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J Biol Chem, Vol. 273, Issue 50, 33735-33740, December 11, 1998
From the Department of Physiology, Johns Hopkins Medical School,
Baltimore, Maryland 21205
UhpT, the sugar phosphate transporter of
Escherichia coli, acts to exchange internal inorganic
phosphate for external hexose 6-phosphate. Because of this operational
asymmetry, we studied variants in which right-side-out (RSO) or
inside-out (ISO) orientations could be analyzed independently to ask
whether the inward- and outward-facing UhpT surfaces have different
substrate specificities. To study the RSO orientation, we constructed a
histidine-tagged derivative, His10K291C/K294N, in which the
sole external tryptic cleavage site (Lys294) had been
removed. Functional assay as well as immunoblot analysis showed that
trypsin treatment of proteoliposomes containing
His10K291C/K294N led to loss of about 50% of the original
population, reflecting retention of only the RSO population. To study
the ISO orientation, we used a His10V284C derivative, in
which a newly inserted external cysteine (Cys284) conferred
sensitivity to the thiol-reactive agent,
3-(N-maleimidylpropionyl)biocytin. In this case,
3-(N-maleimidylpropionyl)biocytin treatment of
proteoliposomes containing His10V284C gave about a 60%
loss of activity, and immunodetection of biotin showed parallel
modification of an equivalent fraction of the original population.
Together, such findings indicate that the UhpT RSO and ISO orientations
are in about equal proportion in proteoliposomes and that a single
population can be generated by exposure of these derivatives to the
appropriate agent. This allowed us to study proteoliposomes with UhpT
functioning in RSO orientation (His10K291C/K294N) or ISO
orientation (His10V284C) with respect to the kinetics of
glucose 6-phosphate transport by phosphate-loaded proteoliposomes and
also the inhibitions found with 2-deoxy-glucose 6-phosphate, mannose
6-phosphate, galactose 6-phosphate, fructose 6-phosphate, and inorganic
phosphate. We found no significant differences in the behavior of UhpT
in its different orientations, indicating that the transporter
possesses an overall functional symmetry.
In Escherichia coli, the uhp locus (for
uptake of hexose phosphates)
coordinates the expression of four proteins responsible for
incorporation of external sugar phosphate. UhpA and UhpB together form
a two-component regulatory system that activates expression of the
transporter, UhpT, after extracellular G6P binds to the membrane
receptor, UhpC (1). UhpT then acts to move sugar 6-phosphate inward in
exchange for internal inorganic phosphate (2) in an electrically
neutral antiport reaction (2, 3). Hydropathy analysis of the UhpT amino
acid sequence (4), the properties of UhpT-PhoA fusions (5, 6), and
comparisons with other members of the major facilitator superfamily (7,
8) all argue that UhpT has 12 transmembrane To address this issue, we used an in vitro preparation in
which purified protein can be studied in proteoliposomes loaded with
inorganic phosphate. Because proteoliposomes might have UhpT in either
(or both) a right-side-out
(RSO)1 or inside-out (ISO)
orientation, we also designed UhpT derivatives in which one or the
other of these orientations could be eliminated by exposure to an
appropriate blocking agent. For example, in one case we reasoned that
the high proportion of lysine and arginine residues at the UhpT inner
surface would confer sensitivity to trypsin (see Fig. 1). To exploit
any such side-specific trypsin sensitivity, we constructed a variant
lacking the sole tryptic cleavage site at the UhpT extracellular
surface (Lys294), so that only RSO molecules would survive
exposure to protease. Alternatively, we inserted cysteine
(Cys284) into the same extracellular loop (see Fig. 1),
creating a mutant inhibitable by low concentrations of MPB, a
thiol-reactive agent without effect on wild type UhpT (9); in this
case, only ISO molecules would remain active after MPB exposure.
Study of these variants suggests that the RSO and ISO forms are present
in about equal proportions in proteoliposomes. This finding, in turn,
made it possible to ask whether the two orientations showed
differential substrate specificity. For this purpose, we conducted a
simple kinetic study of G6P transport by proteoliposomes containing
UhpT in mixed, RSO, and ISO orientations and also evaluated the
inhibitions exerted by alternative UhpT substrates. We found no
substantial differences in the behavior of UhpT in its different orientations, indicating that the transporter possesses functional symmetry.
