From the Laboratoire de Physiologie des Membranes Cellulaires, Université de Nice Sophia-Antipolis and CNRS UMR 6078, Commissariat à l'Energie Atomique (LRC-CEA 16V), Villefranche sur mer, 06230 France
Received for publication, October 1, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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Previous photolabeling and limited proteolysis
studies suggested that one of the four basic residues (Arg-141) of the
N-terminal cytoplasmic loop connecting helices IV and V (loop 4-5) of
the melibiose permease (MelB) from Escherichia coli has a
potential role in its symport function (Ambroise, Y., Leblanc, G., and
Rousseau, B. (2000) Biochemistry 39, 1338-1345). A
mutagenesis study of Arg-141 and of the other three basic residues of
loop 4-5 was undertaken to further examine this hypothesis. Cys
replacement analysis indicated that Arg-141 and Arg-149, but not
Lys-138 and Arg-139, are essential for MelB transport activity.
Replacement of Arg-141 by neutral residues (Cys or Gln) inactivated
transport and energy-independent carrier-mediated flows of substrates
(counterflow, efflux), whereas it had a limited effect on co-substrate
binding. R141C sugar transport was partially rescued on reintroducing a positive charge with a charged and permeant thiol reagent. Whereas R149C was completely inactive, R149K and R149Q remained functional. Strikingly, introduction of an additional mutation in the C-terminal helix X (Gly for Val-343) of R149C restored sugar transport. Impermeant thiol reagents inhibited R149C/V343G transport activity in
right-side-out membrane vesicles and prevented sugar binding in a
sugar-protected manner. All these data suggest that MelB loop 4-5 is
close to the sugar binding site and that the charged residue Arg-141 is involved in the reaction of co-substrate translocation or substrate release in the inner compartment.
The melibiose permease
(MelB)1 of Escherichia
coli is a membrane-bound ion-coupled sugar co-transporter or
symporter that drives cell accumulation of Na+ and melibiose are transported in a 1:1 ratio, and their
order of binding to MelB at the outer surface (Na+ first)
and release in the inner compartment (Na+ last) are
sequential (13). The membrane potential enhances active
Na+-coupled sugar transport by increasing the rate of
Na+ dissociation in the cytoplasm, which is otherwise slow
and rate-limiting for MelB turnover (see review, Ref. 3). Cooperative
changes in MelB conformation upon substrate binding were inferred from the analysis of the intrinsic fluorescence of MelB and by using fluorescence resonance energy transfer spectroscopy (FRET) with a
fluorescent sugar analog (10, 14). Complementary evidence came from the
finding that MelB substrates afford a cooperative protection against
proteolysis of the highly charged cytoplasmic loop 4-5 connecting
helices IV and V of MelB (7).
Molecular biology, biochemical, and spectroscopic approaches have been
extensively used to collect information on the functional organization
of the transporter. This organization involves the presence of a
potential coordination network for the cation recognition and coupling
mechanism, including four aspartic acid residues distributed in the
inner halves of helices I (Asp-19), II (Asp-55 and Asp-59), and IV
(Asp-124) of the N-terminal hydrophobic domain of MelB (6, 15-18).
Analysis of the properties of second-site suppressor mutants supports
the hypothesis of a proximity between the three N-terminal helices (19,
20). Extensive Cys-scanning mutagenesis of residues of membrane domains
combined with chemical modifications of MelB with impermeant thiol
reagents or with second-site mutation analysis has been used to
identify sectors of given membrane domains facing the co-substrate
pathway. They also suggest proximity between helices I, II, IV, VII, X,
and XI (see Ref. 20 and references therein).
To date, most of the previous analyses focused on the membrane domains
of MelB. However, few reports have already pointed out a possible
functional implication of cytoplasmic loop 10-11 connecting helices X
and XI (3, 21-23). In addition, two other observations suggested that
the cytoplasmic loop connecting helices IV and V (loop 4-5) might also
be important for MelB function (see Fig.
1). The first one showed that the arginyl
residue Arg-141 of loop 4-5 is a melibiose-protected site of MelB
labeling by a photoactivable azido phenyl sugar analog (24). The other
one indicated that Arg-141 is part of one of the two trypsin cleavage targets in this loop 4-5 that are cooperatively protected against protease attack in the presence of the physiological substrates (Na+ + melibiose > Na+ Materials--
Synthesis and labeling of
p-nitrophenyl
Bacterial Strains and Cell Growth--
E. coli DW2-R,
a Rec A Plasmid, Site-directed Mutagenesis, and DNA Sequencing--
A
recombinant pK95 Preparation of Membrane Vesicles--
Right-side-out (RSO)
membrane vesicles prepared by an osmotic shock procedure as described
(2) were concentrated to 2 mg of protein/ml and equilibrated in a
medium containing 0.1 M potassium phosphate (pH 7)
lacking NaCl (<20 µM).
Sugar Transport in Cells or RSO Membrane Vesicles--
Freshly
grown cells were concentrated to 2 mg of protein/ml in a 0.1 M potassium phosphate (pH 7) medium that contained NaCl at
a final concentration lower than 20 µM.
