From the
The role of Glu
The lactose transport protein (LacS) of Streptococcus
thermophilus catalyzes the uptake of galactosides in symport with
a proton (Foucaud and Poolman, 1992). In vivo the dominant
transport reaction corresponds with an exchange of lactose for
intracellularly formed galactose without net movement of protons
(Poolman, 1990; Foucaud and Poolman, 1992). The structure of the LacS
protein differs from other secondary transport proteins by the presence
at the carboxyl terminus of a hydrophilic domain that is homologous to
IIA of various phosphoenolpyruvate:sugar phosphotransferase systems
(Poolman et al., 1989). Although the IIA domain of LacS is not
essential for translocation catalysis, phosphorylation of a histidine
residue in the IIA domain by phosphoenolpyruvate and the general energy
coupling proteins of the phosphoenolpyruvate:sugar phosphotransferase
systems reduces the transport activity (Poolman et al., 1992,
1995).
The amino-terminal carrier domain of LacS corresponds with a
polytopic membrane protein that traverses the membrane most likely 12
times. The primary sequence of the hydrophobic carrier domain of LacS
classifies this protein as a member of a family which also includes
MelB of Escherichia coli, Klebsiella pneumoniae, and
Salmonella typhimurium, the glucuronide transporter (GusB) of
E. coli (Liang, 1992), and the xylose transporter (XylP) of
Lactobacillus pentosus (for a review, see Poolman and Konings,
1993). The lactose transport protein (LacY) of E. coli is not
homologous to members of this family of transporters, however, a
stretch of about 20 amino acids around His
The relative
amounts of LacS were estimated by immunoblotting with a anti-IIA domain
antibody. Membranes prepared from strains expressing either the
wild-type or one of the mutant LacS proteins showed no significant
differences in the expression levels of LacS (data not shown).
In this paper the effects on the kinetic parameters of
transport of different substitutions of Glu
The mutant alleles of both permeases differ in those
aspects where also the properties of the wild-type enzymes are
different. Equilibrium exchange of lactose mediated by the wild-type
LacY protein is independent of pH in the range of 5-10 but
becomes dependent on pH in the E325D mutant (Roepe and Kaback, 1990).
Neutral substitutions result in wild-type exchange activities (Carrasco
et al., 1989). Equilibrium exchange of lactose by wild-type
LacS and LacS-E379D exhibits a typical pH optimum around pH 6, whereas
exchange by LacS-E379Q is largely independent of pH
(Fig. 6B). The rate of efflux of lactose down a
concentration gradient at alkaline pH values approaches the rate of
exchange in wild-type LacY (Viitanen et al., 1983), whereas in
wild-type LacS the rate of efflux is much slower than the rate of
exchange (Fig. 6). Remarkably, the rate of efflux is accelerated
in the LacS-E379D, E379A, and E379Q mutants ( Fig. 5and
Fig. 6A), whereas lactose efflux by the LacY-E325A and
E379Q mutants is inhibited and only marginally greater than in
membranes lacking LacY (Carrasco et al., 1989). The apparent
unidirectional transport by the LacY Glu
Taken together, these results indicate that
Glu
To specify which step(s) in the
translocation cycle of the LacS protein are affected by the Glu
The membrane potential inhibits efflux
transport of the wild-type LacS by affecting the
E
The ES leak is most easily
introduced in the scheme for the wild-type enzyme (Fig. 8,
top panel) when the order of binding on the outside is assumed
to be random instead of proton first, substrate second (see above). The
pH profile of the efflux reaction catalyzed by the E379Q mutant
(Fig. 6A) is indicative of a pK for the
proton-binding site at the outer face of the membrane of around 6
(pK
We thank Dr. G. Sulter for assistance in the
preparation of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
in the lactose-H
symport protein (LacS) of Streptococcus thermophilus was
studied by analyzing the kinetic mechanism of transport of wild-type
and Ala
, Asp
, and Gln
mutant
proteins. Glu
forms part of the sequence motif
Lys-X-X-His-X-X-Glu that is present in a number of
sugar transport proteins, including LacY of Escherichia coli.
The E379A and E379Q mutants were defective in the uptake of lactose
against a concentration gradient and lactose-dependent proton uptake,
but catalyzed facilitated influx of lactose down a concentration
gradient and equilibrium exchange with rates similar to that of the
wild-type enzyme. The E379D mutant was partially defective in the
coupled transport of lactose and protons. These results suggest that an
acidic residue at position 379 is required for the coupled uptake of
lactose and protons and are consistent with a mechanism in which
lactose transport in the E379A and E379Q mutants occurs by uniport
rather than proton symport. Lactose efflux down a concentration
gradient in wild-type LacS and LacS-E379D increased with pH with
apparent pK (pK
) values of
8.5 and 8.0, respectively, whereas efflux in the E379Q mutant
increased sigmoidally with a pK
of about
6.0. Imposition of an artificial membrane potential (inside negative)
in membrane vesicles bearing wild-type LacS or LacS-E379Q not only
inhibited the lactose efflux mediated by wild-type but also that of the
mutant enzyme. To associate the role of Glu
with specific
step(s) in the translocation cycle of LacS, the properties of wild-type
LacS and the Glu
mutants have been evaluated by numerical
analysis of simple kinetic schemes for translocation catalysis by
solute H
symport proteins. The properties of the
wild-type enzyme are consistent with a mechanism in which the order of
ligand binding on the inside is substrate first and proton last,
whereas the order is random (or proton first, substrate last) at the
outer surface of the membrane. The wild-type enzyme is asymmetric with
regard to proton binding; the pK for proton binding on the
outside is at least 4 units higher than the pK on the inside.
