(Received for publication, October 11, 1996, and in revised form, November 19, 1996)
From the Biozentrum, University of Basel, CH-4056 Basel, Switzerland
An in vitro assay of iron-ferrichrome
translocation across the FhuA protein of outer membranes from
Escherichia coli has been devised. Upon reconstitution into
large lipid vesicles, bacteriophage T5 binds to this polyvalent
receptor, triggering a conformational change that resulted in channel
opening. This facilitates the translocation of an
iron(III)-siderophore, without the complexities involved in the
in vivo process. Efflux of
55Fe(III)-ferrichrome across FhuA channels was determined
quantitatively by monitoring the release of trapped radioactivity. The
assay is rapid, reliable, and specific, because other bacteriophages, such as 80, fail to trigger channel opening of the FhuA
receptor.
Understanding the function of ligand-gated channel proteins on the basis of their conformational changes has attracted much interest in recent years (1). Although the number of sequences of proteins belonging to this family has increased rapidly (2), structures are available for a mere few of them and at limited resolution (3). This is caused on the one hand by the difficulties in overexpressing these proteins, particularly those from eucaryotic sources (4), and on the other by the problems besetting the preparation of three-dimensional crystals of membrane proteins, which of course are prerequisite for the determination of high resolution structures by x-ray analysis. We have therefore chosen the iron(III)-ferrichrome translocating FhuA protein from Escherichia coli outer membranes (5-9) as a model of ligand-gated proteins. Proteins from this source have proven very useful in the determination of structures at atomic resolution (10-13). The molecular genetics of E. coli, moreover, are well suited for the investigation of the structural basis of their functional properties (14-16).
At limited availability of iron in the medium, its transport into the
cytoplasm requires a complex cascade of events (17). First, ferric ions
are chelated by one of several siderophores (18). Second, the
iron-siderophore complex binds to its specific surface-exposed
receptor. Binding is a necessary but not sufficient condition to
trigger channel opening in a third step. This process requires energy,
which is transduced from the plasma membrane to the channel protein by
a protein complex consisting of at least three components, the products
of the genes tonB, exbB, and exbD (19,
20). The TonB protein appears to play a key role in this energy
transduction by an as yet poorly understood allosteric transition,
which results in the interaction of the TonB protein with a specific
motif, the so-called TonB box, which is found in several channel
proteins (21-23). Fourth, the substrate is scavenged by a
ligand-specific binding protein in the periplasmic space (24). In a
fifth step, the complex is delivered to a specific, active transport
system in the plasma membrane. In the final, sixth step, the oxidized
form of iron is reduced in the cytoplasm to the ferrous state (25, 26).
The rate-limiting step of this cascade appears to be the energy
requirement of the translocation process across the outer membrane. The
function of the TonB complex is needed also for infection of the cell
with the several viruses and bacterial toxins that use FhuA as a
receptor (phages T1, 80, and colicin M), with the only exception
being bacteriophage T5. This virus has been shown to trigger channel
opening and ion flux through the FhuA protein in the absence of energy
transduction (27). It thus circumvents this requirement, whereas the
other ligands may serve as controls.
We are presently crystallizing the FhuA protein. This requires not only that the protein be available in large quantities but also that its native state can be assayed by a routine procedure throughout the lengthy process of crystallization. We here report an assay in which the FhuA protein and 55Fe(III)-ferrichrome were co-incorporated into large lipid vesicles: the former into the membrane and the latter trapped in the internal compartment. Interaction of T5 phage with the receptor protein, monitored by a fluorescent dye that interacts with the ejected phage DNA, caused the release of radiolabeled Fe(III)-ferrichrome through the FhuA channel. Using radioactive iron, efflux from vesicles could readily be quantitated.
FhuA protein was overexpressed in an E. coli BL21(DE3) strain not expressing OmpF, OmpC, PhoE, LamB, and OmpA proteins (gift of Dr. A. Prilipov), using the plasmid pHK763 carrying the fhuA gene (kindly provided by Dr. H. Killmann). The protein was solubilized from outer membranes using octyl-POE1 (Alexis, Läufelfingen, Switzerland) as the detergent and purified in a procedure analogous to that used for porins from E. coli (28, 29). After a final size exclusion chromatography step, SDS-polyacrylamide gel electrophoresis of the heat-treated protein revealed a single band corresponding to a polypeptide with a mass of 80 kDa.
Ferrichrome LabelingFerrichrome, kindly provided by Dr. J. B. Neilands, was labeled with 55Fe(III) according to Weaver
and Konisky (30). The final solution was 2.03·103
M, as determined by its absorbance at 426 nm. The specific
activity was 55 Ci·mol
1.
Phage T5 (the gift of Dr. V. Braun) and
80 (Biozentrum) were isolated and stored as described (27), using
E. coli BE and DH5
(Biozentrum) for
production of bacteriophages T5 and
80, respectively. Using the same
strains, phage titers of T5 and
80 were determined to be
5·1012 pfu/ml and 7·1012 pfu/ml,
respectively.
