(Received for publication, August 30, 1994; and in revised form, December 12, 1994)
From the
The mannose transporter complex acts by a mechanism which
couples translocation with phosphorylation of the substrate. It
consists of a hydrophilic subunit (IIAB) and two
transmembrane subunits (IIC
, IID
). The
purified complex was reconstituted into phospholipid vesicles by octyl
glucoside dilution. Glucose export was measured with proteoliposomes
which were loaded with radiolabeled glucose and to which purified
IIAB
, cytoplasmic phosphorylcarrier proteins, and
P-enolpyruvate were added from the outside. Vectorial transport was
accompanied by stoichiometric phosphorylation of the transported sugar.
Glucose added to the outside of the proteoliposomes was also
phosphorylated rapidly but did not compete with vectorial export and
phosphorylation of internal glucose. Glucose uptake was measured with
proteoliposomes which were loaded with the cytoplasmic phosphoryl
carrier proteins and P-enolpyruvate and to which glucose was added from
the outside. Vectorial import and phosphorylation occurred with a
higher specificity (K
30 ± 6
µM, k
401 ± 32 pmol of
Glc/µg of IICD
/min) than nonvectorial phosphorylation (K
201 ± 43 µM, k
975 ± 88 pmol of Glc/µg of
IICD
/min).
A new plasmid pTSHIC9 for the controlled
overexpression of the cytoplasmic phosphoryl carrier proteins, enzyme
I, HPr, and IIA, and a simplified procedure for the
purification of these proteins are also described.
Uptake of hexoses and hexitols in bacteria is mediated by
membrane protein complexes, the so-called enzymes II of the bacterial
phosphotransferase system (PTS). ()They couple vectorial
translocation with phosphorylation of the transported solute. Coupling
is tight unlike in eukaryotic cells where transport and phosphorylation
of glucose are mediated by a sequential pair of enzymes (transporter
and kinase). However, phosphorylation of cytoplasmic substrates without
transport, e.g. of glucose derived from maltose within the
cell, has also been observed (Thompson and Chassy, 1985; Thompson et al., 1985; Nuoffer et al., 1988). There exist 16
enzymes II of different substrate specificity in Escherichia
coli. They are composed of three functional units (IIA, IIB, IIC,
or IICD; for nomenclature see Saier and Reizer(1992)) which occur
either as protein subunits or domains of a multidomain protein. The IIC
unit spans the membrane several times. It contains the substrate
binding site. The IIA and IIB units are hydrophilic. They sequentially
transfer phosphoryl groups from the ``high-energy''
phosphoryl carrier protein phospho-HPr to the substrate on the IIC
subunit. Phospho-HPr in turn is regenerated by P-enolpyruvate in a
reaction catalyzed by enzyme I. The collective of phosphoproteins which
have additional functions in metabolite transport, chemotaxis, and
metabolic regulation constitute the bacterial phosphotransferase system
(for reviews, see Meadow et al.(1990), Erni(1992), and Postma et al.(1993)).
The mannose transporter of the PTS has a
broad substrate specificity for mannose, glucose, and related hexoses.
In addition, it acts as a host factor required for infection of E.
coli by bacteriophage (Elliott and Arber, 1978). It consists
of three subunits (Fig. 1). The IIC
and
IID
subunits form a tight transmembrane complex which is
sufficient for penetration of phage
DNA (Erni et al.,
1987). For transport and phosphorylation of sugars, the third subunit,
IIAB
, is also required. IIAB
is reversibly
associated with the IIC
-IID
complex (K
5-10 nM). (
)It consists of two hydrophilic domains IIA
and IIB
which are linked through a hinge
peptide-rich in Ala and Pro (Erni, 1989). IIA
and
IIB
each contain one phosphorylation site (His
and His
) which relay the phosphoryl groups from HPr
to the substrate on the IIC
-IID
complex.
The isolated IIB domain together with the IIC
-IID
complex is sufficient for equilibrium phosphoryl exchange between
Glc-6-P and Glc (Erni et al., 1989).
Figure 1:
Hypothetical model of the mannose
transporter and schematic representation of its action. A,
vectorial transport and phosphorylation. Translocation and
phosphorylation are tightly coupled. The transported solute (Glc) does not exchange with free solute (Glc). B,
nonvectorial phosphorylation. Thin lines indicate the flow of
phosphoryl groups from P-HPr via the IIA
domain (dotted) and the IIB
domain (diagonal
hatching) to Glc. The two domains of IIAB
are linked
by an Ala-Pro-rich hinge. One IIC
subunit (vertical
hatching) and two IID
subunits (horizontal
hatching) form the transmembrane
complex.