The properties of the Glu
mutants correspond with a
lowering of the pK on the outside (pK
pK
), and the induction of a leak
pathway in which the binary enzyme-substrate complex becomes mobile.
in LacY can be
identified in LacS (Fig. 1). Conserved residues include a lysine
(position 319 in LacY, 373 in LacS; arrow 1), a histidine
(position 322 in LacY, 376 in LacS; arrow 2), and a glutamic
acid (position 325 in LacY, 379 in LacS; arrow 3). Residues
His
, Glu
(which should be on the same side
of putative
-helix X), and Arg
(putative helix IX)
of LacY have been proposed to participate in proton translocation via a
charge relay mechanism (Püttner et al., 1986, 1989;
Carrasco et al., 1986; Menick et al., 1987; Lee
et al., 1989), which is based on analogies with proton
transfer via Asp, His, and Ser in serine type proteases (Kraut, 1977).
In this view, Glu
and His
are ion-paired
and interact with Arg
; Glu
and Arg
would polarize the imidazole group of His
which
enhances its capacity to act as a proton shuttle. His
is
poised to accept a proton from Glu
and subsequent
transfer of this proton to Arg
, another residue or the
medium would lead to net proton translocation. The role of His
in proton translocation can be questioned since the requirement
for an ionizable histidine residue at position 322 in LacY is not
always needed for galactoside accumulation and galactoside-dependent
proton transport (King and Wilson, 1989a, 1989b, 1990; Franco and
Brooker, 1991). Similarly, some substitutions of Arg
,
i.e. R302S and R302H, result in mutant proteins that do
accumulate galactosides albeit to lower levels and do exhibit
sugar-dependent proton transport (Matzke et al., 1992). The
degree of uncoupling of sugar transport from H
transport not only varies with the substitutions made for
Arg
and His
, but also with the sugar used
in the transport assay (King and Wilson, 1989a, 1989b) and other
parameters such as the pH of the assay medium (Franco and Brooker,
1991). Altogether, one could conclude that Arg
and
His
play an auxiliary role in proton translocation,
e.g. via modulating the pK of an essential nearby
residue. From the analysis of LacY Glu
mutants, it is
clear that an acidic residue at position 325 is required for
proton-linked galactoside transport (Carrasco et al., 1989;
Franco and Brooker, 1994).
Figure 1:
Conserved residues in interhelix loop
X-XI of LacS and other sugar transport proteins. LacY, lactose
transport protein; RafB, raffinose transport protein;
CscB, sucrose transport protein; LacS, lactose
transport protein; MelB, melibiose transport protein;
GusB, glucuronide transport protein; XylP, xylose
transport protein. The subscripts EC, KP,
ST, LB, SY, and LP refer to
E. coli, K. pneumoniae, S. thermophilus,
Lactobacillus bulgaricus, S. typhimurium and
Lactobacillus pentosus, respectively (for details, see Poolman
and Konings, 1993). Lys, His
, and
Glu
of LacS are indicated by the arrows
1-3, respectively.
In this study, we focus on the role in
translocation of Glu in the lactose transport protein of
S.thermophilus. The data indicate that Glu
is required for the coupled uptake of lactose and protons
possibly by modulating the pK of an essential protonatable
residue. The properties of the S.thermophilus LacS(Glu
) mutant proteins are highly diagnostic of
an enzyme with a substrate leak pathway that allows uncoupled transport
at rates similar to or better than the coupled transport by the
wild-type enzyme.
Bacterial Strains, Growth Conditions, and Preparation
of Cell Suspensions
The bacterial strains used are E. coli DW1
{lac(ZY),
mel(AB), strA}; DW2
{lacl
,
lac(ZY),
melA
,
melB, strA}; JM101
{supE, thi
(lac-proAB), [F', traD36, proAB,
lacl
Z
M15]}; CJ236 {dut1,
ung1, thi1, relA1/pCJ105 (Cm
)}; HB101
{hsdS20(r
Bm
B),
recA13, ara14, proA2, lacY1, galK2, rps (Sm
),
xyl5, mtl1, supE44,
,
F
}. E. coli cells were grown
aerobically at 37 °C in Luria broth, M9 mineral medium supplemented
with 4 g/liter casamino acids and 50 mM glucose or lactose or
M9 mineral medium supplemented with 50 mM carbohydrate and
essential nutrients as indicated by the auxotrophic markers (Sambrook
et al., 1989). When appropriate, the medium was supplemented
with carbenicillin (50 µg/ml), ampicillin (100 µg/ml),
chloramphenicol (10 µg/ml), and/or streptomycin (25 µg/ml).
