, has the structure of a secondary transporter been solved
to a resolution affording molecular analysis.
-helix, with roughly two-thirds of its
surface facing a water-filled translocation pathway.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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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) (28) served as host
for tests of expression and function of plasmid-encoded UhpT. Plasmid
pTrc(HisC0S6) encodes the histidine-tagged,
cysteine-less UhpT (15) that served as parent for all UhpT derivatives
described in this study.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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Transport properties of UhpT mutants displaying altered substrate
selectivity
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Fig. 1.
Growth on, and transport of, G6P and
PEP. Transport of G6P (A) and PEP (B) by
UhpT and its derivatives. Cells were grown in M63 minimal medium
containing 0.2% (w/v) glucose as carbon source, and rates of transport
were monitored by a 5-min incubation of cells with labeled substrate,
as described under "Experimental Procedures." Values shown are
means ± S.E. for three independent experiments. C,
RK5000 without ( ) and with plasmids expressing cysteine-less UhpT
(
) and its D388C (
), D388V (
), and D388K/K391C (
)
derivatives were grown in M63 minimal medium with PEP as sole
carbon source. Growth was monitored by changes in optical density at
660 nm. These data, from a single experiment, are representative of
findings made in three independent trials.
-helix. In most cases (positions 384, 387, 395, and 398), the introduction of lysine into the D388C/K319C
background yielded a non-functional protein (data not shown). It was
evident, however, that placement of Lys at either position
388 or 391 altered UhpT substrate selectivity so as to favor PEP. Thus,
the D388K/K391C variant showed a 10-fold decrease in affinity for
2-dG6P at the same time its affinity for PEP increased 40-fold (Table
I). As before, this gain-of-function derivative was able to both
transport and use PEP as sole carbon source for growth (Fig. 1).
UhpT expression and function in single-cysteine TM11 variants
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Fig. 2.
Growth on, and transport of, F6P at varying
pH. RK5000 without ( ) and with plasmids expressing the parental
cysteine-less UhpT (
) and its K391C derivative (
) were grown in
M63 minimal medium with F6P as a carbon source at pH 7 (A) and pH 5.5 (B). Growth was monitored as
described in the legend to Fig. 1. C, transport of F6P at
varying pH was examined for the parental cysteine-less UhpT (
) and
its K391C derivative (
). Data from three separate trials were
normalized to peak values (9.1-13 nmol/min/mg of protein for
cysteine-less UhpT; 2-4.6 nmol/min/mg of protein for its K391C
variant) and are shown as means ± S.E. Transport was measured as
described under "Experimental Procedures" except that assay and
wash buffers used PIPES rather than MOPS.
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Fig. 3.
Effect of pH on kinetic constants for F6P
transport. Km (A) and
Vmax (B) values for parental
cysteine-less UhpT ( ) and its K391C derivative (
). Assays were
performed as described under "Experimental Procedures" except that
assay and wash buffers used MES/MOPS rather than MOPS. Kinetic
constants are means ± S.E. for three independent
experiments.
rather than G6P2
). If so, one
might expect enhanced transport and growth under more acidic conditions
where monovalent sugar phosphate is enriched. To test this possibility,
we measured transport and growth at external pH values from pH 5.5 to
8.25, a range that spans the pK1 of sugar
phosphate (pK1 = 6.1). In such work, we found
that maximal transport by the mutant (tested at 50 µM
substrate) occurred at a pH approximately 0.5 pH units more acidic than
found for the parent protein (pH 6.3 and 6.9, respectively) (Fig.
2C). We also found that the K391C variant, unlike its
parent, showed a marked decrease in function above pH 7. Both
observations are consistent with the idea that the mutant works best
when monovalent sugar phosphate is the predominant species. This idea
was further supported by kinetic work showing that the
Km for sugar 6-phosphate transport by the mutant
increased 4-5-fold at alkaline pH (125-640 µM), whereas
that of the parental protein remained constant throughout the entire pH
range studied (Fig. 3). In similar fashion, growth of the K391C
derivative was pH-dependent. Thus, although the parent
protein supports growth equally well between pH 5.5 and 7.0, the K391C
mutant grows at progressively slower rates as the environment becomes
more alkaline, with complete growth stasis at pH 7 (Fig. 2,
A and B). This finding, coupled with the kinetic
observations (Fig. 3), leads us to suggest that in this variant the
capacity to handle divalent sugar phosphate is compromised. This
interpretation further emphasizes the region in and around these
positions as essential determinants in substrate specificity.