Due to the complexity of the system comprising two cytoplasmic phosphoryl carrier proteins (enzyme I and HPr) in addition to the transporter, sugar phosphorylation rather than vectorial transport activity is measured in routine assays (Kundig and Roseman, 1971). Of all the purified PTS transporters only the mannitol transporter could be reconstituted into phospholipid vesicles, so far (Elferink et al., 1990). The mannose transporter differs from the mannitol transporter in subunit composition and stoichiometry, chemistry of the active site, and protein structure. The molecular mechanism of vectorial solute transport and its coupling to phosphorylation are not understood. As a first step toward the elucidation of this process the mannose transporter was reconstituted into phospholipid vesicles and the vectorial transport and phosphorylation activity was measured. Also addressed was the question of substrate channeling (Srivastava and Bernhard, 1986; Srere, 1987; Welch and Easterby, 1994) between the transport and phosphorylation moieties, the hypotheses of transport regulation by the membrane potential (Reider et al., 1979; Robillard and Konings, 1981, 1982), and the proposition of a coupling of solute uptake with proton extrusion (Scarborough, 1985). To provide the soluble phosphoryl carrier proteins enzyme I and HPr, which are required in large amounts for the loading of proteoliposomes, a recombinant plasmid for their overexpression was constructed and methods for their purification are described.
In parallel experiments the membrane potential formed by
K-efflux from K
-loaded
proteoliposomes was measured with the fluorescence indicator
1,3,3,1`,3`,3`-hexamethylindoldicarbocyanine (NK529, Nippon Kankoh
Shikiso Kenkyusho, Okayama, Japan) as described by Apell et
al.(1985). 170 µl of K
containing purified
proteoliposomes (without [
C]Glc) and 530 µl
of buffer (149 mM NaP
, pH 7.5, 1 mM KP
) containing 37 µM NK529 (added from
2.5 mM stock solution in 1:9 (v/v) ethanol/water) were mixed
in a 1-ml cuvette. After the fluorescence signal reached a constant
value (F
), valinomycin was added to a final concentration
of 40 nM whereupon the fluorescence signal rapidly decreased
to a new constant value (F). The K
concentration was
increased by small increments and the fluorescence change (
F
= F-F
) was monitored. There is a linear relationship
between the membrane potential, which was calculated from the Nernst
equation, and the fluorescence change: 0.64 F/mV. The fluorescence
measurements were carried out with a Perkin-Elmer LS-5B luminescence
spectrometer. The excitation wavelength was set to 620 nm (slit width 5
mm) and the emission wavelength to 680 nm (slit width 5 mm).
Figure 2:
Plasmid map of pTSHIC9 (A) and
Coomassie-stained polyacrylamide gels of enzyme I, HPr, and IIA (B). Lane 1, whole cell lysate from
IPTG-induced cells; lane 2, purified HPr; lane 3,
purified enzyme I; lane 4, purified IIA
(the
double band represents full-length and NH
terminally
processed forms of IIA
(Meadow et al., 1986)).
The molecular mass markers are phosphorylase b (94 kDa),
bovine serum albumin (68 kDa), ovalbumin (43 kDa), carbonic anhydrase
(30 kDa), soybean trypsin inhibitor (20.1 kDa), and
-lactalbumin
(14.4 kDa). The 8-25% polyacrylamide gel was prepared according
to Fling and Gregerson (1986).
Figure 3:
Proteolysis of the
IIC/IID
complex in proteoliposomes. A, 50% of the IID
(D) subunits are
processed to an intermediate (D*) upon incubation of intact
proteoliposomes with subtilisin (S), close to 100% after
solubilization of proteoliposomes with Triton X-100. Digestion of
IIC
(C) to C* is not complete. B, 50% of the IID
(D) subunits are
processed upon incubation of intact proteoliposomes with trypsin (T). No distinct IID
intermediate is formed and
digestion of IIC
(C) to C* is not
complete. The 20% polyacrylamide gels were stained with alkaline
silver.
