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Biophys J, August 2002, p. 899-911, Vol. 83, No. 2
and
*Fachbereich Biologie/Chemie, Universität Osnabrück,
D-49034 Osnabrück, Germany; Botanisches Institut,
Universität Kiel, D-24098 Kiel, Germany; and
Max-Planck-Institut für Biophysik, Abt.
Strukturbiologie, D-60528 Frankfurt am Main, Germany
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The channel properties of Toc75 (the protein import pore
of the outer chloroplastic membrane) were further characterized by electrophysiological measurements in planar lipid bilayers. After improvement of the Toc75 reconstitution procedure the voltage dependence of the channel open probability resembled those observed for
other 
-barrel pores. Studies concerning the pore size of the
reconstituted Toc75 indicate the presence of a narrow restriction zone
corresponding to the selectivity filter and a wider pore vestibule with
diameters of ~14 Å and 26 Å, respectively. Interactions between
Toc75 and different peptides (a genuine chloroplastic transit peptide,
a synthetic peptide resembling a transit peptide, and a mitochondrial
presequence) show that Toc75 itself is able to differentiate between
these peptides and the recognition is based on both conformational and
electrostatic interactions.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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According to the endosymbiont hypothesis
chloroplasts originated from gram-negative, cyanobacteria-like
ancestors, which were ingested by a primitive eucaryotic cell via
endocytosis (Gray, 1992
). During the course of the subsequently evolved
symbiotic relationship the majority of the genes of the symbiont were
transferred to the nucleus of the host (Martin et al., 1998). This
necessitated a mechanism for the import of the now nucleus-encoded
proteins, which are synthesized at free ribosomes in the cytoplasm. The components of the import machinery in the outer chloroplastic membrane
have already been identified to a considerable extent and are now
generally referred to as Toc proteins (translocon of the outer
chloroplastic membrane) (Schnell et al., 1997). For recent reviews, see
Chen and Schnell (1999), Vothknecht and Soll (2000), and Schleiff and
Soll (2000). The major components identified so far are Toc160, Toc64,
Toc34, and Toc75. Toc160 and Toc34 are GTP-binding proteins (Kessler et
al., 1994; Seedorf et al., 1995; Bölter et al., 1998a) and at
least Toc160 and perhaps also Toc34 seem to be the first contact sites
of the preprotein (Kouranov and Schnell, 1997; Ma et al., 1996). Toc64
functions early in preprotein translocation, presumably as a docking
protein for cytosolic cofactors (Sohrt and Soll, 2000).
Toc75 is a protease-insensitive, integral component of the outer
envelope, and on this account it was already early on presumed to be
the actual pore of the import machinery (Perry and Keegstra, 1994).
Furthermore Toc75 was also reported to interact specifically with
preproteins (Ma et al., 1996), thus accounting for the finding that the
initial binding to Toc159 can be bypassed (Chen et al., 2000). Both
notions are further corroborated by our first electrophysiological measurements of the reconstituted Toc75 channel (Hinnah et al., 1997).
The Toc proteins show no homologies to the components of other
protein-transport systems, not even to the Tom proteins (translocon of
the outer mitochondrial membrane), which have an analogous assignment.
Only recently a homologue to Toc75 termed synToc75 has been identified
in the outer membrane of the cyanobacterium Synechocystis
PCC6803 (Reumann et al., 1999; Bölter et al., 1998b). The
functional properties of synToc75 as determined by electrophysiological measurements are nearly indistinguishable from the properties of Toc75
as presented here (Bölter et al., 1998b).
We have previously described patch clamp experiments performed with
reconstituted Toc75 (Hinnah et al., 1997). However, these experiments
were hampered by the fact that only the bath side of a patch is
accessible for exchange of the electrolyte solution and addition of
peptides. Therefore, we started to perform bilayer measurements, but
initially the fusion rates of proteoliposomes with the planar lipid
bilayer were very low (Hinnah et al., 1997). As the used fusion method
is dependent on the presence of open channels (Woodbury and Hall,
1988), this was to be expected considering the rather unusual voltage
dependence of the open probability of Toc75 as revealed by patch clamp
experiments. We have since then changed the reconstitution method.