UhpT function in single-cysteine TM11 variants exposed to PCMBS
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-helix having roughly two-thirds of its surface facing a
water-filled channel. Because of their relatively high
K0.5 values relative to other PCMBS-sensitive positions (Table III), we suggest that Thr393 and
Lys404 lie at the boundary of this surface. We also note
that this region lies adjacent to a stripe containing the uncharged
residues that show low specific activity as single-cysteine variants
(Gly389, Gly392, Tyr396,
Gly399) (Table II), suggesting that this second stripe
abuts a neighboring helix. The significance of these observations is
highlighted by the added finding that PCMBS-responsive residues are
unaffected by the probe when treatment is in the presence of excess
substrate. At the least, such substrate protection indicates that
access to this region is blocked by structural events that accompany substrate binding or transport, consistent with the idea that this
portion of TM11 forms part of the translocation pathway.
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Fig. 4.
-Helical representation of
TM11. Overhead (A) and side (B) view of TM11
(residues 383-404) modeled as an
-helix (3.6 residues per turn).
The cytoplasmic (residue 383) and periplasmic (residue 404) boundaries
are derived from the analysis of hydropathy (39) and the results of
reporter gene fusions in UhpT and GlpT (40, 43). Amino acid residues
accessible to PCMBS are shaded; residues shaded
black are protected from PCMBS attack by either G6P or PEP,
whereas those shaded gray are not. Boxed residues
in A refer to inactive variants (Table II). Dotted
lines in A demarcate the likely boundary between that
region of TM11 facing a water-filled translocation pathway and the
region that contacts either lipid or protein.
This structural model (Fig. 4) offers one way to view this part of the transport pathway, but it does not address the mechanism by which altered substrate selectivity becomes associated with positions 388 and 391. To understand this phenomenon, it is helpful to recall the elements believed to contribute to maintenance of substrate selectivity by the wild type protein. Because UhpT transports sugar phosphates but not sugars, there must be a mechanism that identifies the presence of phosphate at the substrate C6 (or C5) position. Crystallography of enzymes and receptors that interact with Pi or organophosphates shows that arginine residues almost always take part in the recognition of the anionic phosphoryl group (44-48; summarized in Ref. 33). (A prominent exception, triose phosphate isomerase, uses lysine rather than arginine (49)). We believe that in UhpT, this role is played by two arginines (Arg46 and Arg275) at the periplasmic poles of TM1 and TM7, about 30 residues prior to the internal duplication characteristic of all members of the MFS (33, 50); only these arginines are both essential and conserved throughout the UhpT family (33). Much less is known about the interaction between UhpT and the polyol ring of its ligands, but crystallography of carbohydrate-protein complexes suggests that this may occur via aromatic amino acid stacking interactions with the furanose/pyranose ring and by hydrogen bonding with at least some of the offshooting hydroxyls (51-53). These interactions are presumed to occur within the hydrophobic core of UhpT, because the arginines required for recognition of the phosphoryl group are located near the periphery. Certainly, the finding that an uncompensated positive charge at position 388/391 allows UhpT to process substrates carrying an additional anionic charge (e.g. PEP) is consistent with the idea that a substrate orients within UhpT with its anionic phosphate pointed toward the periplasm and the C1 position toward the cytoplasm (15).