Cells, Plasmids, and Proteins--
In His10UhpT, the
N terminus of the wild type protein has a polyhistidine extension
allowing metal affinity purification (10). Its histidine-tagged
derivative, His10K291C/K294N, was generated by double
stranded mutagenesis (Chameleon®, Stratagene), using an
oligonucleotide specifying two mutations, K291C and K294N, as well as a
silent BstBI site for purposes of identification; in the
His10V284C derivative, a single mutation (V284C) removing an AccI site was introduced. The nature of each mutant was
verified by sequencing (Core Facility, Johns Hopkins University). As
host for plasmids encoding His10UhpT and its derivatives,
we used E. coli strain RK5000, which carries a deletion
within the chromosomal uhp locus (4). Expression of
His10UhpT and its derivatives was under the control
trc promoter (10).
Purification and Reconstitution--
To purify
His10UhpT and its derivatives (10), membrane vesicles
(20-40 mg of protein) (11) were solubilized at 4 °C by addition of
8 ml of buffer A (200 mM NaCl, 100 mM potassium
phosphate, pH 8, 50 mM G6P, 10% (v/v) glycerol, 1.5%
(w/v) n-dodecyl- Transport Assay--
To assay transport, washed proteoliposomes
were diluted 10-fold with assay buffer and preincubated at 23 °C for
3 min before adding 50 µM [14C]G6P. At the
indicated times, 100-µl aliquots were filtered on 0.22-µm GSTF
Millipore filters and washed twice with 5 ml of assay buffer. To assess
competition between G6P and alternate substrates, the test substrate
was added together with 50 µM [14C]G6P for
2 min before filtration.
SDS-PAGE and Immunoblot Analysis--
After SDS-PAGE (12),
protein was visualized by systems directed to either the UhpT N or C
terminus. In one case, we used horseradish peroxidase-conjugated
nickel-nitrilotriacetic acid (Qiagen) to monitor the N-terminal
polyhistidine; alternatively, we used a rabbit polyclonal antibody
directed against the UhpT C terminus (9, 10). Signals were developed by
enhanced chemiluminescence (Amersham Pharmacia Biotech) and quantitated
as described (8). To detect modification by MPB, we used low SDS
(<0.15%) during sample preparation and electrophoresis to preserve
the UhpT-MPB-streptavidin complex (9).
Chemicals--
Detergents were purchased from
Calbiochem-Novabiochem. E. coli phospholipid was from Avanti
Polar Lipids, Inc., trypsin (sequencing grade, tosylphenylalanyl
chloromethyl ketone-treated) was from Worthington Biochemicals, and MPB
was from Molecular Probes, Inc. D-1-[14C]G6P
(54 mCi/mmol) was obtained from NEN Life Science Products.
Early work showed that addition of trypsin to sensitized intact
cells leads to cleavage of wild type UhpT but not of a variant lacking
Lys294 (9). However, subsequent studies in proteoliposomes
appeared to contradict this finding, because even mutants lacking
Lys294 had some deficit after exposure to protease (see
below). A simple explanation for this discrepancy is that UhpT exists
in mixed orientation in proteoliposomes, with some molecules inserted
RSO and others ISO. Thus, the partial trypsin sensitivity of
Lys294 variants would reflect cleavage of the ISO
orientation but not the RSO population. This hypothesis prompted us to
develop two UhpT variants, His10K291C/K294N and
His10V284C (see "Experimental Procedures"), whose
properties would allow us to evaluate the proportion of RSO and ISO
forms in proteoliposomes and to characterize the properties of each orientation.
Comparison of His10UhpT with
His10K291C/K294N and His10V284C--
The
variants studied in this work each have a polyhistidine N-terminal
extension to facilitate protein purification (10). The parental
molecule, His10UhpT, was otherwise wild type in sequence. In one derivative, His10K291C/K294N, Lys291 and
Lys294 were replaced by cysteine and asparagine,
respectively, thereby eliminating tryptic cleavage
site(s)2 exposed in the
extracellular loop between TM7 and TM8 (Fig.