Toc75 is now reconstituted by dialysis in the presence of the detergent
Mega9 or alternatively with the detergent Triton and subsequent removal
of the detergent by adsorption to BioBeads. This resulted in a markedly
altered voltage dependence of the open probability and sufficiently
high fusion rates (for details see below). The latter now allowed for a
detailed characterization of further electrophysiological properties of
the Toc75 channel, in particular the effects of different (transit-) peptides on the channel conductance and selectivity. The experiments presented here deal mainly with the following questions. 1) Does the
Toc75 channel specifically recognize transit peptides, and is it
able to distinguish between a genuine transit peptide, a synthetic
peptide of similar structure, and a mitochondrial presequence? 2) What
kind of interaction between transit peptides and the Toc75 channel
takes place (electrostatic interaction or interaction based on
conformational information)?
Moreover, experiments have been performed to determine more
precisely the pore size of Toc75. The previously proposed diameter of 8 to 9 Å (Hinnah et al., 1997) has repeatedly been subject to debate
(Chen and Schnell, 1999; Schatz, 1998) as it seemed rather small in
comparison to the diameters of other protein-conducting channels (Hill
et al., 1998; Kunkele et al., 1998; Hanein et al., 1996). Indeed, this
value was only a first estimation whose calculation was based solely on
the conductance of the channel and was moreover dependent on several
assumptions (Hille, 1992; Hinnah et al., 1997). Now we present a
reconsidered value for the diameter of the constriction zone (and an
additional value for the vestibules of the channel), which is in better
agreement with the diameters of other protein conducting channels and
would allow the translocation of partially folded preproteins.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Expression and purification of Toc75
Heterologous expression in Escherichia coli, and
purification of Toc75 was performed as already described in Hinnah et
al. (1997). The reconstitution of in this way prepared Toc75 was
performed with Mega9 (see below).
Additionally Toc75 has been expressed and purified as described in Rogl
et al. (1998) with the following modifications: insoluble parts have
been removed by centrifugation at 100,000 × g instead of gel filtration and afterwards the preparation was concentrated with
centripreps (Amicon, Beverly, MA) to 1 mg/mL in 20 mM Tris/HCl, pH 8.0, 10% glycerol, 1 mM EDTA, and 0.1% Triton X-100. This preparation was
reconstituted with Triton X-100 (see below). Both preparations of Toc75
were used and showed no significant differences.
Reconstitution
Reconstitution with Mega9
Small unilamellar liposomes were obtained from purified azolectin (type IV-S, Sigma, St. Louis, MO) as described (Hinnah et al., 1997), and Mega9 was added up to a concentration of 80 mM. Purified Toc75 at a concentration of 0.7 to 1.2 mg/ml in 6 M urea, 10 mM Tris/HCl, pH 6.9 was also supplemented with Mega9 up to a final
concentration of 80 mM. Both solutions were mixed adjusting the lipid
to protein ratio to 0.5 to 0.8 mg of protein/10 mg of lipid. The
mixture was then dialyzed overnight against 5 l of 10 mM MOPS/Tris, pH
7.0 as described previously (Hill et al., 1998).
Reconstitution with Triton X-100
The Toc75 solution with 1% Triton X-100 was mixed with a liposome solution (see above) yielding a final concentration of 0.5 mg of Toc75/10 mg of lipid with 0.7% Triton X-100. The mixture was incubated with ~20:1 (w/w BioBeads/Triton X-100) BioBeads (Holloway, 1973) for 2 h with gentle stirring.