What determines whether substrates on the translocation pathway will be transported? Early work indicated that the exchange reactions mediated by UhpT are electrically neutral in nature (27, 54), and the abrupt alteration in selectivity that accompanies lysine insertion at positions 388 or 391 reinforces the idea that maintenance of electrostatic neutrality is an essential criterion. We also believe that this same view can help interpret the behavior of the K391C variant, which shows a distinct acid shift in the pH optimum for growth and transport (Figs. 2 and 3). We speculate that in this mutant the uncompensated electronegative center, Asp388, acts as a resident fixed anion, so that preservation of an electrostatic neutrality would require a selectivity biased toward monovalent sugar phosphate. A consequence of this bias would be that, as observed, the pH optimum for sugar phosphate transport and growth would shift in the acid direction. However, at the sugar phosphate concentrations used for growth studies, the levels of monovalent substrate would exceed the Km for transport, even at pH 7. Therefore, we believe that the growth phenotype is best explained by changes in the protonation state of Asp388. At more alkaline pH, the anionic charge associated with this position would preclude transport of divalent sugar phosphate, because electrostatic neutrality could not be achieved. Protonation of the resident anion would be favored as external pH is lowered, thereby restoring the parental character of the translocation pathway. This interpretation would be unrealistic if the pKa of Asp388 is as low as that found for aspartate in model compounds (pKa of 4) (55). On the other hand, it is known that the pKa of aspartate may take on a significantly higher value, depending on the nature of its local environment. It remains feasible, then, that the pKa of Asp388 falls within the range of pH values we have tested (Figs. 2 and 3).
The assumption that substrate preference is influenced by the protonation state of Asp388 also allows us to reconcile the contradictory findings that at pH 7 the K391C variant transports but does not grow on sugar phosphate (Fig. 2). UhpT, as do other Pi-linked antiporters, carries out both heterologous (Pi:sugar phosphate) and homologous (Pi:Pi, sugar phosphate:sugar phosphate) exchanges (25, 27, 38, 56). One might conclude that heterologous exchange is the preferred reaction in vivo, because homologous exchanges do not usually lead to net substrate fluxes. However, the affinity of UhpT for sugar phosphate is 10-40-fold higher than for Pi, suggesting that homologous exchange must be preferred over heterologous reaction (56). Furthermore, because E. coli maintains a slightly alkaline cytoplasmic pH over a wide range of external pH values (57), one expects a reaction in which net sugar phosphate accumulation arises from the electroneutral exchange of two monovalent sugar phosphates (external) for a single divalent species (internal) (25). We suggest that such an asymmetric exchange is not possible for the K391C mutant placed at relatively alkaline pH, because the deprotonated state of Asp388 would not permit use of the divalent anionic substrate. Instead, this mutant would only be able to carry out exchange using monovalent sugar phosphate. This might then appear as near normal levels of transport, but the overall 1-for-1 stoichiometry of such a reaction would not support growth.
Our observations also indicate that a large tract of TM11 lines the
UhpT translocation pathway. That UhpT TM7 also lies on the
translocation pathway (20) is in agreement with the general helix-packing model proposed by Goswitz and Brooker (23), which suggests that both of these helices are within the core of potential pathway-lining segments in MFS transporters. This model, as well as
mutagenesis studies in LacY and the H+/sucrose symporter
CscB (17, 58, 59), suggests that TM7 and TM11 may be neighbors in UhpT,
an idea that can be tested by disulfide cross-linking using the
single-cysteine libraries currently available.
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ACKNOWLEDGEMENT |
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We thank Robert Kadner for the gift of strain RK5000.
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FOOTNOTES |
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* This work was supported in part 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@ bs.jhmi.edu.
Published, JBC Papers in Press, May 10, 2001, DOI 10.1074/jbc.M102017200
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ABBREVIATIONS |
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The abbreviations used are: MFS, major facilitator superfamily; TM, transmembrane segment; G6P, glucose 6-phosphate; MOPS, 4-morpholinepropanesulfonic acid; F6P, fructose 6-phosphate; PEP, phosphoenolpyruvate; PCMBS, p-chloromercuribenzosulfonate; 2-dG6P, 2-deoxyG6P; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); MES, 2-(N-morpholino)ethanesulfonic acid.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Poolman, B., and Konings, W. N. (1993) Biochim. Biophys. Acta 1183, 5-39[Medline] [Order article via Infotrieve] |
2. | Maloney, P. C. (1994) Curr. Opin. Cell Biol. 6, 571-582[Medline] [Order article via Infotrieve] |
3. | Henderson, P. J. F. (1993) Curr. Opin. Cell Biol. 5, 708-721[Medline] [Order article via Infotrieve] |
4. | Marger, M. D., and Saier, M. H. (1993) Trends Biochem. Sci. 18, 13-20[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Pao, S. S.,
Paulsen, I. T.,
and Saier, M. H.
(1998)
Microbiol. Mol. Biol. Rev.