1). Using this variant, we anticipated
that only RSO molecules would survive exposure of proteoliposomes to
trypsin. In a second derivative, His10V284C, we placed a
cysteine in the same extracellular loop (Fig. 1) to provide a target
for the thiol-reactive agent, MPB (9); here, we expected MPB
modification of Cys284 to inhibit the RSO but spare ISO
proteins.
Initial experiments showed that each variant could be purified in high
yield, and a simple kinetic study of [14C]G6P transport
showed comparable behavior for both wild type and mutant proteins (Fig.
2); maximal velocities were within a 2-fold range, and in each case we recorded a Michaelis constant (Km) of about 50 µM. We confirmed that
treatment of His10K291C/K294N proteoliposomes with trypsin
and His10V284C proteoliposomes with MPB gave partial
inhibitions (about 50% in each case) as expected if both RSO and ISO
populations were present (data not shown; see below). In those cases,
we also found that the Km for G6P transport was
largely unaffected by treatment with trypsin or MPB. Thus, with
His10K291C/K294N we noted Km values of
64 and 67 µM, respectively, for the control and
trypsin-treated material, whereas with His10V284C, we found
Km values of 32 and 39 µM for control
and MPB-treated preparations. These findings would suggest that the RSO
and ISO orientations have comparable kinetic responses to G6P
(Km values of 67 and 39 µM,
respectively), if it can be shown that trypsin and MPB act in the
manner predicted. In the experiments described below, therefore, our
first goal was to provide direct evidence that these probes function as
expected.
RSO Molecules Remain after Exposure of His10K291C/K294N
Proteoliposomes to Trypsin--
The tryptic cleavage site at the UhpT
extracellular surface is absent in His10K291C/K294N,
leading one to expect that only the lysine/arginine-rich intracellular
surface of ISO forms would be sensitive to external trypsin. To test
this prediction we studied trypsin sensitivity with proteoliposomes
containing either His10UhpT or His10K291C/K294N
(Fig. 3). Both proteins showed partial
sensitivity at low levels of trypsin (<10-100 µg/ml), whereas at
higher levels (>100 µg/ml), although His10UhpT continued
to display sensitivity, His10K291C/K294N never fell below
about 55% of its initial activity (Fig. 3A). These findings
are consistent with degradation of both RSO and ISO forms of
His10UhpT but loss of only the ISO population of
His10K291C/K294N, and direct tests fully support this
interpretation. For example, immunoblots using a C-terminal antibody
confirmed that trypsin had acted on the entire His10UhpT
population but on only a subset of the His10K291C/K294N
present (Fig. 3B, lanes 1-4). A similar
conclusion came after examination of duplicate gels probed with the
nickel-nitrilotriacetic acid-conjugated peroxidase designed to detect
the polyhistidine N terminus (Fig. 3B, lanes 5-8). Thus, cleavage at Lys294 in the RSO population
should generate an 18-kDa N-terminal fragment and a 33-kDa C-terminal
peptide, and, in fact, both products are visualized in the appropriate
blots (arrows in Fig. 3B, lanes 2 and
6). (The 18-kDa fragment shows poor recovery, perhaps
because small hydrophobic proteins are not well retained on
nitrocellulose during transfer from the parent gel.) In this same
experiment, we also monitored transport of [14C]G6P (Fig.
3C), and those data, too, supported the idea that trypsin
targets both RSO and ISO forms of His10UhpT but only ISO molecules of His10K291C/K294N. Moreover, from the residual
activity in His10K291C/K294N (55%), one would conclude
that the RSO and ISO forms were present in equal proportion; as noted
later, this reduction in activity is quantitatively matched by loss of
immunoreactivity at 50 kDa (see below). Together, these data indicate
that RSO and ISO orientations are present in about equal amounts after reconstitution of UhpT and that preparations containing only the RSO
population can be generated by trypsin treatment of
His10K291C/K294N.
ISO Molecules Remain after Exposing His10V284C
Proteoliposomes to MPB--
Work with His10K291C/K294N
(Fig. 3) gave positive evidence for RSO molecules in proteoliposomes.
To document the activity of the remaining ISO population, we next
studied His10V284C, in which a V284C substitution confers
high sensitivity to the thiol-reactive agent, MPB (9).