Planar lipid bilayers
Planar lipid bilayers were produced by the painting technique
(Mueller et al., 1963). A solution of 80 mg/ml azolectin (type IV-S,
Sigma) in n-decan (analytical grade, Merck, Rahway,
NJ) was applied to a hole (100-500 µM diameter) in a Teflon septum, separating two bath chambers (volume 3 ml each). Both chambers were
equipped with magnetic stirrers. Through continuously lowering and then
reraising of the solution level, the lipid layer across the hole was
gradually thinned out until a bilayer was formed. This formation was
monitored optically and by capacitance measurements. The resulting
bilayers had a typical capacitance of 0.5 µF/cm2 and a resistance of >100 G
. The
noise was 3 pA (r.m.s.) at 5-kHz bandwidth. After the formation of a
stable bilayer in 20 mM KCl, 10 mM MOPS/Tris, pH 7.0 (in both
chambers = symmetrical conditions), the solutions were changed by
perfusion to asymmetrical conditions 250 mM/20 mM KCl, 10 mM MOPS/Tris,
pH 7.0, cis/trans. An osmotic gradient of a
channel-permeant solute, is, among the absolute necessity of the
channel in the proteoliposome being in the open state (Woodbury and
Hall, 1988), a prerequisite for fusion of proteoliposomes with the
bilayer (Cohen et al., 1989). To promote attachment of the
proteoliposomes to the bilayer, CaCl2 was added to the cis chamber to a concentration of 10 to 20 mM (Niles
and Cohen, 1987; Zimmerberg et al., 1980). Proteoliposomes were then added to the cis chamber directly below the bilayer, causing
a slow flow of proteoliposomes along the bilayer surface. After fusion
the electrolytes were changed to the final composition. Because we did
not observe any asymmetric response of the Toc75 channels incorporated
into the planar bilayer so far we did not have any experimental access
to verify whether the channels were incorporated with random
orientation or whether preferentially oriented Toc75 channels reveal
symmetric pore properties. However, the later possibility is likely
because also with only a single channel incorporated no asymmetric
responses were detected at all.
The Ag/AgCl electrodes were connected to the chambers through 2 M KCl-agar bridges. The electrode of the trans compartment was directly connected to the headstage of a current amplifier (Axon Gene Clamp 500, Axon Instruments, Union City, CA). Reported membrane potentials are always referred to the trans compartment. The amplified currents were digitized at a sampling interval of 0.2 ms, filtered with a low-pass-filter at 1 kHz (Frequency Devices 902, Haverhill, MA), and fed into a Digidata1200 A/D converter (Axon Instruments) to store on the hard disk of an IBM compatible PC. For analysis, a WINDOWS-based analysis software ("SCIP" single channel investigation program) developed in our laboratory was used in combination with Origin 6.0 (Microcal Software Inc.).
Experiments
"Voltage ramp" refers to the continuous increase of voltage with a rate of 10 mV/s. It is important to note that every voltage ramp starts at 0 mV. In the diagrams of the results section two ramps are always depicted together: one from 0 mV to a positive voltage and the other from 0 mV to a negative voltage. The sequence of the ramps was regularly switched but proved to be of no consequence for the experiments. The only exceptions are the experiments with TrOE33 where the sequence is especially mentioned and important for the interpretation of the experiments.
Calculation of pore size
The pore size has been calculated according to Hille (1992, p.
294f):
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with a very short constriction
zone flanked by wide vestibules), and 
is resistivity of the
solution, 49.5 cm for a 250-mM KCl solution, but taking into account
the correction factor of Smart et al. (1997) the value is 247.5 cm.
Polyethylene glycol method
This method allows the estimation of the pore size, according to
Sabirov et al. (1993), Krasilnikov et al. (1992), and Bezrukov and
Kasianowicz (1997).
The effect of the presence of 20% of PEG (polyethylene glycol) of different molecular weight on the channel conductance was measured. PEG are sphere-like, neutral polymers and their hydrodynamic radii are given in Table 1. The principle of the measurement is as follows. The electric conductivity of the bulk solution is lowered in the presence of PEG. At low hydrodynamic radius the PEG can enter the channel and lower the conductance of the channel by the same factor as the bulk conductivity, but as their radius increases the PEG are progressively excluded from the channel interior and the conductance begins to recover.