Overnight cultures or exponentially growing cells (OD
= 0.4-0.8; Erlenmeyer flasks) were harvested by
centrifugation, washed twice, and resuspended to a final protein
concentration of about 40 mg/ml in 100 mM potassium phosphate
2 mM magnesium sulfate (KPM buffer) or 30 mM K-citrate, 30 mM K-piperazine-N,N`-bis(2-ethanesulfonic acid), 30 mM K-2(N-cyclohexylamine)ethanesulfonic acid, 2 mM MgSO
(CPC buffer) (pH 6.5, unless indicated
otherwise). Concentrated cell suspensions were stored on ice until use.
Isolation of Membranes
For the isolation of
right-site-out membrane vesicles cells were grown in 10-15-liter
fermentors with vigorous aeration using M9 mineral medium supplemented
with 4 g/liter casamino acids and 50 mM glucose. The cells
grew exponentially up to optical densities at 660 nm of 2.5. Cells were
harvested at OD of 2.0-2.5, and right-site-out
membrane vesicles were prepared according to Kaback(1971). Inside-out
membrane vesicles were prepared by a 2-fold passage of the cells
through a French pressure cell (20,000 pounds/square inch) as described
(Poolman et al., 1983).
Transport Assays
Transport experiments were
performed at 30 °C unless specified otherwise.
(i) Active Transport
Cells (0.6-1.2 mg/ml)
in KPM buffer containing 10 mMD-Li-lactate as the
electron donor were preenergized for 2 min in the presence of oxygen.
At time 0, radiolabeled substrate was added, and at appropriate time
intervals the uptake reaction was stopped by addition of 2 ml of
ice-cold 100 mM LiCl. Cells were filtered (0.45-µm
cellulose nitrate filters (Millipore Corp.)) and washed with 2 ml of
ice-cold LiCl. For the estimation of intracellular concentrations, a
specific internal cell volume of 3 µl/mg of protein was used.
(ii) Efflux and Exchange in Intact Cells
Cells
were incubated overnight at 4 °C with the appropriate concentration
of radiolabeled galactoside and in the presence of deoxyribonuclease I
(20 µg/ml). The next day, potassium azide and carbonylcyanide
m-chlorophenylhydrazone were added to final concentrations of
30 mM and 50 µM, respectively, and the cells were
incubated for another 2 h at room temperature essentially as described
(King and Wilson, 1990). Aliquots of 1 µl of concentrated cell
suspension (50-80 mg/ml) were diluted into 500 µl of KPM or
CPC buffer containing no substrates (efflux) and unlabeled galactosides
(exchange), respectively. The transport reaction was stopped by rapid
filtration as described above.
(iii) Efflux and Exchange in Membrane Vesicles
For
efflux and exchange in the presence of artificially imposed diffusion
potentials, membrane vesicles were resuspended to a final concentration
of 30-40 mg of protein/ml in the buffers specified below prior to
loading with 5 mM [C]lactose or 2
mM
[
C]methyl-
-D-thiogalactopyranoside.
To generate a
, valinomycin was added to the membrane
suspension to a final concentration of 2 nmol/mg of protein. The
membranes were washed and resuspended in 120 mM potassium
P
or 120 mM sodium phosphate (NaP
), pH
8.0, containing 2 mM MgSO
. Potassium-loaded
membranes were diluted into the same buffer (no gradient) or in
NaP
buffer (
, interior negative). Sodium-loaded
membranes were diluted into the same buffer (no gradient) or in
potassium-containing buffers (
, interior positive).
Measurement of Proton Transport
Sugar-induced proton uptake was measured essentially as
described by Henderson and Macpherson(1986). Cells were washed and
resuspended in an equal volume of 150 mM KCl plus 2
mM glycylglycine, pH 6.5. The cells were starved for 3 h at 37
°C (shaking cell suspension), washed two more times, and
resuspended in the KCl-glycylglycine medium to a final protein
concentration of 60-70 mg/ml. Proton transport was measured with
a pH electrode in 150 mM KCl, 30 mM potassium
thiocyanate plus 2 mM glycylglycine, pH 6.2, at a cell density
of 3 mg/ml. The cell suspension was made anaerobic by flushing with
argon. Proton uptake was induced by addition of 5 mM sugar.
Immunoblotting
The amount of wild-type and various mutant LacS proteins in
the membrane was estimated by immunoblotting with an anti-carboxyl
terminus antibody directed against the synthetic peptide
NH-Cys-Glu-Lys-Val-Glu-Ala-Leu-Ser-Glu-Val-Ile-Thr-Phe-Lys-Lys-Gly-Glu
(COOH-terminal acid)). The synthetic peptide (Multiple Peptide Systems,
San Diego) was conjugated to keyhole limpet hemocyanin through the
cysteine at the amino terminus of the peptide (Harlow and Lane, 1988).