(Cys291 in His10K291C/K294N is not a target for
such agents (9).) Proteoliposomes containing His10UhpT were
not responsive to MPB at concentrations as high as 200 µM, but we found a clear sensitivity on the part of
His10V284C, whose activity was reduced by 60% (Fig. 4A), as anticipated if only
the RSO population of His10V284C is inhibitable by the
maleimide. With this assumption, the data indicate 50% inhibition of
the RSO molecules at 10-15 µM MPB. Matos et al. (9) had found a considerably higher apparent sensitivity for
this same mutation (50% inhibition at 1 µM MPB), but
this may be expected because they exposed for both longer time (10 min
versus 2 min) and at higher temperature (23 °C
versus 4 °C). In this experiment (Fig. 4), we had also
exposed MPB-treated and untreated proteoliposomes to the impermeant
thiol-reactive probe, [2-(trimethylammonium)-ethyl]methane-thiosulfonate (13). In that
case, inhibition of activity (by 55%) was found only for the
preparation not previously exposed to MPB (data not shown), supporting
the idea that MPB reactivity was confined to the RSO population.
To show MPB modification directly, we used streptavidin to generate a
His10V284C-biotin-streptavidin complex whose formation is
reflected by loss of immunoreactivity at 50 kDa and its partial recovery at 80-90 kDa (9) (Fig.
4B).3 We found no
evidence of such a complex after MPB treatment of His10UhpT
proteoliposomes (Fig. 4B, lanes 1 and
2) unless endogenous cysteines had been exposed by a
preincubation with SDS. By contrast, denaturation was not required for
formation of the complex in parallel trials with His10V284C
(Fig. 4B, lanes 4 and 5). Assays of
[14C]G6P transport in this same experiment confirmed that
MPB modification was, in fact, associated with inhibition of function
(Fig. 4C and see below).
Distribution of RSO and ISO Orientations in
Proteoliposomes--
In several experiments, we assessed the relative
loss of sugar phosphate transport caused by treatment with trypsin or
MPB and compared this with the fraction of protein that remained
unmodified as monitored by immunoblots. Both measurements showed
parallel change (Table I), and use of
either data set leads one to conclude that proteoliposomes contain RSO
and ISO orientations in roughly equal proportion.
Functional Equivalence of RSO and ISO Orientations--
In the
preliminary work comparing His10UhpT with its derivatives,
His10K291C/K294N and His10V284C, we noted no
significant change of Km for sugar phosphate
transport when proteoliposomes were treated to generate the single
orientations (see above). This may now be interpreted as evidence that
the two orientations have similar kinetic features during G6P
transport. To extend this analysis, we measured apparent inhibition
constants (Ki values) derived by competition between
[14C]G6P and a number of alternate substrates. The
experiments shown in Fig. 5 give examples
of such work using a substrate of relatively high affinity (fructose
6-phosphate, Ki The orientation of proteins incorporated into proteoliposomes has
been analyzed in several cases (15-21), and those findings suggest a
variable result depending not only on the method of reconstitution but
perhaps also on the protein in question and the detergent or
phospholipid employed (reviewed in Ref. 20; see also Ref. 21). A simple
argument developed by Rigaud (20) and others (21) suggests that if
reconstitution proceeds by insertion of protein into preformed
detergent-destabilized liposomes, one might anticipate a population of
uniform orientation due to preferential insertion of the hydrophobic
sector into the lipid bilayer. Because the cytoplasmic surface of most
membrane proteins is considerably more hydrophilic than the
extracellular face, this approach predicts recovery of proteins in a
largely ISO orientation, as observed with the Ca2+-ATPase
(19), the sodium/proline symporter (22), and the chimeric LacS protein
(21). By contrast, if reconstitution proceeds from mixed micelles, as
in the method used here, the final population is more likely to be of
mixed orientation; this latter expectation may also be favored if, as
here, the use of high lipid/protein ratios avoids protein packing
considerations by ensuring only a single transporter per proteoliposome
(3). Despite such arguments, it is clear that direct tests must be used
to evaluate each case.