Import of [C]Glc was measured with
proteoliposomes which were loaded with purified soluble PTS proteins
and P-enolpyruvate. [
C]Glc was added to start
the uptake reaction (Fig. 4). 80% of the glucose originally
present at a concentration of 5 µM was concentrated in the
proteoliposomes and accumulation was accompanied by phosphorylation (Fig. 4A). The k
and K
of vectorial transport by proteoliposomes were
401 ± 32 pmol of Glc/µg of
IIC
-IID
D/min and 30 ± 6
µM, respectively (Fig. 4, B and C). Assuming that the molecular mass of the functional unit of
the mannose transporter is 90 kDa and that only 50% are correctly
oriented and therefore active, a turnover number of 1.2 s
can be calculated. No uptake of [
C]Glc
could be detected, when P-enolpyruvate, enzyme I, HPr,
IIAB
, and [
C]Glc were added to the
outside of the proteoliposomes (results not shown), indicating that
transport occurs only if the solute ([
C]Glc) and
the phosphoryl transfer components are on opposite sides of the
membrane.
Figure 4:
Uptake and phosphorylation of glucose. A and B, IIC-IID
containing proteoliposomes are loaded with IIAB
,
the phosphoryl carrier proteins, and P-enolpyruvate. The uptake
reaction is started (arrow) by addition of
[
C]Glc to the outside. Open symbols,
uptake; closed symbols, phosphorylation. A background of 5
pmol of Glc has been subtracted. A, 0.1 µM IIC
-IID
,
IIC
-IID
:phospholipid ratio =
1:30,000, 5 µM Glc (
,
), 50 µM Glc (
,
). B, 0.04 µM IIC
-IID
,
IIC
-IID
:phospholipid ratio 1:75,000, 3.8
µM (
), 7.5 µM (
), 15 µM (
), 28 µM (
), 56 µM (
), and 112 µM Glc (
). C,
Lineweaver-Burk plot of initial rates of uptake activity from B. k
= 1.2 ± 0.1
s
and K
= 30
± 6 µM. Shown are means and S.D. of three
independent transport experiments done with a single batch of
reconstituted proteoliposomes.
Observations with intact bacteria indicated that PTS
transporters not only catalyze vectorial transport with phosphorylation
but also phosphorylation of substrates in the cytoplasm (nonvectorial
phosphorylation, e.g. of glucose generated in the cell from
maltose). When [C]Glc is added together with the
soluble phosphoryl carrier proteins to the outside of
IIC
-IID
containing proteoliposomes, Glc-6-P
is formed rapidly without concommitant transport (Fig. 5A). The k
and K
of nonvectorial phosphorylation by
proteoliposomes are 975 ± 88 pmol of Glc-6-P/µg of
IIC
-IID
/min and 201 ± 43
µM, respectively (Fig. 5B).
Phosphorylation without transport is therefore 2-3 times faster
than vectorial phosphorylation.
Figure 5:
Nonvectorial phosphorylation of glucose. A, P-enolpyruvate and the phosphoryl carrier proteins are
added to the outside of IIC-IID
containing
proteoliposomes (0.1 µM IIC
-IID
,
IIC
-IID
:phospholipid ratio 1:30,000). The
reaction was started by addition of [
C]Glc to
15.1 µM (
), 38.5 µM (
), 76.3
µM (
), 151.0 µM (
), 298.5
µM (
), and 579.0 µM (
) final
concentration. B, Lineweaver-Burk plot of initial rates of
phosphorylation activity from A. k
= 2.9 ± 0.3 s
and K
= 201 ± 43
µM. Shown are means and S.D. of three independent
experiments done with a single batch of reconstituted
proteoliposomes.
This nonvectorial phosphorylation
raises two questions. First, passive diffusion of solute followed by
nonvectorial phosphorylation could in principle be misinterpreted as
true vectorial phosphorylation in the reconstituted system. Second,
transport coupled to phosphorylation and nonvectorial phosphorylation
could be competing reactions. The two problems were addressed by
measuring the efflux of [C]Glc in the presence
and absence of nonlabeled external glucose.