H+-coupled sugar transport was assayed in this medium by
adding either [3H]melibiose (20 mCi/mmol) or
[14C]TMG (3 mCi/mmol) at a final concentration of 0.4 or
0.8 mM. The assay was stopped after the given times by
diluting and immediate filtration of the samples using glass fiber
filters (7). When Na+- or
Li+-dependent sugar transport was measured,
NaCl or LiCl was added at a final concentration of 10 mM
just before the addition of the radioactive substrates. Transport in
RSO membrane vesicles was assayed by a similar procedure except for the
presence of reduced phenazine methosulfate as an energy donor (2). The radioactivity retained on the filters was counted in a Packard scintillation counter.
Entrance Counterflow Activity--
RSO membrane vesicles (20 mg/ml) were previously loaded with 30 mM melibiose for
12 h at 4 °C. 2-µl aliquots of sugar-loaded vesicles were
diluted 100-fold in a buffer containing [3H]melibiose at
a concentration of 0.1 mM (40 mCi/mmol). The
extra-vesicular unlabeled sugar present in the membrane aliquots raised
the final concentration of sugar of the diluting medium to 0.4 mM (10 mCi/mmol). The intra-vesicular accumulation of
radioactivity was terminated at the given times by a
dilution/filtration procedure similar to that used for transport assays.
Binding Assays--
Western Blot Analysis and Protein Determinations--
Samples
containing solubilized RSO membrane protein (~20 µg) were subjected
to SDS, 12% polyacrylamide gel electrophoresis and transferred onto
polyvinylidene difluoride membranes. The electroblotted proteins were
probed with a mouse anti-C-terminal MelB antibody followed by
incubation with a horseradish peroxidase-linked sheep antibody and
visualized by enhanced chemiluminescence (7). Results were expressed as
percent of the signal recorded from membranes carrying the Cys-less
permease (0.4-0.6 nmol/mg of protein, see Table I). Cell or membrane
protein was estimated by a modified Folin procedure, with bovine serum
albumin as a standard.
Fluorescence Assays--
A LS 50 PerkinElmer fluorometer was
used to measure the Na+-dependent FRET signals
(Eex, 297 ± 5 nm;
E Identification of R149C/V343G Second-site Revertants--
A
strategy developed by Ding and Wilson was used (28). Briefly,
DW2-R-melA+ cells expressing R149C permease grew initially
as white colonies on 1% melibiose MacConkey agar plates (Difco). After
3-5 days incubation at 37 °C, small red areas appeared and were
picked and re-streaked for colony purification. After isolation of the plasmid DNA, mutations responsible for fermentation recovery were identified by sequencing the entire gene using primers annealing at
about 250-bp intervals.
Transport and Binding Properties of K138C, R139C, R141C, or R149C
Permeases--
Throughout this study the C-less MelB permease
retaining a wild-type transport phenotype was used as control (see Fig.
2, Table I, and Ref. 26). The functional
consequences of individual replacements
of each four positively charged residues of loop 4-5 by a cysteine was
assessed by measuring Na+-dependent
[3H]melibiose accumulation in E. coli
DW2-R cells expressing each mutant. Fig. 2A shows that K138C
and R139C cells accumulated [3H]melibiose in 10 min to an
extent close to or higher than C-less MelB cells. Western blot analysis
suggested that the difference in transport could be accounted for by
variation of the cell membrane permease contents (R139C > C-less
MelB > K138C, data not shown). The sugar (melibiose, TMG) and
ionic selectivity profiles (Na+, Li+, or
H+) of K138C and R139C permeases were similar to that of
C-less MelB (data not shown). In contrast, neither R141C nor R149C
permeases catalyzed significant cell accumulation of melibiose (Fig.
2A) or of TMG (data not shown). Because R141C and R149C
membrane permease contents were 65 and 74% that of C-less MelB
membrane, respectively (Fig. 2B), these two mutants must
have a catalytic defect.
To assess more precisely the catalytic defect(s) of R141C and R149C
permeases, their Na+-dependent sugar binding
activity was measured in RSO membrane vesicles using the high affinity
radiolabeled sugar analog
FRET spectroscopy of RSO membrane vesicles incubated in the presence of
A Positive Charge Is Required at Position 141 for Co-substrate
Translocation--
Entrance counterflow is an experimental assay where
a very large increase in inward movement of radioactive sugar can be
triggered by diluting de-energized RSO membrane vesicles loaded with
unlabeled sugar (30 mM) into a medium containing labeled
sugar at 0.4 mM. It is accepted that the influx of the
labeled substrate during the initial phase of the trans-stimulation
process is primarily associated to shuttling of the loaded ternary
complex (MelB-ion-sugar) across the membrane and does not include
contribution of the empty carrier (3, 29). A transient uptake of
[3H]melibiose with a peak at about 1 min was observed in
control C-less MelB membranes (Fig.
4A) but not in R141C vesicles.
In addition, downhill efflux of [3H]melibiose from
sugar-loaded R141C membrane vesicles was completely inhibited (data not
shown). All these data can be explained assuming an impaired
reorientation of the loaded R141C carrier or sugar release and
probably also sugar binding at the cytoplasmic surface.