In our work with UhpT, a simple detergent-dilution protocol was used
for reconstitution at a high lipid-to-protein ratio (3, 23, 24), with
the expectation that this antiporter might be recovered in mixed
orientation in proteoliposomes. The availability of specifically
engineered variants has allowed experimental verification of this
prediction. Thus, use of His10K291C/K294N, which lacks the
sole extracellular tryptic cleavage site (9), enabled an unambiguous
demonstration that about half the recovered activity arises from
molecules of a RSO orientation (Fig. 3 and Table I). Access to the ISO
population was offered by use of a second variant, His10V284C, whose MPB-reactive cysteine allowed inhibition
of the RSO orientation without effect on ISO molecules. It is also likely that such maneuvers, especially use of trypsin, are of general
utility. Most membrane proteins are enriched for tryptic cleavage sites
(lysine and arginine) on their cytoplasmic surfaces (25), so this
surface should be targeted preferentially by trypsin. By contrast, a
rarity of lysine and arginine at the extracellular surface suggests
this surface will have few, if any, tryptic cleavage sites; those that
are present may well be susceptible to mutagenesis. For these reasons,
trypsin may serve as a simple diagnostic tool for determining the
orientation of reconstituted protein and perhaps also as the basis of a
method for generating a preparation of uniformly RSO orientation as
illustrated here and in unpublished work in the OxIT, the
oxatate/formate antiporter of oxalobacter (26).
Use of His10K291C/K294N or His10V284C, along
with appropriate treatments with trypsin or MPB, made it possible to
characterize the UhpT RSO and ISO orientations in a direct way. Our key
finding has been that the RSO and ISO forms are functionally
equivalent in the handling of substrates of physiological
relevance (Table II). On the one hand, as noted earlier, the absence of
a Km shift for G6P after conversion of a population
of mixed to single orientations argues that the two UhpT surfaces
have roughly comparable kinetic features. We also complemented this
observation with a more extended survey to measure the apparent
inhibition constants for a variety of UhpT substrates (Table II).
Neither approach revealed substantial differences in the behavior of
the RSO or ISO orientations, and this distinguishes UhpT from several
bacterial porters in which a clear kinetic bias is evident (21, 22). Nevertheless, the finding of a functional symmetry is fully
consistent with the biological role of UhpT. Although this anion
exchanger may be thought of in the context of sugar phosphate
accumulation, under some conditions UhpT must also function in the
extrusion of excess phosphate (14). Parenthetically, we note this work did not examine the possibility that function might be affected by a
lipid asymmetry. Although there is no reason to suspect that such tests
would alter our basic conclusion, it is clear that for the LacY
protein, phospholipid composition can affect the coupling between
lactose and proton symport (27).
The finding that UhpT has functional symmetry is compatible with the
suggestion of a single substrate binding site alternately accessible
from either membrane surface (28, 29), as well as the proposal of a
simple Ping Pong kinetic mechanism (28) in which rate-limiting events
are not imposed by the pathways substrates use to move into and out of
the binding site (29). This last point is of special interest. Current
evidence suggests that the pathway is lined by several residues at the
center of TM7 (29, 30), and by two required arginine residues,
Arg46 and Arg275, located in equivalent
positions with respect to the N- and C-terminal halves of the molecule
(8). Because arginine is commonly found in binding sites that accept
phosphate or organic phosphates (cf. Ref. 8), it is presumed that
Arg46 and Arg275 contribute to the anionic
selectivity mechanism that ensures an overall electroneutral exchange
(14, 28). If so, one might expect these residues to lie at the midpoint
of the translocation pathway, yet present models of topology (5, 6)
place them at its external border (8). This, along with our present
finding of functional symmetry of the RSO and ISO forms, suggests
that the main rate-limiting events in anion exchange must occur after substrates interact with the binding site and/or selectivity mechanism; such events are presumably associated with changes of protein conformation required to close the pathway after substrate enters from
one surface, trapping substrate in an occluded state and then opening
the pathway to the other surface, enabling transmembrane passage
(29).
We thank Dr. R. J. Kadner for the gift
of strain RK5000. We also acknowledge helpful discussions with Drs. K. Alessi, R. Garnepudi, and J. Hall.
*
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.