To test for passive and
IIC-IID
-mediated facilitated diffusion,
(proteo)liposomes were loaded with [
C]Glc and
purified by spin column chromatography (without the hexokinase
treatment described under ``Materials and Methods''). By
adding hexokinase and ATP, a constant amount of Glc could be converted
to Glc-6-P in the external compartment of pure liposomes as well as of
proteoliposomes containing the IIC
-IID
complex. This amount did not change when the soluble phosphoryl
carrier proteins were added as long as either P-enolpyruvate or the
IIAB
subunit were omitted (Fig. 6). The amount of
Glc-6-P did, however, increase immediately above this background when
P-enolpyruvate (Fig. 7B) was added, and this increase
was accompanied by a stoichiometric decrease of trapped glucose (Fig. 7A). This result indicates that (i) export of
[
C]Glc concomitant with phosphorylation is
catalyzed only by the complete phosphotransferase system, (ii) passive
and IIC
-IID
-dependent facilitated diffusion
of [
C]Glc do not occur on the time scale of up
to 1 h, and (iii) a small amount of [
C]Glc which
was not removed by spin column purification alone remains accessible
from the outside. Henceforth, residual nontrapped glucose was removed
with hexokinase prior to spin column purification as described under
``Materials and Methods.'' When IIAB
, enzyme I,
HPr, and P-enolpyruvate were added to the hexokinase-treated and spin
column-purified proteoliposomes, rapid export of trapped
[
C]Glc concomitant with phosphorylation could be
measured (Fig. 7). Export of glucose and phosphorylation are
strictly coupled in a 1:1 molar ratio.
Figure 6:
Nonspecific carry over (adsorption) and
passive diffusion of Glc out of proteoliposomes. Proteoliposomes were
loaded with [C]Glc and purified by spin column
centrifugation (time 0). Hexokinase (1 mg/ml) and ATP (4.0 mM)
were added after 20 min (arrow) and the formation of Glc-6-P
was monitored by ion exchange chromatography. Pure liposomes without
IIC
-IID
(
); proteoliposomes with
IIAB
, enzyme I, HPr, but without P-enolpyruvate (
)
proteoliposomes with enzyme I, HPr, P-enolpyruvate but without
IIAB
(
). A constant amount of Glc-6-P is formed
from external glucose that does not further increase with
time.
Figure 7:
Export
and phosphorylation of glucose. IIC-IID
containing proteoliposomes (0.1 µM IIC
-IID
,
IIC
-IID
:phospholipid ratio 1:30,000) are
loaded with [
C]Glc (2.85 mM). The
export reaction is started (arrow) by addition to the outside
of IIAB
, HPr, enzyme I, and P-enolpyruvate without
(
,
) and with (
,
) 17.5 mM unlabeled
Glc. A, export of trapped [
C]Glc. B, vectorial phosphorylation, formation of Glc-6-P. External
glucose does not compete with export and phosphorylation of
[
C]Glc (compare triangles and circles).
, diffusion of [
C]Glc
observed when P-enolpyruvate is omitted from the incubation mixture.
Shown are means and S.D. of three independent experiments done with a
single batch of reconstituted
proteoliposomes.
Are nonvectorial
phosphorylation, as described above (Fig. 5) and vectorial
transport and/or phosphorylation of transported substrate (Fig. 7) competing reactions? To address this question export
and phosphorylation of trapped [C]Glc was
measured in the presences of increasing amounts of external
[
C]Glc. As shown in Fig. 7, a 6-fold
molar excess of external [
C]Glc neither slows
down the rate of [
C]Glc export (Fig. 7A) nor its phosphorylation (Fig. 7B). This indicates that (i) nonvectorial
phosphorylation and vectorial transport are not competing reactions and
(ii) that [
C]Glc does not equilibrate with
[
C]Glc between the translocation and
phosphorylation reaction and coupling between these two steps therefore
must be tight (Fig. 1). No difference was observed whether
[
C]Glc was added simultaneously with the
cytoplasmic phosphoryl carrier proteins (Fig. 7) or whether it
was added before (results not shown). Reconstituted
IIC
-IID
does catalyze exchange
equilibration between internal and external glucose.
The purified mannose transporter of the bacterial
phosphotransferase system was reconstituted by detergent dilution into
proteoliposomes and two activities, vectorial transport concomitant
with phosphorylation and nonvectorial phosphorylation were measured.
Transport is coupled to phosphorylation in a 1:1 ratio. The K of the transport reaction is 30 ± 6
µM. A turnover number of 1.2 ± 0.1 s
can be calculated, assuming that the minimal functional unit
consists of one IIC
and two IID
subunits
(Rhiel et al., 1994), and that at most 50% of the complexes
are correctly oriented and active (probably an overestimate). The
turnover number of the PTS transporter for mannitol is 4 s
(Elferink et al., 1990) and similar numbers can be
calculated from reported experiments with the purified
H
/lactose transporter (3.5 s
,
Newman et al.(1981); 0.6 s
, Consler et
al.(1993)), the intestinal Na
/glucose transporter
(4 s
, Peerce and Clarke(1990)), and the histidine
transporter (1.1 min
, Bishop et al.(1989)).