Immunization of rabbits and collecting blood samples were performed
using standard procedures (Harlow and Lane, 1988). Inside-out or
right-site-out membrane vesicles of E. coli DW2 (or HB101)
carrying plasmid pSKE8 and expressing wild-type or mutant LacS protein
were used to estimate the expression of the lactose transport protein.
Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (12.5% polyacrylamide) and transferred to
polyvinylidene difluoride membranes by semidry electrophoretic
blotting. Processing of the polyvinylidene difluoride membranes
(antibody binding: serum dilutions of 10,000-40,000-fold), and
immunoblot detection using a secondary alkaline phosphatase-labeled
anti-rabbit IgG was performed as described (Harlow and Lane, 1988).
Site-directed Mutagenesis and DNA Sequencing
The mutagenic primers used are E379A,
5`-CACCGTGATGCT-TCGTTAACTTTGTCA; E379D,
5`-CACCGTGATGATTCGTTAACTTTGTCA; and E379Q,
5`-CACCGTGATCAATCGTTAACTTTGTCA. The nucleotides changed by the
site-directed mutagenesis are underlined. The nucleotide changes
resulting in the desired amino acid substitution are indicated in bold.
The other (silent) mutations were made to create a new HincII
site. Site-directed mutagenesis was carried out by the Kunkel method
(Kunkel et al., 1987). Single-stranded uracil-containing DNA
of pSKE8 was isolated from E. coli CJ236
(dut, ung
)/pSKE8
after infection with helper phage M13KO7 (Sambrook et al.,
1989). Closed circular heteroduplex DNA with the desired mutations was
synthesized in vitro as described (Kunkel et al.,
1987) and transformed to E. coli JM101
(ung
). Plasmid DNA was isolated from a number
of transformants and plasmids bearing the desired mutation(s) were
identified by digestion with HincII. The plasmids with an
extra HincII site were transferred to E. coli HB101,
and the phenotype (growth on MacConkey-lactose agar and mineral media,
transport activity) of at least two independent isolates was determined
(see ``Results''). Subsequently, mutations were verified by
nucleotide sequencing of double-stranded DNA using the dideoxy chain
termination method (Sanger et al., 1977) and a set of primers
complementary to a region of lacS located 50-100 bases
downstream or upstream of the mutation site. The nucleotide sequence of
the entire lacS-E379Q gene was determined.
Mutagenesis
Fig. 1
shows the alignment of
the amino acid sequence of putative interhelix loop X-XI of the lactose
transport protein (LacS) of S.thermophilus and
homologous regions in other sugar transport proteins. The glutamate
residue at position 379 of LacS is conserved in each of the aligned
sequences (arrow 3). Glu of the lactose
transport protein (LacS) was replaced by site-directed mutagenesis with
aspartate, glutamine, or alanine. Each mutant was isolated
independently at least twice, and the phenotypes were compared with
those of the wild-type. Plasmids with the mutations were used to
transform E. coli HB101 (lacY1), and transformants
were streaked on lactose MacConkey agar plates or mineral medium plates
supplemented with lactose. The lac phenotype of each of the
position 379 mutants on both types of media was indistinguishable from
that of the wild-type strain, i.e. HB101/pSKE8.
Uphill and Downhill Transport
To measure uphill
transport of lactose, each of the plasmids, bearing lacS wild-type or a mutant allele, was used to transform E. coli DW2 (lacZY). The wild-type protein accumulated
lactose 13-14-fold within 4 min, LacS-E379D accumulated
2-3-fold, while no accumulation was observed with the mutants in
which Glu
was replaced by alanine or glutamine. The
LacS-E379A and LacS-E379Q exhibited an initial rate of lactose uptake
that was significantly higher than the strain with the plasmid control
(Fig. 2, left panel).
Figure 2:
Uphill and downhill transport of lactose.
For uphill transport, E. coli DW2/pSKE8 wild-type and various
LacS mutants were grown in Luria broth supplemented with carbenicillin
(50 µg/ml). Cells were washed and resuspended in KPM, pH 6.6, to a
final protein concentration of 60-70 mg/ml. Transport of lactose
(19 µM, final concentration) was assayed in the presence
of the electron donor D-Li-lactate and at a final protein
concentration of 0.6-0.7 mg/ml. For downhill transport, E.
coli HB101/pSKE8 wild-type and various mutants were grown in Luria
broth supplemented with carbenicillin (50 µg/ml) and 0.1
mM isopropyl-1-thio--D-galactopyranoside. Cells
were treated as described above, and transport of lactose (47
µM, final concentration) was assayed in the absence of
exogenous electron donor. The experiments were performed at 30
°C.