The abbreviations used are:
RSO, right-side-out; G6P, glucose 6-phosphate; ISO, inside-out; MOPS, 4-morpholinepropanesulfonic acid; MPB, 3-(N-maleimidylpropionyl)biocytin; PAGE, polyacrylamide gel
electrophoresis; TM, transmembrane 2
Prior study concluded that Lys294
was the only trypsin-accessible residue at the UhpT extracellular
surface (9). This is true for the conditions originally tested (9), but
Lys291 also serves as a cleavage site at the high levels of
trypsin (1 mg/ml) used in these experiments.
3
We noted here, as reported before (9), that
reactivity of this complex to the anti-UhpT antibody was inefficient,
likely because of steric hindrance caused by bound streptavidin. As a result, formation of the complex with streptavidin is judged by loss of
immunoreactivity at 50 kDa.
Functional Symmetry of UhpT, the Sugar Phosphate Transporter of
Escherichia coli*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-helices and that its N and C termini lie in the cytoplasm (see Fig. 1). Accordingly, the
structure of UhpT (and related transporters) is strongly asymmetric along an axis perpendicular to the membrane surface. This fact, along
with the biochemical asymmetry of the in vivo reaction, in
which external sugar phosphate can exchanged for internal phosphate, raises the question of whether UhpT has an inherent bias in substrate preference at its extracellular and intracellular surfaces.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-maltoside, 0.2% (w/v) E. coli phospholipid, 5 mM 2-mercaptoethanol) and mixed with 0.4 ml of nickel-nitrilotriacetic acid-agarose equilibrated with
buffer A. After overnight incubation, the mixture was placed in a
Poly-PreP® column (Bio-Rad) and washed with 15 ml of buffer B (buffer
A prepared at pH 7 containing 50 mM imidazole and 1.5% n-octyl--D-glucopyranoside in place of 1.5%
n-dodecyl--maltoside). Before elution, the column was
washed with 0.5 ml of buffer B in which
n-dodecyl--maltoside replaced
n-octyl--D-glucopyranoside; bound protein
(0.3-0.5 mg) was eluted in 0.5 ml of buffer B with 200 mM
imidazole added in place of 200 mM NaCl. Purified protein (5-10 µg) was reconstituted by detergent dilution into
proteoliposomes loaded with 100 mM potassium phosphate (pH
7) as described (10), isolated and washed by centrifugation, and
finally resuspended (4 °C) in 1 ml of assay buffer (100 mM potassium sulfate, 50 mM MOPS/K , pH 7). To
study the UhpT RSO orientation, proteoliposomes with
His10K291C/K294N were resuspended with or without 1 mg/ml trypsin and then incubated at 33 °C for 1 h before adding 0.3 mM phenylmethylsulfonyl fluoride to stop the reaction. To
examine the ISO orientation, proteoliposomes with
His10V284C were resuspended with or without 0.2 mM MPB before a quench with 20 mM
2-mercaptoethanol. After either treatment (trypsin or MPB), the
suspension was expanded to 20 ml with iced assay buffer and
centrifuged, and proteoliposomes were finally resuspended in 1 ml of
assay buffer.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Topology of UhpT. The presumed topology
of UhpT is shown (4, 8, 28). Vertical columns represent the 12 transmembrane -helices (TM1-TM12). The locations of possible
tryptic cleavage sites (arginine and lysine) are shown as black
circles. The extracellular loop between TM7 and TM8 contains two
lysines that have been altered to asparagine and cysteine in the
His10K291C/K294N variant. The cysteine inserted at position
284 in the His10V284C variant is also shown. Previous work
(9) showed these residues each lie in the extracellular loop connecting
TM7 and TM8.
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Fig. 2.
Kinetics of His10UhpT and its
derivatives. His10UhpT ( 
) and its derivatives,
His10K291C/K294N (
) and His10V284C (
)
were purified by metal chelate affinity chromatography (10) and
reconstituted in phosphate-loaded proteoliposomes as described under
"Experimental Procedures." Initial rates of [14C]G6P
transport were measured by 2-min incubations with substrate. Maximal
velocities of 2.7, 1.3, and 3.1 µmol/min/mg protein were found for
His10UhpT, His10K291C/K294N, and
His10V284C, respectively. A Km of 50 µM was derived for each protein.