Nonvectorial phosphorylation has an approximately 7-fold higher K
and a 3-fold faster k
than the transport reaction. It is likely that solute
translocation requires more extensive protein motion than the binding
and release reaction during nonvectorial phosphorylation, and that the
former reaction therefore is slower than the latter. A similar
difference of catalytic constants was also observed with the mannitol
transporter (Elferink et al., 1990).
Although nonvectorial
phosphorylation on the one hand and phosphorylation coupled with solute
translocation on the other hand are mediated by the same protein
complex, no cross-inhibition between the two reactions could be
observed. Neither the export of [C]Glc out of
proteoliposomes nor phosphorylation of the exported solute could be
inhibited by Glc added to the outside of the proteoliposomes.
Noncompetition between nonvectorial and vectorial phosphorylation
indicates that the transported glucose is not in diffusion equilibrium
with glucose in the bulk phase. Transport and phosphorylation are
either mechanistically coupled or the rate of phosphorylation of bound
glucose is much faster than the rate by which unphosphorylated glucose
could dissociate (dissociation being a precondition for exchange; Fig. 1). This is a clear example of tight channeling by a
multifunctional enzyme which catalyzes two sequential reactions
(Srivastava and Bernhard, 1986; Srere, 1987). No competition between
translocation and nonvectorial phosphorylation could be detected within
the experimental error of the export assay. However, the initial rate
of export could not be measured accurately because export is fast and
due to the small intravesicular volume of very short duration. Small
differences might therefore have gone unnoticed.
The results
summarized above differ in three respects from recent observations with
the mannitol transporter which indicated facilitated diffusion,
exchange, and uncoupling of transport and phosphorylation. (i) Purified
IICBA reconstituted into proteoliposomes mediated
facilitated diffusion in a way which was saturable and specific for
mannitol (Elferink et al., 1990). (ii) Addition of unlabeled
Mtl to the exterior of inside-out membrane vesicles resulted in the
complete exchange of [
H]Mtl bound to the interior
(Lolkema et al., 1991). (iii) Over 50% of the radiolabeled
mannitol bound to the periplasmic side of IICBA
containing inside-out oriented membrane vesicles exchanged with
unlabeled mannitol before it became phosphorylated, indicating that
mannitol dissociates after translocation at a rate comparable to that
of phosphorylation. The differences could have several reasons. (i) The
two transporters are very different with respect to amino acid
sequence, active site residues (histidine versus cysteine in
the second phosphorylation site; Pas and Robillard(1988), Erni et
al.(1989), and Pas et al.(1991)), and subunit composition
(4 domains in 3 subunits versus 3 domains in 1 subunit) and
the molecular mechanism of their action might be correspondingly
different. (ii) Facilitated diffusion catalyzed by the
IIC
-IID
complex went unnoticed because it
was indistinguishable from passive diffusion of L-glucose and
diffusion of D-glucose out of protein-free liposomes (results
not shown).
The proteolysis data are not easily reconciled with the
proposition of Beneski et al.(1982) that the II complex is symmetrically oriented in the membrane. They observed
that II
interacts with 2-deoxyglucose and phosphorylate
this sugar when the phosphoryl carrier proteins are located either
inside of right-side out membrane vesicles (resulting in uptake and
phosphorylation) or outside the vesicles (nonvectorial
phosphorylation). If the two surfaces exposed toward the aqueous phases
were identical, proteolysis should proceed to 100% and not only 50% as
observed (Fig. 3). However, functional symmetry found by Beneski et al.(1982) does not necessitate a structural symmetry of the
complex. Both faces of an asymmetrical complex could in principle have
independent sugar phosphorylation activity, and noncompetition between
vectorial transport and nonvectorial phosphorylation could then be
explained. However, it appears more likely to us that some
randomization of sidedness might have occurred during preparation of
membrane vesicles by Beneski et al.(1982).
The present reconstitution method is useful whenever nonvectorial phosphorylation and vectorial transport must be investigated in parallel and when interference with PTS activity by other membrane components has to be excluded. It will be used as an assay complementary to in vivo uptake studies and in vitro measurement of nonvectorial phosphorylation.