Chemical modification of single-Cys mutant with positively charged
methanethiosulfonate derivatives (25) provides means to incorporate a
positive charge at the Cys position in the mutants. Fig. 4 illustrates
the effect of MTSEA+ or MTSET+ derivatives on
the activity of R141C RSO membrane vesicles. Significant rescue of
active transport (Fig. 4B), melibiose counterflow (Fig. 4A),
or efflux (data not shown) was observed on reacting R141C vesicles with
MTSEA+ (1 mM) but not with MTSET+
(1 mM). The selective reactivation of R141C permease by the
permeant MTS reagent added externally to RSO membrane vesicles is
readily explained by first taking into account that MTSEA+
traverses the E. coli membrane much more efficiently than
MTSET+ at the concentration of 1 mM used here
(30) and, second, that the thiol-reagent must cross the membrane to
react with Arg-141 exposed to the cytoplasmic medium (7).
Analysis of R141K or R141Q permeases brought additional insights into
the mechanism by which this charged arginine residue might be involved
in MelB function. Fig. 5 and Table I
indicate that R141Q behaved as R141C. Thus, R141Q failed to catalyze
H+-, Na+-, or Li+-coupled transport
of [3H]melibiose or [14C]TMG (Fig 5).
However the mutant still bound Arg-149 Is Close or at the Sugar Binding Site--
All attempts to
rescue R149C sugar binding or transport by reacting the mutant with
either MTSEA+ or MTSET+ failed (see Fig.
6A). The strong aggregation
tendency of purified R149C precluded any study of its accessibility to
the SH reagents by currently available labeling procedures implying
affinity purification of the permease. Analysis of R149K and R149Q were
more instructive. Fig. 5 indeed shows that cells expressing the two
last mutants catalyzed significant
Na+-dependent [3H]melibiose
accumulation. The positive charge of Arg-149 is, therefore, not an
absolute requirement for active transport. However, some of the R149Q
and R149K properties were significantly different from those of C-less
MelB. Thus, although R149Q and R149K affinity for the coupling
Na+ ions was not significantly modified (Fig. 5 and Table
I), their affinity for
Additional insight into the role of Arg-149 was obtained by examining
the functional properties of second-site revertants of R149C. Twenty
independent clones that recovered fermentation activity on MacConkey
plates were isolated from four different experiments. Analysis of their
nucleotide sequence indicated that 15 of them were true revertants. In
contrast, the five remaining ones systematically carried a second
substitution of a glycine for the valine 343 located in the inner half
of helix X in addition to the original R149C mutation in loop 4-5 (see
Fig. 1). Despite many attempts, no other distinct second-site
suppressor mutation was isolated. Fig. 6 shows that R149C/V343G RSO
membrane vesicles accumulated melibiose up to 10% of the levels
observed in C-less MelB vesicles. Na+-dependent
accumulation of [14C]TMG was about half that of
[3H]melibiose in these vesicles (data not shown).
Remarkably, although R149C/V343G had limited capacity to concentrate
the substrates, it displayed sugar affinity and Na+
activation constants very close or identical to those of C-less MelB
(Table I). Moreover, the revertant incubated with
Dns2-S-Gal displayed a FRET signal comparable with the one
recorded from C-less MelB (data not shown). One may finally note that
the double mutant has a comparatively better affinity for
Finally, the effects of various thiol reagents on the transport and
sugar binding properties of R149C/V343G RSO membrane vesicles were
analyzed. First, we observed that reacting the RSO membrane vesicles
with impermeant thiol reagents such as MTSET+ (Fig. 6) or
PCMBS The results presented above provide evidence that mutagenesis of
two of four basic residues, Arg-141 and Arg-149, located in the inner
loop 4-5 of MelB permease from E. coli impaired the Na+-sugar symporter function. The data suggest that Arg-141
primarily takes part in the reaction of co-substrate translocation,
whereas the nearby Arg-149 residue may be located close or at the sugar binding site and most likely contributes to its structural
organization. Together with the established properties of loop 4-5,
these data bring evidence for a role of this extra-membranous
cytoplasmic domain into the translocation process catalyzed by the
ion-coupled sugar transporter.
The conclusion that only Arg-141 and Arg-149 are important for MelB
function relies on the observation that individual replacement of
either arginine with a cysteine totally inactivated MelB transport, whereas Cys substitution of the neighboring Lys-138 or Arg-139 residues
had no noticeable effect. It is worth noting that Arg-141 and Arg-149
but not Lys-138 or Arg-139 are among the most conserved residues in the
putative inner loop 4-5 of several homologous transporters composing
the galactoside-pentoside-hexuronide family (6, 31, 32). Detailed
analysis of the properties of Arg-141 and Arg-149 mutants suggest that
these two residues contribute differently to MelB activity.