62,
1-34 |
6. | Paulsen, I. T., Sliwinski, M. K., and Saier, M. H. (1998) J. Mol. Biol. 277, 573-592[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Fu, D.,
and Maloney, P. C.
(1997)
J. Biol. Chem.
272,
2129-2135 |
8. | Voss, J., He, M. M., Hubbell, W. L., and Kaback, H. R. (1996) Biochemistry 35, 12915-12918[CrossRef][Medline] [Order article via Infotrieve] |
9. | Voss, J., Hubbell, W. L., Hernandez-Borrell, J., and Kaback, H. R. (1997) Biochemistry 36, 15055-15061[CrossRef][Medline] [Order article via Infotrieve] |
10. | Williams, K. A. (2000) Nature 403, 112-115[CrossRef][Medline] [Order article via Infotrieve] |
11. | Lee, J.-I., Varela, M. F., and Wilson, T. H. (1996) Biochim. Biophys. Acta 1278, 111-118[Medline] [Order article via Infotrieve] |
12. |
Kaback, H. R.,
Frillingos, S.,
Jung, H.,
Jung, K.,
Prive, G. G.,
Ujwal, M. L.,
Weitzman, C.,
Wu, J.,
and Zen, K.
(1994)
J. Exp. Biol.
196,
183-195 |
13. | Varela, M. F., and Wilson, T. H. (1996) Biochim. Biophys. Acta 1276, 21-34[Medline] [Order article via Infotrieve] |
14. |
Franco, P. J.,
and Wilson, T. H.
(1999)
J. Bacteriol.
181,
6377-6386 |
15. |
Hall, J. A.,
Fann, M.-C.,
and Maloney, P. C.
(1999)
J. Biol. Chem.
274,
6148-6153 |
16. | Varela, M. F., Brooker, R. J., and Wilson, T. H. (1997) J. Bacteriol. 179, 5570-5573[Abstract] |
17. | King, S. C., Hansen, C. L., and Wilson, T. H. (1991) Biochim. Biophys. Acta 1062, 177-186[Medline] [Order article via Infotrieve] |
18. |
Someya, Y.,
Kimura-Someya, T.,
and Yamaguchi, A.
(2000)
J. Biol. Chem.
275,
210-214 |
19. | Yan, R.-T., and Maloney, P. C. (1993) Cell 75, 37-44[Medline] [Order article via Infotrieve] |
20. |
Yan, R.-T.,
and Maloney, P. C.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
5973-5976 |
21. | Matsuzaki, S., Weissborn, A. C., Tamai, E., Tsuchiya, T., and Wilson, T. H. (1999) Biochim. Biophys. Acta 1420, 63-72[Medline] [Order article via Infotrieve] |
22. |
Fu, D.,
and Maloney, P. C.
(1998)
J. Biol. Chem.
273,
17962-17967 |
23. |
Goswitz, V. C.,
and Brooker, R. J.
(1995)
Protein Sci.
4,
534-537 |
24. |
Venkatesan, P.,
and Kaback, H. R.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9802-9807 |
25. | Maloney, P. C., Ambudkar, S. V., Anantharam, V., Sonna, L. A., and Varadhachary, A. (1990) Microbiol. Rev. 54, 1-17 |
26. | Maloney, P. C., and Wilson, T. H. (1996) in Escherichia coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C. , Curtiss, R. , Ingraham, J. L. , Lin, E. C. C. , Low, K. B. , Magasanik, B. , Reznikoff, W. S. , Riley, M. , Schaechter, M. , and Umbarger, H. E., eds) , pp. 1130-1148, American Society of Microbiology, Washington, D. C. |
27. |
Sonna, L. A.,
Ambudkar, S. V.,
and Maloney, P. C.
(1988)
J. Biol. Chem.
263,
6625-6630 |
28. | Weston, L. A., and Kadner, R. J. (1987) J. Bacteriol. 169, 3546-3555[Medline] [Order article via Infotrieve] |
29. | Ho, S. N., Hunt, H. D., Morton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene 77, 51-59[CrossRef][Medline] [Order article via Infotrieve] |
30. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
31. | Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual , pp. 486-510, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
32. |
Matos, M.,
Fann, M.-C.,
Yan, R.-T.,
and Maloney, P. C.