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The conductance ratio
G20/G0
is plotted against the hydrodynamic radius of the PEG, and the data are
fitted according to Bezrukov and Kasianowicz (1997) with the following
(modified) equation:
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the characteristic radius of the channel. G0 is the
conductance in 100mM KCl solution, G20
is the conductance in 100 mM KCl solution with 20% PEG, 
'/ ratio
of bulk conductivities in the presence and absence of polymer is 0.553, w0 is the "cutoff" polymer radius,
which is the characteristic radius of the channel, is the maximal
conductance, and P3 is set as 1.
TrOE33
TrOE33 is the transit peptide of the 33-kDa subunit of the
oxygen evolving complex, which is associated with photosystem II at the
lumenal side of the thylakoid membrane (Hashimoto et al., 1997). The
transit peptide is accordingly bipartite, N terminus (amino acids
1-43) is the stroma targeting domain and C terminus follows the
thylakoid targeting domain (amino acids 44-81), which accounts for the
transport into the lumen of the thylakoid after removal of the stroma
targeting domain by the CCPase (Cline et al., 1993). Only the
N-terminal domain is relevant for the experiments presented here and
shows a composition typical for stroma targeting domains: N terminus an
uncharged region, followed by a region rich in positively charged amino
acids (bold and underlined), and the whole sequence is rich in
hydroxylated amino acids (bold).
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-helical regions (see below), contrasting the
general opinion that transit peptides are perfect random coils (von
Heijne and Nishikawa, 1991).
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TrOE33 was expressed in E. coli and was in a concentration of 0.3 to 0.5 mg/mL in 100 mM NaCl, 50 mM imidazol, 8 M urea, and 20 mM Tris/HCl, pH 8.6.
CoxIV
CoxIV is a synthetic peptide identical to the mitochondrial
presequence of the cytochrome oxidase subunit IV. It carries 4.8 positive charges at pH 7.0 (5 at pH 4.0) and is rich in hydroxylated residues. The structure is predicted to be an amphiphilic -helix, characteristic for mitochondrial presequences (von Heijne et al., 1989). CoxIV fused to cytochrome oxidase subunit IV was
transport-competent in import studies with isolated mitochondria
(Allison and Schatz, 1986).
CoxIV comprises 23 amino acids and the sequence is as follows:
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SynB2
SynB2 is an artificial peptide whose sequence is derived from
CoxIV (Allison and Schatz, 1986). It possesses an identical charge of
4.8 positive charges at pH 7.0 but is in contrast to CoxIV enriched in
polar amino acids (R, Q, S):
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). SynB2 is not transport competent in import studies with isolated mitochondria (Allison and Schatz, 1986).
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CoxIV and SynB2 were a kind gift of Prof. N. Pfanner (Freiburg). The lyophilisated peptides were dissolved at a concentration of 10 mM in 10 mM MOPS/Tris, pH 7.0.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Heterologous expression of Toc75 in E. coli and
purification were performed as described before (Hinnah et al., 1997).
The most frequent current transition of the reconstituted Toc75 channel
corresponded to the largest conductance (Fig.
1 A) and is therefore named
main conductance in the following. This trace (Fig. 1 A)
also best illustrates the slow and occasional closures of Toc75
channels and the absence of openings at a high membrane potential. As
derived from the current voltage relationship this main conductance has
a value of 1.32 nS ± 38 pS in 1 M KCl symmetrical solution (Fig.
1 C) (the conductances cited in the text are the mean values
of five independent measurements with 3-12 active Toc75 channels per
each bilayer). Furthermore the Toc75 channel displayed two smaller and
rarely encountered subconductances (Fig. 1 B).
Subconductance 1 is represented by transitions from open to
closed* (Fig. 1 B) (or from closed to open*, not shown) and
has a value of 860 ± 41 pS (1 M KCl symmetrical), and
subconductance 2 is constituted by the transition from closed to
closed*, Fig. 1 B (or from open to open*, not shown) with a
value of 460 ± 33 pS. The complete transition from open to
closed, representing the main conductance, is noticeable in Fig. 1
B, thus corroborating the classification of the
subconductances (Laver and Gage, 1997).