Although the substitution of a short (Cys) or larger (Gln) neutral
residue for Arg-141 abolished sugar transport, it had no major effect
either on the Na+-dependent recognition process
of Despite variable changes of MelB transport activity produced on
replacing Arg-149 by a Cys, a Lys, or a Gln residue, all the mutations
systematically modified MelB sugar recognition properties. Thus, the
inactive R149C permease no longer bound sugars. Also, R149K and R149Q,
which still catalyzed Na+-dependent active
melibiose transport, displayed a selective and drastic reduction of
their affinity for Analysis of the properties of the second-site suppressor mutant
R149C/V343G provided further clues to the mechanism by which Arg-149
participates in the process of sugar binding. The presence of an
additional mutation (V343G) led to partial recovery of transport activity despite the absence of the native arginine at position 149. This second mutation also restored an affinity for the substrates and a
sugar binding site microenvironment comparable with that of the C-less
permease. These findings provided independent confirmation that there
is no absolute need for a charged residue at position 149 for sugar
transport and recognition by MelB. Moreover, because the rescuing Val
The observed inhibition of both R149C/V343G sugar transport and binding
activity by MTSET+ or PCMBS The two major conclusions drawn above are that Arg-141 is primarily
involved in reorientation of the loaded carrier or in the process of
sugar release at the inner membrane surface and that Arg-149 is a
domain of the sugar binding site close to or at the inner membrane
surface. Are these proposals consistent with known biochemical
characteristics of Arg-141 and flanking regions of loop 4-5? It has
previously been shown that Arg-141 is a melibiose-protected site of
MelB labeling by a photoactivable azidophenyl sugar analog (24). It
must be recalled that no residue in loop 4-5 and of the flanking helix
V other than 141 was photolabeled, suggesting that Arg-141 is a
specific labeling target in this MelB region. However, the reactive
azido is at a short distance from the galactosyl moiety that dictates
MelB sugar specificity (36). This means that the Arg-141 local region
may well be adjacent to the sugar binding site rather than necessarily
within the site. The observations that (i) Arg-141 is only 8 residues
apart from Arg-149 in the primary amino acid sequence (Fig. 1), (ii)
Arg-149 is located close to the inner water-membrane interface (Fig.
7), and (iii) Arg-141 is exposed to the inner aqueous medium (7) are in
line with this interpretation. The two later findings further suggest
that proximity between the Arg-141 domain of loop 4-5 and the Arg-149
domain of the sugar binding site occurs at the inner surface membrane
level. Positioning Arg-141 in a domain immediately after the sugar
binding site would also explain that replacing this arginine by neutral
residues effects translocation or release of the sugar into the
cytoplasm rather than binding of the external the sugar substrate to
MelB. Finally, the finding that MelB substrates affords cooperative
protection against loop 4-5 digestion by trypsin (Na+ and
melibiose > Na+
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosides (melibiose,
raffinose) or
-galactosides
(methyl-1-thio-
-galactopyranoside (TMG)) using a favorable
electrochemical potential gradient for Na+,
Li+, or H+ (1-3). The MelB symporter (473 amino acids, 53 kDa) has 12 helical membrane domains (4-8). A
recombinant transporter bearing a His6 tag
(Mel-His6 permease) can be purified in large amounts and
exhibits ion-dependent sugar binding and transport
properties similar to MelB in its native environment once reconstituted
in artificial lipid membranes (9-11). Recently, two-dimensional
crystallization of Mel-His6 permease was achieved, and a
projection map at 8 Å resolution was derived by electron
crystallography (12). The projection map suggests an asymmetric protein
unit hosting 12 potential transmembrane
-helices that are
distributed in two domains lining a central and curve-shaped cleft.
melibiose)
(7). To elucidate the role of Arg-141 and loop 4-5, we analyzed the
functional consequences of replacing Arg-141 by different residues
(Cys, Lys, Gln) and compared them with the effects produced by the
mutagenesis of the three other basic residues of this loop (Lys-138,
Arg-139, and Arg-149). Complementary information was obtained by
reacting the newly introduced Cys residues with methanethiosulfonate
derivatives (25) and isolation of second-site suppressor mutants. The
obtained data suggest that the cytoplasmic loop 4-5 of MelB is close
to the sugar binding site and may participate directly in co-substrate
translocation by MelB.
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Fig. 1.
Secondary structure model of melibiose
permease (5). Helices are represented by rectangles, a
number of which are labeled with roman letters. Top and
bottom numbers in the rectangles correspond to
the first and last residues in the helices. The zoomed-in area shows
the amino acid composition of cytoplasmic loop 4-5 connecting
membrane-spanning segments IV and V. Shadowed letters
indicate the positively charged residues subjected to individual
cysteine replacement and lettered according to their position on the
MelB sequence. The second-site mutation present in helix X (V343G) of
the revertant R149C/V343G described below is indicated in bold
face.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-6-[3H]galactopyranoside
(
-[3H]NPG) or
6-O-
-D-[3H]galactopyranosyl-D-glucose
and synthesis of the fluorescent sugar analog (Dns2-S-Gal)
were carried out by Dr. B. Rousseau and Y. Ambroise (Département de Biologie Joliot Curie/CEA-Saclay, France). [14C]TMG
was from Isotopchim (France). The methanethiosulfonate derivative reagents (MTSEA+, MTSET+) were purchased from
Toronto Research Chemicals, Inc. Stock solutions (0.2-0.5
M) were prepared in distilled water, stored in the frozen state, and used immediately after thawing. Stock solutions of N-ethylmaleimide (Sigma) were prepared at 0.2 M
in ethanol.
derivative of strain DW2 (
melB
lacZY), was
transformed with the given plasmids (9). Freshly transformed cells were
grown at 30 °C in M9 medium supplemented with glycerol (0.5 g/liter)
and casamino acids (0.2%) as the carbon source, thiamine (0.5 mg/ml),
and ampicillin (100 µg/ml) until an
A600 of 1-1.2 was reached. Cells were
then washed and resuspended in 0.1 M potassium phosphate
buffer (pH 7).