For downhill uptake of lactose,
E. coli HB101 was used as host, and cells were grown in the
presence of 0.1 mM
isopropyl-1-thio--D-galactopyranoside in order to express
the
-galactosidase activity maximally. Uptake of lactose down its
concentration gradient was observed with LacS (wild-type) but also with
the Glu
mutants (Fig. 2, right panel). In
fact, the rates of lactose uptake by the Glu
mutants were
significantly higher than those of the wild-type.
Coupling of Lactose and H
The apparent inability of the LacS-E379Q and
E379A mutants to transport lactose against a concentration gradient
(Fig. 2, left panel) could be due to a defect in the
coupling of lactose and proton transport. Lactose uptake by E. coli DW2/pSKE8 wild-type LacS and LacS-E379Q was measured for prolonged
time intervals (up to 30 min, sampling every 1-2 min) and at pH
5.0, 5.7, 6.6, and 7.5. Fig. 3shows that highest accumulation
levels are observed at pH 5.7 (lactose Transport
/lactose
is 33 for wild-type LacS). For LacS-E379Q some accumulation can
be observed at pH 5.0 and 5.7, but not at pH 6.6 and 7.5.
Figure 3:
Steady state accumulation levels of
lactose in E. coli DW2/pSKE8 LacS (wild-type), and LacS-E379Q.
For experimental procedures see legend to Fig. 2 (uphill transport),
except that cells were washed and resuspended in KPM of the indicated
pH and transport was assayed at a final lactose concentration of 3.6
µM. Uptake of lactose was monitored for 30 min and samples
were taken every 1-2 min.
Lactose-induced H uptake was measured with a pH
electrode. Addition of 5 mM lactose to a suspension of E.
coli DW2/pSKE8 wild-type LacS resulted in a transient
alkalinization of the medium which is consistent with a lactose
H
symport mechanism (Fig. 4). The alkalinization
was reduced in the LacS-E379D mutant and was not observed with strains
carrying either the control plasmid (LacS
),
LacS-E379A, or LacS-E379Q. From these experiments it is tentitatively
concluded that lactose and H
uptake in the LacS-E379Q
(and E379A) mutant(s) is uncoupled and that lactose uptake proceeds via
uniport.
Figure 4:
Lactose-induced proton uptake by LacS
(wild-type), LacS-379D, and LacS-E379Q. E. coli DW2/pSKE8 was
grown in M9 mineral medium supplemented with 1 g/liter casamino acids,
0.001% (w/v) thiamine, 20 mM glycerol, and 50 µg/ml
carbenicillin. Lactose (30 µl of a 500 mM solution) was
added at the time indicated by the arrow. The pH of the
suspension was monitored with an electrode; small aliquots of 10
mM KOH were used to calibrate the pH
recordings.
Efflux Down a Concentration Gradient and Equilibrium
Exchange
The experiments presented above describe uphill
(p-driven) and downhill (driven by the lactose
concentration gradient,
pLac; [lactose]
< [lactose]
due to high
-galactosidase activity) lactose uptake. The lactose transport
protein also catalyzes reaction(s) in the opposite direction, e.g. efflux down a concentraton gradient as well as exchange (Foucaud
and Poolman, 1992). Fig. 5shows efflux and equilibrium exchange
of lactose at pH 6.6 (pH
= pH
) by
wild-type LacS and the various Glu
mutants. Lactose
efflux down a concentration gradient was slow for the wild-type
protein, much faster for LacS-E379D, and fastest for LacS-E379A and
LacS-E379Q (Fig. 5, left panel). This indicates that
substitution of Glu
for Asp, Ala, or Gln alters the
rate-determining step(s) of the efflux reaction, possibly by shifting
the protonation equilibrium at the outer surface of the membrane to the
deprotonated form of the carrier molecule or allowing a more rapid
return of the unloaded carrier to the inner surface of the membrane.
The differences in the rates of lactose exchange catalyzed by the
wild-type and mutant proteins were relatively small (Fig. 5,
right panel), suggesting that the rate-determining steps in
the translocation cycle for exchange are different from those in efflux
transport.
Figure 5:
Efflux down a concentration gradient and
equilibrium exchange of lactose by wild-type and mutant LacS proteins.
E. coli DW2/pSKE8 wild-type and various LacS mutants were
grown in Luria broth supplemented with carbenicillin (50 µg/ml).
Cells were washed and resuspended in KPM, pH 6.6, to a final protein
concentration of 60-70 mg/ml. Loading of the cells with
[C]lactose (5 mM, final concentration)
was performed as described under ``Experimental Procedures.''
Aliquots of cells (1 µl) were diluted 500-fold into KPM, pH 6.6,
supplemented with 50 µM CCCP and without (downhill efflux)
or with 5 mM lactose (equilibrium exchange). Efflux (left
panel) and exchange (right panel) were assayed at 20
°C. Inset, non-equilibrium exchange of lactose by E.
coli DW2/pSKE8 LacS (wild-type) and LacS-E379D. The assay
temperature was kept at 10 °C. The experimental data were fitted to
the Michaelis equation. Activity is given as nmol/min
mg of
protein.