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The results of the preceding patch-clamp measurements lead to the
assumption that the conductance of 145 pS at 150 mM KCl symmetrical
represented a value close to the saturation value (Hinnah et al.,
1997). However, these experiments were hampered by the fact that high
salt concentrations reduced the stability of the G-seals and so a
detailed study was not feasible. Consequently we now reexamined the
single-channel conductance at numerous different KCl concentrations
(0.1-3 M; data not shown) and obtained somewhat different results. The
single-channel conductance revealed a linear current voltage
relationship (Fig. 1 C) and increased almost linearly with
concentration in the range of 0.1-1 M KCl, and only above 1 M KCl the
single-channel conductance gradually began to saturate. The calculation
of the saturation value of the conductance using a modified
Michaelis-Menten equation (Hille, 1992, p. 362) resulted in
Gmax = 6.2 nS and
KM = 2.49 molal.
The reversal potential of the Toc75 channel in the presence of a salt
gradient of 250 mM/20 mM KCl, 10 mM MOPS/Tris, pH 7.0, cis/trans and trans/cis was
48 mV (n 
120) and 
48 mV (n = 20), respectively. Using these values the permeability ratio calculated by
the Goldman-Hodgkin-Katz-voltage-equation was
PK+/PCl
= 14.3, denoting a marked cation selectivity. The permeability of Toc75
for different monovalent cations as calculated from reversal potentials
of measurements under bi-ionic conditions (n = 2-3 for
each ion) reflects the aqueous mobility sequence of these cations:
Cs+ (p = 1.14) > Rb+ (p = 1) 
K+ (p = 1) > Na+ (p = 0.65) > Li+ (p = 0.57). This reflects an
unrestricted passage of these cations through the pore thus indicating
a comparably large pore diameter.
When the pH was lowered to pH 5.0 (250/20 mM KCl, 10 mM NAc/HAc, pH
5.0, cis/trans) the reversal potential was
considerably reduced to 35 mV (n = 4) (Fig.
2 A), resulting in a
permeability ratio of
PK+/PCl = 6.3. Further decrease to pH 4.0 (250/20 mM KCl, 10 mM NAc/HAc, pH
4.0, cis/trans and
trans/cis) resulted in reversal potentials of 13 mV (n = 5) (Fig. 2 B) and 14 mV
(n = 2, data not shown), respectively. The resulting
permeability ratio is
PK+/PCl = 1.9, so the Toc75 channel is only weakly cation selective at pH 4.0. This dependence of the selectivity on the pH of the aqueous phase
demonstrates that the cation selectivity is apparently caused by an
excess of negatively charged groups inside the pore or at the pore
mouths. At pH 4.0 these groups should be mostly protonated and
uncharged, therefore unable to convey a selectivity to the channel.
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As already mentioned briefly, the voltage dependence of the open probability differs markedly from the one previously determined in patch clamp experiments.
In the absence of a membrane potential the Toc75 channels were
constantly open (Fig. 1 D). In response to membrane
potentials of opposite polarity, conductance behavior was symmetrical
with respect to channel closure. The open probability was reduced
symmetrically at higher membrane potentials; the half-maximal effect
was reached at 81 mV and at 90 mV, respectively. Beyond ±120 mV
the open probability approached zero.
In a first approximation the pore size of Toc75 has been calculated
using an Ohmic model of conductance according to Hille (1992). The
resulting diameter of 8 to 9 Å for the constriction zone (Hinnah et
al., 1997) was based on the assumption that the conductivity of the
electrolyte solution within the pore is equivalent to the conductivity
of the bulk solution. However, Smart et al. (1997) have demonstrated
that the conductivity of the electrolyte solution within the pore has
to be considered to be reduced by the factor 5. Taking this correction
factor into account the recalculated value for the constriction zone
now equals d = 15.4 Å (for details see Material and Methods).
To substantiate the calculated result, the permeability of the
Toc75 channel for the large organic cations
tetraethylammonium (TEA+)
(cross-section 6 Å) and tetrabutylammonium
(TBA+) (cross-section 10 Å) was measured.