(1996)
J. Biol. Chem.
271,
18571-18575 |
33. | Fann, M.-C., Davies, A. H., Varadhachary, A., Kuroda, T., Sevier, C., Tsuchiya, T., and Maloney, P. C. (1998) J. Membr. Biol. 164, 187-195[CrossRef][Medline] [Order article via Infotrieve] |
34. | Miller, J. H. (1972) Experiments in Molecular Genetics , p. 431, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
35. | Rosenberg, H. (1987) in Ion Transport in Prokaryotes (Rosen, B. P. , and Silver, S., eds) , pp. 205-248, Academic Press, Inc., San Diego, CA |
36. | Hofstee, B. H. J. (1959) Nature 184, 1296-1298[Medline] [Order article via Infotrieve] |
37. | Dixon, M. (1953) Biochem. J. 55, 170-171 |
38. | Maloney, P. C., Ambudkar, S. V., Thomas, J., and Schiller, L. (1984) J. Bacteriol. 158, 238-245[Medline] [Order article via Infotrieve] |
39. | Friedrich, M. J., and Kadner, R. J. (1987) J. Bacteriol. 169, 3556-3563[Medline] [Order article via Infotrieve] |
40. | Island, M. D., Wei, B.-Y., and Kadner, R. J. (1992) J. Bacteriol. 174, 2754-2762[Abstract] |
41. |
Fu, D.,
Sarker, R. I.,
Bolton, E.,
and Maloney, P. C.
(2001)
J. Biol. Chem.
276,
8753-8760 |
42. |
Mueckler, M.,
and Makepeace, C.
(1999)
J. Biol. Chem.
274,
10923-10926 |
43. | Gott, P., and Boos, W. (1988) Mol. Microbiol. 2, 655-663[Medline] [Order article via Infotrieve] |
44. | Leucke, H., and Quiocho, F. A. (1990) Nature 347, 402-406[CrossRef][Medline] [Order article via Infotrieve] |
45. | Johanson, R. A., and Henkin, J. (1985) J. Biol. Chem. 260, 1465-1474[Abstract] |
46. | Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187[Medline] [Order article via Infotrieve] |
47. |
Lundqvist, T.,
and Schneider, G.
(1989)
J. Biol. Chem.
264,
7078-7083 |
48. | Evans, P. R., Farrants, G. W., and Hudson, P. J. (1981) Philos. Trans. R. Soc. Lond. B Biol. Sci. 293, 53-62[Medline] [Order article via Infotrieve] |
49. | Lodi, P. J., Chang, L. C., Knowles, J. R., and Komives, E. A. (1994) Biochemistry 33, 2809-2814[Medline] [Order article via Infotrieve] |
50. | Griffith, J. K., Baker, M. E., Rouch, D. A., Page, M. G. P., Skurray, R. A., Paulsen, I. T., Chater, K. F., Baldwin, S. A., and Henderson, P. J. F. (1992) Curr. Opin. Cell Biol. 4, 684-695[Medline] [Order article via Infotrieve] |
51. | Sharff, A. J., Rodseth, L. E., and Quiocho, F. A. (1993) Biochemistry 32, 10553-10559[Medline] [Order article via Infotrieve] |
52. | Vyas, N. K. (1991) Curr. Opin. Struct. Biol. 1, 732-740 |
53. | Quiocho, F. A., Spurlino, J. C., and Rodseth, L. E. (1997) Structure 5, 997-1015[Medline] [Order article via Infotrieve] |
54. | Ambudkar, S. V., Sonna, L. A., and Maloney, P. C. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 280-284[Abstract] |
55. | Kyte, J. (1995) Structure in Protein Chemistry , p. 59, Garland Publishing Inc., New York |
56. |
Fann, M.-C.,
and Maloney, P. C.
(1998)
J. Biol. Chem.
273,
33735-33740 |
57. | Padan, E., Zilberstein, D., and Rottenberg, H. (1976) Eur. J. Biochem. 63, 533-541[Abstract] |
58. | He, M. M., Voss, J., Hubbell, W. L., and Kaback, H. R. (1995) Biochemistry 34, 15661-15666[Medline] [Order article via Infotrieve] |
59. | Frillingos, S., Sahin-Toth, M., Lengeler, J. W., and Kaback, H. R. (1995) Biochemistry 34, 9368-9373[Medline] [Order article via Infotrieve] |