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Fig. 3.
Effects of trypsin on proteoliposomes
containing His10UhpT and His10K291C/K294N.
Proteoliposomes were treated with trypsin as described under
"Experimental Procedures." A, after exposure to trypsin
at the indicated concentrations, the initial rate of G6P transport was
estimated by a 5-min incubation with labeled substrate. Rates are
expressed as a fraction of the initial value. B,
proteoliposomes treated with 1 mg/ml trypsin were processed for
SDS-PAGE, and in duplicate gels protein was visualized by either the
C-terminal-specific antibody (lanes 1-4) or by the
N-terminal polyhistidine detection system (lanes 5-9). In
each gel, His10UhpT is shown at the left
(lanes 1, 2, 5, and 6);
His10K291C/K294N is shown at the right
(lanes 3, 4, 7, and 8);
control liposomes (no protein) treated with trypsin are shown in
lane 9. The arrow and arrowhead
indicate, respectively, the N-terminal 18-kDa fragment and C-terminal
33-kDa fragment expected of tryptic cleavage at Lys294 in
His10UhpT. C, in the same experiment,
[14C]G6P transport was measured for the control
(open symbols) and trypsin-treated (black
symbols) preparations of His10UhpT (left
panel) and His10K291C/K294N (right panel).
Values are the means ± S.E. of triplicate measurements.
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Fig. 4.
Effects of MPB on proteoliposomes containing
His10UhpT and His10V284C. Proteoliposomes
were exposed to MPB as described under "Experimental Procedures."
A, after exposure to MPB at the indicated concentrations,
the initial rates of G6P transport were measured as described in the
legend to Fig. 3. Rates are normalized to the untreated control value.
B, proteoliposomes exposed to 200 µM MPB were
processed for SDS-PAGE, and samples containing His10UhpT
(lanes 1-3) or His10V284C (lanes
4-6) were analyzed by immunoblot in the presence (lanes
2, 3, 5, and 6) or absence
(lanes 1 and 4) of 8 µg/ml streptavidin (9). In
two cases (lanes 3 and 6) proteoliposomes were
solubilized with 0.5% SDS before labeling with MPB and streptavidin.
C, in the same experiment, [14C]G6P transport
was measured for the control (open symbols) and MPB-treated
(black symbols) preparations of His10UhpT
(left panel) and His10V284C (right
panel). Values are the means ± S.E. of triplicate
measurements.
Distribution of RSO and ISO orientations in proteoliposomes
0.2 mM) and one of
relatively low affinity (phosphate, Ki 3-5
mM); a number of other experiments of this sort are
summarized by Table II. Collectively,
those data show that the two UhpT orientations respond in equivalent
fashions to the five substrates tested and that the ratio of derived
Ki values (RSO/ISO) varies no more than 2-fold for
substrates of physiological significance (14). Such tests were
conducted using proteoliposomes loaded with inorganic phosphate, but in
two additional experiments (not shown) we studied
His10V284C (± MPB) in G6P-loaded proteoliposomes. In that
case, apparent Ki values for inorganic phosphate were the same as before (Fig. 5 and Table II). Together with the data
noted in Fig. 5 and Table II, these findings lead us to conclude that
the two orientations of UhpT are functionally equivalent with respect
to processing of fructose 6-phosphate, mannose 6-phosphate, 2-deoxoxyG6P, galactose 6-phosphate, and inorganic phosphate.
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Fig. 5.
Apparent Ki values for
fructose 6-phosphate and inorganic phosphate.
[14C]G6P transport was measured in the absence and
presence of fructose 6-phosphate (top panels) or inorganic
phosphate (bottom panels) using proteoliposomes containing
UhpT of mixed orientation (open symbols) or uniform
orientation (closed symbols). The protein used to obtain RSO
or ISO orientations is indicated on the individual graphs. Dotted
lines intersect the abscissa at the apparent Ki
value. Given a purely competitive interaction with G6P, for these assay
conditions the apparent Ki should be about 80% of
the true Ki.
Apparent Ki values of UhpT substrates measured with
proteoliposomes containing protein of mixed, RSO, or ISO orientation
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
ACKNOWLEDGEMENTS
FOOTNOTES
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.
-helix.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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