AHB plasmid with a cassette containing the
melB gene (9) encoding a permease devoid of its four native cysteine (C-less MelB) was constructed with the appropriate mutagenesis primers and subsequently used for control experiments or for permease engineering. This C-less carrier displayed a valine instead of Cys-235
and a serine instead of Cys-110, Cys-310, and Cys-364 (26). Individual
Cys replacements were engineered by polymerase chain reactions using
the appropriate primers (27-30 nucleotides, Genosys) and the
recombinant plasmid harboring the C-less carrier melB gene
as a matrix. Plasmids were isolated using the QIAprep Spin Miniprep Kit
(Qiagen). Sequencing was performed using 35S-labeled dATP
with the sequencing kit (Amersham Biosciences).
-[3H]NPG binding to RSO
membrane vesicles was assessed by a flow dialysis procedure as
described in Damiano-Forano et al. (13). RSO membrane
vesicle suspensions (10-20 mg/ml) were initially equilibrated with
-[3H]NPG (0.8 mCi/mmol), and the medium NPG
concentration was varied between 0.1 and 10 µM by the
stepwise addition of unlabeled ligand. The maximal number of binding
sites (Bmax) and apparent binding constant
(KD) were calculated graphically from Scatchard plots of the data. The activation constant for NPG binding by Na+ (KNa) was determined using plots
of the apparent KD values for NPG as a function of
the reciprocal concentration of sodium chloride in the range of 0.1-10
mM. Melibiose inhibitory constants (Ki)
were estimated by progressive displacement of bound
-[3H]NPG (0.8 mCi/mmol, 1 µM) by
stepwise addition of sugar (0.5-20 mM). These
Ki values were used to calculate the melibiose binding constants (KD) (27).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Cell sugar transport by K138C, R139C, R141C,
or R149C MelB mutants. A, [3H]melibiose
transport by E. coli DW2-R cells transformed with given
recombinant plasmids. Control experiments were conducted using the
C-less MelB permease. Cell transport was assayed using cells (2 mg of
protein/ml) equilibrated in 0.1 M potassium phosphate (pH
7). Transport was started immediately after the addition of NaCl and
[3H]melibiose (20 mCi/mmol) at final concentrations of 10 and 0.4 mM, respectively, and terminated at the indicated
time points by a rapid dilution followed by immediate filtration.
B, permease contents of R141C and R149C. Western blot
analysis of the permease expression levels in RSO membrane vesicles was
carried out with anti-C-terminal antibody as described under
"Experimental Procedures."
NPG binding constants of the C-less permease, of the permease carrying
different substitutions at position 141 or 149, and of the second-site
revertant R149C/V343G
-free, 0.1 M potassium phosphate buffer (pH 7)
containing carbonyl cyanide m-chlorophenylhydrazone (5 µM) and monensin (0.75 µM) as described in
the Fig. 3 legend.
-[3H]NPG binding was assayed in a flow
dialysis cell as described in Damiano-Forano et al.
(13).
-[3H]NPG binding was determined in
vesicle suspensions initially equilibrated with
-[3H]NPG
(0.8 mCi/mmol), and the medium NPG concentration was varied in the
range of 0.1-10 µM by the stepwise addition of unlabeled
ligand. The maximal number of binding sites (Bmax)
and apparent NPG binding constant (KD) were
calculated graphically from Scatchard plots of the data. The activation
constant for NPG binding by Na+ (KNa) was
determined from plots of the apparent KD for NPG as
a function of the reciprocal concentration of sodium chloride in the
range of 0.1-10 mM. Values are the means of 3 experiments,
and S.E. were generally <20%. NS, not significant.
-[3H]NPG as a ligand (13).
The upper curve in Fig.
3A illustrates typical binding
variations recorded from de-energized C-less MelB membrane vesicles.
The drop of free
-[3H]NPG concentration observed on
adding NaCl (20 mM) reflects enhanced
-[3H]NPG binding to C-less MelB membranes, whereas the
subsequent rise occurring upon the addition of unlabeled NPG in excess
(250 µM) was due to the release of bound ligand
(upper curve). Fig. 3A also shows that R141C
membranes retained Na+-dependent NPG binding
activity (middle curve), whereas R149C membranes did not
(lower curve), even when the NaCl concentration was raised
up to 1 M (data not shown). Additional binding experiments indicated that R141C permease had at most 3-4 times less affinity for
-[3H]NPG (KD, Table I) or for
melibiose (2 mM instead of 0.6 mM, not shown)
than C-less permease. In contrast, no significant change in activation
of sugar binding to the two mutants by sodium was observed
(KNa, Table I).
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Fig. 3.
Na+-dependent
-[3H]NPG binding and FRET signal recorded from C-less
MelB, R141C, or R149C RSO membrane vesicles. A,
Na+-dependent
-[3H]NPG binding
to RSO membrane vesicles harboring each permease was assayed by means
of a flow dialysis technique (13). RSO membrane vesicles concentrated
to about 10 mg of protein/ml (C-less MelB) or 15-20 mg of protein/ml
(R141C, R149C) were equilibrated at 20 °C in Na+-free
potassium phosphate (100 mM, pH 7) buffer containing
-[3H]NPG (0.8 mCi/mmol, 1 µM) carbonyl
cyanide m-chlorophenylhydrazone (ClCCP) (5 µM) and monensin (0.75 µM) in the upper
chamber of the dialysis cell. When indicated by arrows, NaCl
and unlabeled
-NPG were successively added at a final concentration
of 20 mM or 250 µM, respectively.
Ordinate, radioactivity (cpm) collected from the lower
chamber of the dialysis cell used as a reporter of the concentration of
free
-[3H]NPG in the membrane suspension.