For the preparation of lipid vesicles, 2 µCi of [14C]dipalmitoylphosphatidylcholine (DuPont NEN) was added to 40 mg of lipids (4:1 phosphatidylcholine:phosphatidylglycerol, Avanti Polar Lipids Inc.) in chloroform. Liposomes were obtained by using the reverse phase evaporation technique in HEPES buffer (20 mM HEPES, 0.15 M NaCl, pH 7.2) as described by Szoka and Papahadjopoulos (31). The final lipid concentration was 10.9 mM. FhuA protein and 55Fe-ferrichrome were incorporated by resolubilization of the liposomes, addition of the desired constituents, and subsequent detergent removal (32, 33). 14C-Labeled vesicles were mixed with octylglucoside (0.6 M), 7 µCi of 55Fe-ferrichrome, 20 µg of purified FhuA protein, and HEPES buffer to yield final volumes of 330 µl, containing 5 mM, and 40 mM of lipid and OG, respectively. The mixture was gently agitated for 30 min at room temperature. 27 mg of BioBeads SM2 (Bio-Rad) were enclosed in a small dialysis bag and added to the solution. The reaction vessel was agitated for 2 h at room temperature. A second batch of BioBeads SM2 (27 mg) was added, and the incubation was continued overnight at 4 °C. Residual ferrichrome was removed from the vesicles by two sequential gel filtration chromatography steps on a 1 × 9 cm Sephacryl S-200 HR (Pharmacia Biotech Inc.) column using the above mentioned HEPES buffer. Fractions were collected and aliquots counted for 14C and 55Fe during 20 min in a Packard 2200CA counter. Proteoliposomes containing 55Fe-ferrichrome were stored at 4 °C and used for assays within 24 h.
Fluorescence StudiesEjection of phage DNA was measured at 37 °C in a SLM 8000C fluorimeter (SLM-Aminco, Urbana, IL), using a fluorescent quaternary ammonium dye derivative (YO-PRO 1, Molecular Probes Inc.), which intercalated into double-stranded DNA (34). The excitation and emission wavelengths used were 490 and 509 nm, respectively. Calibration of the fluorescent signal caused by the release of phage DNA was performed by incubating an aliquot of phage T5 stock solution with an equal volume of 9 M LiCl at 50 °C for 10 min and subsequent dilution of known amounts of phages in HEPES buffer (35). Reconstituted FhuA protein (0.6 µg) was mixed with 5·109 phage T5 pfu in 1.5 ml of HEPES buffer containing 1 mM of CaCl2, 1 mM MgSO4, and 2 µM YO-PRO 1. The increase of the fluorescence signal was recorded as a function of time. After reaching a plateau, 20 units of DNase I (Boehringer Mannheim) was added. To solubilize the vesicles, octyl-POE was added to a final concentration of 1%. In one control experiment, liposomes devoid of FhuA protein were used. In another control, 0.6 µg of detergent-solubilized FhuA protein was used in 1% octyl-POE.
Translocation AssaysFerrichrome efflux was monitored by
separating free from entrapped 55Fe-ferrichrome on a
Sephacryl S-200 column following incubation of the vesicles with
different ligands. A ratio of one pfu/liposome was used for incubations
with phages. DNase I treatment was used to reduce the viscosity of the
sample, because ejected phage T5 DNA interfered with chromatography.
Translocation assays were performed by adding the following components
to a total volume of 250 µl: (a) 60 µl of
proteoliposomes from the peak fraction of the excluded volume (see Fig.
1A), 80 µl of phage T5 stock solution, 30 units of DNase
I, 2.5 µl of 0.1 M MgSO4, 0.5 µl of 0.5 M CaCl2, and HEPES buffer were incubated for 60 min at 30 °C. (b) As in procedure (a), but 65 µl of phage 80 stock solution was used instead of phage T5.
(c) As in procedure (a), except that no phage was
added. (d) 60 µl of proteoliposomes, 25 µl of 0.6 M OG, and HEPES buffer were incubated for 20 min at room
temperature. Samples were loaded onto a Sephacryl S-200 column and
eluted with HEPES buffer, and subsequent fractions were counted for
14C and 55Fe as described above. The stability
of the liposomes following interaction with phage T5 was assessed by
gel chromatography using entrapped [3H]dextran (70 kDa
average mass, Amersham Corp.). No efflux of 3H label from
proteoliposomes was observed.