Under conditions of exchange with 5 mM
[C]lactose inside and 5 mM
[
C]lactose outside the cell, the exchange
reaction of the Glu
mutants was consistently faster than
that of the wild-type. Since the release of
[
C]lactose from the cell is monoexponential, at
least initially, with a slope that depends upon the external lactose
concentration, an apparent K
for the exchange
reaction can be estimated. The K
values of
the wild-type LacS and LacS-E379D were 10 and 2.5 mM,
respectively (Fig. 5, right panel, inset). The
maximal rates of exchange were the same for the wild-type and E379D
mutant protein. Since the wild-type and Glu
mutant
proteins are expressed to the same level in E. coli membranes,
the differences in the equilibrium exchange reactions shown in
Fig. 5
, right panel, can be explained by the lower
K
of the E379D mutant relative to the
wild-type protein.
pH Dependence of Efflux Down a Concentration Gradient and
Equilibrium Exchange
The pH dependence of the first-order rate
constant (k) of the efflux reaction of the wild-type, E379D,
and E379Q mutant proteins is shown in Fig. 6A. The pH
profile of the E379D mutant is shifted to lower pH values by at least 2
units, whereas the profile of the E379Q mutant is shifted even further
(pK of about 6.0). This suggests that residues at position 379
directly affect the deprotonation of the carrier molecule at the outer
surface of the membrane, perhaps by modulating the pK of a
nearby group that is on the pathway of proton translocation.
Figure 6:
pH
dependence of efflux down a concentration gradient (A) and
equilibrium exchange (B) of lactose by wild-type and mutant
LacS proteins. Experimental procedures are the same as described in the
legend of Fig. 5 except that CPC buffer was used and the external pH
was varied between 4.1 and 9.8. The temperature was kept at 25 and 15
°C for the efflux and exchange assay, respectively. First-order
rate constants of efflux (A) and exchange (B) are
plotted as a function of pH.
Equilibrium exchange of galactosides in membrane vesicles of S.thermophilus, expressing LacS, exhibits an optimum
between pH 6-7 (Foucaud and Poolman, 1992). Similar results were
obtained with the product of the lacS gene cloned in E.
coli (Fig. 6B). Moreover, the pH profile of the
exchange reaction of the wild-type and E379D mutant LacS proteins was
similar. In contrast to the wild-type protein and LacS-E379D, the E379Q
mutant was much less affected by pH in the range of 4 to 10.
Effect of Membrane Potential on Efflux Down a
Concentration Gradient and Equilibrium Exchange
Neutral
substitutions of Glu result in a carrier protein that is
unable to transport lactose against a concentration gradient. To test
whether a membrane potential (
) has a kinetic effect on the
apparent uniport of lactose, efflux down a concentration gradient by
the wild-type and E379Q mutant in the presence and absence of a
was compared. Fig. 7, left panel, shows, as
expected, that lactose efflux in membrane vesicles bearing wild-type
LacS is retarded by a
(inside negative). Comparable results
were obtained with LacS-E379D (data not shown), but also (at least
qualitatively) with membrane vesicles derived from strains expressing
LacS-E379Q (Fig. 7, right panel). These results indicate
that also for the LacS-E379D and LacS-E379Q enzymes one or more steps
in the translocation cycle are affected (kinetically) by the membrane
potential. Equilibrium exchange of lactose by LacS wild-type and
position-379 mutants was not affected by the
(data not
shown).
Figure 7:
Effect of membrane potential ()
and sodium gradient (
pNa) on lactose efflux down a concentration
gradient in right-site-out membrane vesicles of E. coli DW2/pSKE8 expressing the wild-type or LacS-E379Q protein. Membrane
vesicles were washed and resuspended in 20 mM potassium
phosphate, 100 mM potassium acetate, pH 8.0. Concentrated
membrane vesicles (35 mg of protein/ml) were diluted 100-fold into the
appropriate buffers, pH 8.0, to generate a
(inside
negative) or
pNa (control +Na
, 20
mM NaCl on the outside) or no gradient (control
-Na
) as described under ``Experimental
Procedures.'' The internal lactose concentration was 5
mM, and efflux was assayed at 30
°C.
in the
lactose transport protein of S.thermophilus have
been analyzed. The properties of LacS Glu
mutants reveal
some striking similarities and differences with the corresponding
mutations in LacY (). The mutations reduce the ability of
both enzymes to accumulate sugars against a concentration gradient.