B, Na+-dependent variation of the
FRET signal recorded from C-less MelB, R141C, or R149C RSO membrane
vesicles incubated in the presence of the fluorescent sugar analog
Dns2-S-Gal. De-energized RSO membrane vesicles (100 µg of
protein/ml) were incubated with Dns2-S-Gal (10 µM) and excited at 297 ± 5 nm (14). The FRET signal
(average 10 scans) was recorded before and after the addition of NaCl
(20 mM) to the suspension, and the
Na+-dependent signal was calculated by
subtracting the spectra.
-galactoside fluorescent analog Dns2-S-Gal provides the
means to assess ion-induced structural changes of MelB and related
variation of the sugar binding site microenvironment (14). The specific
Na+-induced, tryptophan-mediated fluorescence signal
(Emax at 465 nm) recorded from C-less MelB membrane
vesicles excited at 297 ± 5 nm (upper spectrum, Fig.
3B) was similar to that previously recorded for wild-type
MelB membrane vesicles. More interesting, R141C permease retained
comparable Na+-dependent FRET signal
(middle spectrum), indicating that Na+ ions
still induced a cooperative modification of the sugar binding site
structure in this mutant. In contrast, no significant FRET signal was
recorded from R149C membranes (lower spectrum). From all
these data, we conclude that the R141C mutation introduces a defect in
the transport cycle at a step that follows co-substrate binding,
whereas the R149C mutation directly impairs sugar binding.
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Fig. 4.
Restoration of MelB entrance counterflow or
transport in R141C RSO membrane vesicles with the permeant thiol
modifier MTSEA+. A, entrance counterflow
activity. R141C (squares) or C-less (circles) RSO
membrane vesicles were concentrated to about 20 mg of protein/ml in 0.1 M potassium phosphate buffer (pH 7) containing NaCl (10 mM), monensin (0.75 µM), and carbonyl cyanide
m-chlorophenylhydrazone (ClCCP) (5 µM) and equilibrated overnight at 4 °C in the presence
of melibiose at a final concentration of 30 mM. Counterflow
was assayed by diluting the loaded membrane vesicles 200-fold into the
same saline medium containing [3H]melibiose (20 mCi/mmol)
at a final concentration of 0.4 mM. At the indicated time,
the reactions were terminated by rapid filtration. When tested,
MTSEA+ was added to the loaded vesicle suspension at a
final concentration of 1 mM 5 min before dilution
(closed squares). B, [3H]melibiose
accumulation. Active transport was assayed on well aerated aliquots of
RSO membrane vesicles harboring C-less (open circles) R141C
(open squares) permeases (50 µl, 2 mg protein/ml) prepared
in 0.1 M potassium phosphate buffer (pH 7) containing NaCl
(10 mM) and reduced phenazine methosulfate (0.8 mM). Transport reactions at 23 °C were started by the
addition of [3H]melibiose (20 mCi/mmol) at a final
concentration of 0.8 mM and terminated by a rapid dilution
followed by immediate filtration. When tested, MTSEA+
(filled symbols) or MTSET+ (gray
symbols) were added at a final concentration of 1 mM
for 2 min before the addition of MelB substrates.
-[3H]NPG in a
Na+-dependent fashion (Table I). Consistently,
R141Q membranes displayed Na+-dependent FRET
signal similar to that of R141C (data not shown) and no longer
catalyzed carrier-mediated movements of sugar (counterflow, efflux,
data not shown). The data in Table I and Fig. 5 also show that R141K
permease retained ion-coupled melibiose or TMG transport and
Na+-dependent NPG binding activity. However, it
is worth noting that all Arg-141 substitutions led to limited reduction
of sugar affinity (3-4-fold) in comparison to C-less MelB (Table
I).
View larger version (19K):
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Fig. 5.
- or
-galactoside transport by MelB permease carrying a
substitution of a lysine or a glutamine for either Arg-141 or
Arg-149. E. coli DW2-R cells expressing the R141K,
R141Q, R149K, or R149Q mutants or the control C-less permease were
prepared in Na+-free 0.1 M potassium phosphate
(pH 7) containing less than 20 µM sodium salts as
described under "Experimental Procedures." H+-coupled
[3H]melibiose transport or [14C]TMG (3 mCi/mmol, 0.8 mM) were assayed in this Na+-free
medium (50-µl aliquots, 100 µg of proteins) at 23 °C.
Na+- or Li+-coupled transport of
[3H]melibiose (20 mCi/mmol, 0.8 mM) were
measured in the presence of 10 mM NaCl or LiCl. Histograms
show the amount of labeled sugars accumulated in 10 min (means of 3 determinations ± S.E.).
-galactosides was severely reduced (about
10-fold, Table I) and that for
-galactosides was beyond detection
limit. Overall, although a charged residue is not absolutely required at position 149 for active transport, the charged arginyl side chain
seems to provide optimal interactions for co-substrate binding.
View larger version (18K):
[in a new window]
Fig. 6.
Inhibition of
Na+-dependent sugar transport of R149C/V343G in
RSO membrane vesicles by MTSET+. RSO membrane vesicles
(2 mg of protein/ml) harboring the revertant permease were equilibrated
in Na+-free, 0.1 M potassium phosphate buffer
(pH 7) and incubated for 5 min in the presence of MTSET+ (1 mM). [3H]Melibiose transport (20 mCi/mmol,
0.8 mM) was then assayed as described in Fig. 2 except for
the presence of reduced phenazine methosulfate. Control experiments
were carried out using C-less RSO membrane vesicles. Histograms
represent the amount of labeled sugar accumulated in 10 min (means of 3 experiments ± S.E.).