Proteoliposomes reconstituted with FhuA protein were freed from
extraneous 55Fe-ferrichrome by gel filtration
chromatography in two steps, of which the second reveals a negligible
peak of free ferrichrome (Fig. 1A). When
stored at 4 °C, the vesicles remained stable over a period of
24 h. Upon the addition of phage T5 (1 per 30 proteoliposomes), ejection of DNA was revealed by the increase of the fluorescence intensity of the dye YO-PRO 1(see Fig. 2), which
intercalated into double-stranded DNA (36). An instantaneous rise, seen
in all curves, could be attributed to the interaction of the dye with
free DNA originating from the small fraction of lysed T5 phages present
in the added phage stock solution. The subsequent gradual rise (Fig. 2,
curve A) was attributed to two components. Phage DNA ejected
in the medium interacts with the fluorescent dye; the contribution of
phage DNA injected into the vesicle interior is more difficult to
quantitate. When the curve reached a plateau, all T5 phages had bound
to a receptor protein molecule, as supported by the observation that
the addition of proteoliposomes after 1400 s had no further effect
on the fluorescence signal (Fig. 2, arrow 1). Treatment of
the solution with DNase I (Fig. 2, arrow 2) caused a rapid
decrease of the fluorescence intensity of curve A by about
30%. This showed that the fraction of phage DNA existing in solution
was immediately accessible to enzymatic hydrolysis. The
liposome-entrapped DNA could also be degraded but only upon solubilization of the vesicles with a detergent (Fig. 2, arrow 3). This result represented strong evidence that phage DNA did enter the liposomes. Solubilization of the vesicles performed before
addition of DNase I (Fig. 2, dotted line) caused a rapid increase in fluorescence intensity. The value reached corresponded to
the level seen if detergent-solublized FhuA protein was present without
liposomes (control experiment; Fig. 2, curve C). This result
appeared to account for the quenching of the DNA-dye complex in the
liposome interior and its release upon the addition of a detergent. The
residual fluorescence, seen in curves A and C, was due to oligonucleotides resulting from DNase I activity (37).
Translocation of Fe(III)-ferrichrome was shown in Fig. 1
(B-D). Phage T5 triggered channel opening of the FhuA
protein reconstituted in lipid vesicles, facilitating the efflux of
approximately 85% of the labeled ferrichrome (Fig. 1B). The
control experiments showed that with phage 80, less than 15% of the
55Fe label was released, an amount also observed if no
phage was added under otherwise identical conditions. The addition
of octylglucoside, causing the solubilization of the
proteoliposomes, released >95% of the Fe(III)-ferrichrome into the
medium (Fig. 1D). The remaining 55Fe label was
eluted in the excluded volume adsorbed to lipid/protein/detergent aggregates.
We have designed an in vitro assay system that allows
the quantitative determination of translocation of an
iron(III)-siderophore complex through FhuA channels. The energy
requirement of the in vivo translocation process has been
circumvented by exploiting the properties of phage T5, which upon
binding to the membrane-incorporated FhuA protein, causes DNA ejection.
This process is specific, because it was not observed with other
ligands such as phage 80. A significant difference in the time
courses of T5 phage DNA ejection with FhuA protein, either
reconstituted into liposomes or in its detergent-solubilized form, was
observed, for which three explanations may apply: (a) The
effective concentration of binding sites exposed to the solution is
half that of the total protein if the inserted membrane protein is
oriented randomly. (b) Accessibility of surface-exposed
binding sites may be considerably reduced, due to a taut conformation of the protein in the lipid bilayer (38). (c) When phage T5 DNA is injected into vesicles, the fluorescence probe YO-PRO 1 may give
rise to a reduced signal. This may be due to limited accessibility of
dye to the vesicles, quenching of the signal, or both. The observation
that the fluorescence intensity rose significantly upon solubilization
of the vesicles by detergent revealed that phage DNA was indeed trapped
in the liposomes. The results from these experiments provide strong
evidence that the FhuA protein was reconstituted in the vesicles in an
active form and that it allowed DNA to cross the lipid bilayer. This
should now give the opportunity to address the question of the
specificity of transported solutes and of the mechanism by which
macromolecules cross biological membranes.
Our choice of gel filtration chromatography to quantitate ferrichrome
efflux is based on the accuracy and reproducibility with which
potential adsorption of ferrichrome to vesicles and leakage can be
distinguished from actual efflux. Thus, we could determine that
ferrichrome efflux following incubation with phage 80 amounts to
about 15% of the total release of 55Fe label from the
vesicles. This tallied with the value of another control experiment in
which no phage was added at all and thus represented the background
leakage from liposomes under the reaction conditions used. The value of
85% of ferrichrome released during incubation with phage T5 can be
assigned to leakage on the one hand and to a fraction of about 80% of
liposomes that encountered a phage particle on the other. Keeping the
ratio of phage-to-liposome constant, efflux could be maximized by
adjusting the ratio of incorporated FhuA protein per vesicle to a value
between 5 and 10. Now that these values have been established, it will
be possible to use filter assays for the routine determination of
efflux with large sets of samples.
In conclusion, iron(III)-ferrichrome transport across the outer
membrane of E. coli, the physiologically relevant in
vivo function of the FhuA protein, could be assessed in an
in vitro system using bacteriophage T5 as the triggering
ligand. This stratagem avoids the problems involved in the energy
transduction from the plasma membrane to the channel protein. The assay
allows the specificity of the gating ligand (phage T5 versus
80) to be assessed and should now provide a tool to determine the
vectoriality and specificity of the transported solutes, as well as the
question of DNA transfer. As to our structural studies of the receptor
protein (29), its functionality can now be monitored at any stage
during purification and crystallization. In addition, it opens the
possibility for kinetic studies of the transport rate through the FhuA
protein and sets the stage for reconstitution of the entire
translocation cascade.