Neutral substitutions abolish uphill transport completely whereas the
Glu to Asp substitutions result in reduced accumulation levels. For
both types of mutants, and both in LacS and LacY, the rate of downhill
uptake is not much affected. Also, the apparent affinity constants for
transport or sugar binding are only moderately affected by the
Glu
and Glu
mutations in LacS and LacY,
respectively.
mutants has not
been fully explained but suggests that translocation of the unloaded
carrier is highly rate-determining during downhill efflux but not
during downhill influx.
in LacS and Glu
in LacY play direct
roles in the energy coupling mechanisms of the corresponding proteins
and that the glutamate residues are closely associated with the pathway
for proton transport in each of the proteins. Without the negative
charge at position 379 (LacS) and 325 (LacY), the proteins behave as
sugar uniporters at least when uptake transport is assayed. A similar
role for Glu
in LacS and Glu325 in LacY is remarkable
because the overall polarity of the sequences around these residues is
quite different, which, in turn, suggests a different position of the
residues in the secondary structure models of the two proteins. The
sequence motif
Lys
-X-X-His
-X-X-Glu
of LacY is present in the middle of putative transmembrane
-helix
X, which is supported by a large number of lacY-phoA (LacY-alkaline phosphatase) fusions (Calamia and Manoil, 1990,
1992). Given the similarity in function this would imply that the motif
Lys
-X-X-His
-X-X-Glu
of LacS is present in a similar transmembrane segment. However, the
region around Lys
, His
, and His
in LacS is much more hydrophilic (Fig. 1), and, hydropathy
profiling predicts this region to be in a cytoplasmic loop between
-helices X and XII. The functional similarities between
Glu
of LacS and Glu
of LacY (this study),
and His
of LacS and His
of LacY (Poolman
et al., 1992), suggest that the hydrophilic region containing
the motif
Lys
-X-X-His
-X-X-Glu
of LacS is located in the interior of the protein as well and forms
part of the translocation pathway.
mutations, the kinetic behavior of the sugar H
symporter has been simulated. The procedure for analyzing the
steady state kinetic behavior of hypothetical enzymes has been
described previously (Lolkema, 1993). The kinetics of the efflux and
exchange reactions catalyzed by wild-type LacS have been studied
extensively (Foucaud and Poolman, 1992; this work). For instance,
exchange is characterized by a bell-shaped pH profile that has its
optimum around pH 6. The pH optimum for efflux is at much higher pH
values (>8) and, moreover, efflux is much slower than exchange.
Based on these and other observations, it has been concluded that the
binding order on the inside of the membrane is substrate first, proton
second. Simulations of kinetic schemes with random and various orders
of binding showed that the suggested ordered binding on the inside
exclusively resulted in the observed experimental behavior, provided
that binding at the outside was not of the same order
(i.e. either random or proton first, substrate second) and
that the pK of the proton binding site on the outside was at
least 4 pH units higher than on the inside (not shown). The random
order on the outside was chosen (see below) in the kinetic scheme
presented in Fig. 8, top panel, that together with the
set of rate constants described in the legend to the figure mimics
wild-type LacS. The pH profile of exchange is a consequence of the
ordered binding at the inner side of the membrane and the optimum is
mainly determined by the pK of the proton binding site at this
side of the membrane (pK
= 6;
Fig. 8
, top panel). At pH values below the
pK
, the rate goes down because the
carrier remains protonated at the inside which inhibits the exchange
between bound and free substrate. At pH values above the
pK
, the exchange rate goes down because
the carrier deprotonates which reduces the fraction of the enzyme in
the ternary complex. The maximal rate of exchange at the pH optimum is
determined by the rate constants for the translocation of the ternary
complex (k
and k
).
Efflux requires protonation of the carrier at the inside of the
membrane (pK
= 6) and
deprotonation of the carrier at the outside (pK
= 10). The two pK values involved are such that
these requirements are difficult to meet, which results in low turnover
rates. Maximal turnover is observed at a pH value in between the two
pK values. At pH values below the optimum, the rate decreases
because the carrier remains protonated at the external face of the
membrane, whereas at pH values above the optimum, the rate decreases
because the carrier does not become protonated at the internal face of
the membrane.
Figure 8:
Schematic representation of the reactions
involved in LacS-mediated galactoside transport. E,
H, and S represent the carrier
protein, proton, and ligand (galactoside), respectively. The
subscripts o and i refer to the outer and inner
surface of the membrane, respectively. In the model, the formation of
the ternary complex (ESH) via binary EH and
ES are indicated; the association-dissociation of substrate
and proton at the inner surface of the membrane is ordered (see text).
Since information on cooperativity between H
and sugar
binding is not available, the rate constants for H
binding to the free and sugar liganded carrier were chosen to be
identical (likewise for sugar binding). A membrane potential was
included in the simulations by affecting k
,
k
, k
, and
k
as described previously (Lolkema and Poolman,
1995). Top panel, wild-typeLacS protein. The
affinity constant for the substrate is 1 mM, the
pK
and pK
are 10 and 6,
respectively; the corresponding rate constant are:
k
= 1,000
mM
s
,
k
= 1,000
s
, k
=
100,000 µM
s
, k
= 10 s
,
k
= 20
s
, k
=
200 s
, k
= 1,000 µM
s
, k
=
1,000 s
, k
= 1,000
mM
s
,
k
= 1,000 s
,
k
= 2000 s
,
k
= 2 s
.