-galactosides (KD) or for Na+ ions on
the basis of the KNa value than any of the
individual single R149C or V343G mutants (Table I).
(not shown) before the transport assay
inhibited Na+-dependent melibiose transport by
75 and 50%, respectively. Permeant thiol reagents like
MTSEA+ or N-ethylmaleimide inhibited the
revertant transport activity by more than 90% (data not shown).
Comparatively, sugar transport in control C-less membrane vesicles was
at most reduced by less than 5% by MTSEA+ or not affected
by the other reagents. To identify the step of the R149C/V343G
transport cycle inhibited by MTSET+ inhibited, we analyzed
the effect of this reagent on the
-[3H]NPG binding to
RSO membrane vesicles and looked for an eventual protection by the
co-substrates (Fig. 7). To this end,
revertant RSO membrane vesicles were incubated in a medium without
substrate, in media supplemented with either Na+ (20 mM) or
-[3H]NPG (4 µM)
alone, or finally, in a medium supplemented with both substrates.
Except for the control, MTSET+ was then added to all
samples at a final concentration of 1 mM. After 5 min, the
binding activity was assayed by the flow dialysis procedure in the
presence of
-[3H]NPG and Na+ ions at a
final concentration of 1 µM and 20 mM,
respectively. The effect of the thiol reagent was estimated from the
amount of bound ligand released in the medium upon the addition of
melibiose in excess (20 mM). Fig. 7 shows that the amount
of bound
-[3H]NPG released upon the addition of
melibiose was comparable in membranes not incubated with
MTSET+ (open circles) and in membranes treated
with the thiol reagent in the presence of both Na+ and
-[3H]NPG (closed circles). Compared with
this amount (100%), only 25% bound
-[3H]NPG was
released when the membranes were incubated with MTSET+ in
the absence of substrates (closed triangles) or when the
medium contained only Na+ ions (open triangles).
Finally, a significantly higher amount of
-[3H]NPG
(50%, open diamonds) was released when the
MTSET+ treatment was carried out in the presence of
-[3H]NPG alone. Comparatively,
-[3H]NPG binding to C-less membranes was insensitive
to MTSET+ in all conditions tested (not shown). Taken
together, these findings suggest that the sugar substrate affords
protection against inactivation of sugar binding to the second site
revertant by MTSET+. The more efficient
-[3H]NPG protection observed in
Na+-containing and than in Na+-free media is
most likely linked to the higher rate of sugar binding to MelB permease
in the presence of Na+ ions (13). Finally, the lower extent
of sugar binding inactivation in sugar-free medium was observed in RSO
vesicles reacted with 0.2 mM PCMBS
(data not
shown). Overall, these observations make it likely that the residue at
position 149 is located at or close to the sugar binding site and in a
hydrophilic environment accessible from the outer medium.
View larger version (24K):
[in a new window]
Fig. 7.
Selective protection against inactivation by
MTSET+ of -[3H]NPG binding to R149C/V343G
RSO membrane vesicles by the sugar. De-energized RSO membrane
vesicles (20 mg of proteins/ml) were prepared in Na+-free
medium (pH 7) containing 0.1 M potassium phosphate and
separated in 5 aliquots (300 µl each). The following additions were
made before MTSET+ treatment: closed circles,
-[3H]NPG (4 µM) and Na+ (20 mM); open diamonds,
-[3H]NPG (4 µM); open triangles, Na+ (20 mM); closed triangles, no substrates. Except for
the control (open circles), all samples were incubated for 5 min with MTSET+ at a final concentration of 1 mM. The samples were then diluted four times, and when
needed, the missing ion and/or sugar substrates were adjusted to a
final concentration of 20 mM or 1 µM,
respectively. After concentrating the protein to 20 mg/ml in the
diluting medium,
-[3H]NPG binding was assayed by the
flow dialysis procedure. When indicated by arrows, melibiose
was added at a final concentration of 20 mM to the membrane
suspensions. The dotted line refers to the rate of decay of
free
-[3H]NPG recorded from membrane vesicles devoid
of MelB permease.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
-galactosides or on the associated cooperative
modification of the sugar binding site environment. In contrast, R141C
and R141Q no longer catalyzed energy-independent carrier-mediated flow
of sugar in the inward (initial rate of counterflow) or in the outward
(downhill efflux) direction. As emphasized under "Results," the
influx of the labeled substrate during the initial phase of counterflow
is part of an exchange process that only involves sugar loading/release
at each membrane surface and shuttling of the loaded (MelB-ion-sugar) complex (3, 29). Because the R141C and R141Q mutants still bind the
co-substrates at the outer surface, the mutant counterflow defect is
most likely associated to a defective reorientation of the loaded
carrier. Alternatively, the defect may arise from a reduction of the
rate of release of the labeled sugar into the inner compartment (and/or
a reduction of the rate of binding of internal sugars). It would be
more difficult to link the lack of counterflow activity to an
alteration of the binding properties of Na+ ions to MelB at
the cytoplasmic membrane surface because the sugar exchange reaction
occurs without concomitant binding and release of the coupling ion on
the internal surface (33). Finally, the finding that only MelB carrying
a positively charged residue (Arg, Lys, or R141C reacted with
MTSEA+) at position 141 mediated co-substrate translocation
highlights the importance of the charge. It raises the possibility of
electrostatic interactions between Arg-141 and unknown negatively
charged residues elsewhere in MelB. However, more complex
hydrogen-bonding interactions like those observed in the
arabinose-binding protein structure cannot be excluded (34). Screening
of second-site suppressor revertants of R141C, so far unsuccessful,
should help to discriminate among these interpretations.
-galactosides or no longer recognized
-galactosides. Incidentally, because R149Q retained transport
activity, contribution of the residue at position 149 to MelB activity
is probably involved in hydrogen bond interactions rather than
electrostatic ones. Implication of Arg-149 in the sugar binding
reaction can, therefore, be suspected.
Gly mutation occurred on a small apolar residue located on the
hydrophobic face of helix X distant from the R149C mutation in loop
4-5, it seems more likely that the second mutation compensates for a
structural defect prevailing in the R149C mutant. Taken together, the
change in sugar recognition properties of the different Arg-149
mutants, the rescuing effect of a distant mutation in a permease still
carrying the R149C mutation, and an unusual instability of R149C
permease upon purification could be accounted for by assuming that
Arg-149 is involved in the structural organization of the sugar binding
site rather than in a direct binding interaction with the sugar.
provides insight
into the relative localization of Arg-149 with respect to the sugar
binding site. Thus, accessibility of the cysteine at position 149 to
the two hydrophilic and impermeant thiol reagents in RSO membrane
vesicles suggests that Cys-149 in the revertant and, by extension,
Arg-149 in the C-less permease faces an intra-protein polar region
communicating with the outer medium. Moreover, because the sugar itself
selectively protected against inactivation of the sugar binding
reaction by MTSET+ or PCMBS
, this polar
region most likely corresponds to the sugar binding domain. Similar
observations on several transporters have been already used as an
indication that the specific region harboring the target residue is
either directly involved in binding, lining the binding site, or at
least conformationally coupled to the binding of substrates (20, 29,
35). In this context, it should be mentioned that a current topological
model of MelB predicts Arg-149 localization at the junction of
cytoplasmic loop 4-5 and helix V (Ref. 5; see Fig. 1). Any attempt to
relocate Arg-149 within the adjacent membrane domain (helix V) is at
best limited to the very inner extremity of this helix due to the
strong helix-breaker motif PYP preceding Arg-149 in the MelB sequence
(see Fig. 1). On this basis, one can predict that the sugar binding
site region including Arg-149 lies down close or at the inner membrane
surface. It is finally worth mentioning that Cys-scan mutagenesis of
aligned residues in the polar sector of helices V and X suggests that the two helix inner-half portion lines the sugar binding site (manuscript in
preparation).2
melibiose) has been used to
suggest that this loop undergoes structural rearrangement upon
substrate binding (7). Loop 4-5 or part of it including the Arg-141
region may be regarded as a functional mobile domain, accessible to the
bulk of the cytoplasm when the symporter is unloaded, and becomes
inaccessible upon interaction with the nearby membrane sugar binding
site once the carrier is loaded. Such a conformational transition of
the Arg-141 region may favor either reorientation of the loaded
carrier, transient interaction of Arg-141 with the sugar after
reorientation of the permease, or finally, sugar release into the inner
compartment. Consistently, previous fluorescence and mutagenesis
studies of residues sitting in the N-terminal region of loop 4-5 and
in the preceding helix IV suggest that this area of the transporter
might behave as a hinge involved in the coupling mechanism of MelB
(37). More work is, however, needed to assess the validity of these speculations. In conclusion, the data reported in this study provide ground evidence for the existence of a close relationship between loop
4-5 and the sugar binding site and strongly suggest that Arg-141 in
the cytoplasmic loop 4-5 is catalytically involved in the process of
Na+-coupled sugar translocation by the MelB symporter.
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ACKNOWLEDGEMENTS |
---|
We thank Raymonde Lemonnier for excellent technical assistance and Kerstin Meyer for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by European Commission Grant Bio4-CT97-2119 and grants form the CNRS and the French Atomic Energy Commission.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.
A post-doctoral fellow of the European Commission
(Bio4-CT97-2119).
§ To whom correspondence should be addressed. Tel.: 33-4-93-76-52-12; Fax: 33-4-93-76-52-19; E-mail: leblanc@oceane.obs-vlfr.fr.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M210053200
2 C. Basquin, M. Abdel Dayem, R. Lemonnier, T. Pourcher, and G. Leblanc, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
MelB, melibiose
permease,
(6-O--D-galactopyranosyl-D-glucose);
TMG, methyl-1-thio-
-galactopyranoside;
-NPG, p-nitrophenyl
-D-6-galactopyranoside;
FRET, fluorescence resonance energy transfer;
Dns2-S-Gal, 2'-(N-dansyl)aminoethyl-1-thio-
-D-galactopyranoside;
MTSEA+, (2-aminoethyl)methanethiosulfonate hydrobromide;
MTSET+, (2-(trimethylammonium)ethyl) methanethiosulfonate
hydrobromide;
PCMBS
, p-chloromercury benzoic acid;
RSO membranes, right-side-out membranes.
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