Bottom panel, LacS-E379Q protein. The pK
has been lowered from 10 to 6 and an ES leak pathway
(translocations k
, k
)
has been introduced relative to the scheme for wild-type LacS; the
following adjustments to the rate constants have been made:
k
= 1,000 µM
s
, k
= 1,000 s
, k
= 20
s
, k
= 20
s
, k
= 20
s
, k
= 20
s
, k
= 20
s
. In order to lower the pK
,
both the k
and k
were
altered; the behavior of LacS-E379Q can also be simulated by a similar
change in pK which is achieved through adjustment of 1 of the
rate constants. For the other equilibria we have chosen to alter both
the forward and reverse rate constants. The changes in
k
, k
, and
k
were made in order to meet the
requirements of thermokinetic balancing (Walz and Caplan,
1988).
In a previous report (Lolkema and Poolman, 1995), we
analyzed the phenotype of uncoupled transport mutants in which either
the binary enzyme-sugar complex or the enzyme-H complex can freely reorient its binding sites (the
``ES leak'' and ``EH leak,''
respectively). Mutants of Glu
in LacS are of the
ES leak type by the following criteria: (i) sugar accumulation
increases with decreasing pH (Fig. 3), (ii) the rate of efflux
increases sigmoidally with pH (Fig. 6A), and (iii)
efflux is inhibited by the membrane potential (Fig. 7) under
conditions where uphill transport cannot be observed ( Fig. 2and
Fig. 3
). Apparently, the Glu
mutations induce the
isomerization of the binary enzyme-sugar complex (Fig. 8,
bottom panel).
E
transition. This
transition is common to the pathway of coupled and uncoupled (uniport)
transport (Fig. 8). To ascertain equilibration of internal and
external substrate by the uniport pathway, the ES leak
transition has to be
dependent as well. As a result,
affects the kinetics of the uniport cycle, but not the
equilibrium position. A
(inside negative) slows down efflux
through its effect on the
E
E
step but
accelerates efflux by affecting the
ES
ES
transition. The
E
E
transition becomes
more and more rate limiting as the
increases (Lolkema and
Poolman, 1995), and, consequently, not only efflux by the wild-type but
also that by an enzyme with an ES leak is inhibited by a
(inside negative).
= 6). At pH values below the
pK, the rate of efflux decreases because the carrier remains
protonated at the outside. At higher pH values efflux proceeds via the
uncoupled pathway involving translocation of the binary E:S complex
which is independent of pH. Apparently, the E379Q mutation results in a
lowering of the pK of the external proton-binding site.
Simulation of the kinetics after introduction of the ES leak
and lowering of the pK for proton binding on the outside
results in the observed experimental phenomena, i.e. exchange
that is largely independent of pH (Fig. 9, right panel),
an efflux reaction that is stimulated relative to the wild-type enzyme
(Fig. 9, left panel), uncoupled uptake above pH 6.5 and
poorly coupled uptake below pH 6.5, and inhibition of efflux at high pH
by a membrane potential, inside negative (not shown).
Figure 9:
Simulation of the maximal rate of efflux
down a concentration gradient and equilibrium exchange of LacS
wild-type (Fig. 8, top)and LacS-E379Q (Fig. 8,
bottom). The maximal rates of efflux and exchange were
analyzed as a function of pH using the kinetic parameters indicated in
the legend to Fig. 8.
The analysis
indicates that neutral substitutions of Glu in LacS lower
the activation energy for translocation of the binary enzyme-sugar
complex (ES leak) and lower the pK of the proton
binding site at the outside from above 10 to 6, which is equal to the
pK at the inside. The lowering of the
pK
suggests that in the wild-type enzyme
the pK of the proton translocating residue(s) is modulated by
Glu
. Assuming a single site in the proton pathway, this
would imply that Glu
raises the pK of this site
when the carrier is in the conformation with the binding sites facing
outward. A lowering of pK in the mutants is consistent with
the substitutions made (Glu
Gln and Glu
Ala), i.e. a negative charge raises the pK of a nearby group by stabilizing the protonated form. By removing
the negative charge by mutation, or by a conformational change that is
associated with the exposure of the binding sites to the inside, the
pK is expected to be lowered. However, the observed shift in
pK of more than 4 pH units upon substituting Glu
for Gln is extremely large, and the shift may therefore not only
be due to electrostatic effects (Russell and Fersht, 1987). In
principle, a relatively minor conformational change in the E379Q mutant
may have moved the essential protonatable group from a hydrophobic (low
dielectric medium) to a more hydrophilic environment thereby lowering
the pK even further. This would imply that in the wild-type
enzyme the proton-binding site is in a more hydrophilic environment in
the inside conformation than when it is on the outside. The properties
of the E379D mutant are intermediate to those of the wild-type and
E379Q protein which could reflect a different positioning of the
carboxylate group relative to that in the wild-type protein. Finally, a
single point mutation that results in a largely uncoupled transporter
and a significant shift in pK suggests a mechanistic relation
between coupling efficiency and the pK of the proton
translocating group.
Table:
Properties of the LacS Glu and
LacY Glu
mutants
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.