The reversal potentials under bi-ionic conditions were used to
calculate the permeability ratios according to the
Goldman-Hodgkin-Katz-voltage-equation. For 250 mM TEA-Cl
cis/250 mM KCl trans we obtained a reversal potential of Erev = 25 ± 0.4 mV (n = 4) and for 100 mM TBA-CL cis/100 mM
KCL trans we obtained Erev = 43 ± 1.0 mV (n = 3). The Toc75 channel is
permeable by both cations,
PK+/PTEA+ = 3.6 (n = 4) and
PK+/PTBA+ = 4.2 (n = 3). The permeability ratios roughly reflect
the mobility of TEA+/TBA+
in aqueous solution in comparison with the mobility of
K+. Hence the pore has to be considerably larger
than 10 Å to allow for this relatively unrestricted diffusion.
To gain further information on the pore dimensions of the channel, the
ability of differently sized soluble nonelectrolytes to partition into
the Toc75-channel was measured (PEG method; for details see Material
and Methods) (Sabirov et al., 1993; Krasilnikov et al., 1992; Bezrukov
and Kasianowicz, 1997). The results are depicted in Fig. 2
C, however, the interpretation of the experimental data is
not straight forward and yet still controversial. The data are fitted
according to Bezrukov and Kasianowicz (1997) and the resulting
characteristic mean radius of the channel is
rmean = 10.8 ± 1 Å (d = 21.6 ± 2 Å). However, obviously this fit is ill suited to describe our PEG data of Toc75.
As outlined in detail by Smart et al. (1997) it seems rather improbable
that a large pore like Toc75 has a single homogenous radius along its
entire length (for further details, see Discussion). We therefore tend
to interpret the data according to Smart et al. (1997) assuming that
Toc75 has a constriction zone flanked by wide vestibules on both sides.
The minimal radius of the channel (=radius of the constriction zone) is
likely to be in the region where
G20/G0
starts to rise (representing the starting point of PEG exclusion from
the pore Fig. 2 C). Whereas the region where G20/G0
approaches the asymptotic end value (denoting the now nearly complete
exclusion of PEG from the channel Fig. 2 C) is likely to be
the end radius. To identify this minimal and end radius, the
experimental data were fitted by a mathematically suited logistic equation (see legend to Fig. 2 C) and from the second
derivative of the best fit (see insert to Fig. 2 C) we
obtained values of rminimum = 6.8 Å and rend = 12.8 Å, respectively. The
minimum diameter of the Toc75 channel would than be d = 13.6 Å and the one of the wide pore mouths d = 25.6 Å.
Transit peptides: TrOE33, SynB2, and CoxIV
To address the questions mentioned in the introduction we carried out experiments with three different peptides: the genuine chloroplastic transit peptide TrOE33 (transit peptide of the oxygen evolving complex of 33 kDa); the mitochondrial presequence CoxIV (Cytochromoxidase IV); and the artificial peptide SynB2, which is derived from CoxIV and carries an equal amount of positive charges but adopts a different structure (for details see Materials and Methods).
SynB2
The most obvious effect of the addition of 1 µM SynB2 to the trans compartment (250/20 mM KCl cis/trans) was a complete voltage-dependent block of the channel (Fig. 3 B). The block is only noticeable at a positive potential, the interpretation being that at this potential the positively charged peptide is forced into the pore thus plugging it and abolishing the ionic current (for details see Discussion). This explanation is sustained by the fact that the block takes the form of distinct closures (Fig. 3 B). Then each closure represents one single Toc75 channel that is "plugged" by SynB2 and the reopening denotes the exit of the SynB2 molecule(s). The block is completely reversible as shown by the unaltered current at a negative potential (Fig. 3, A and B) or without a potential (data not shown). When the concentrations in the chambers were reversed (20 mM/250 mM KCl, cis/trans) and SynB2 was added to the cis compartment the Toc75 channels were accordingly blocked at a negative potential (data not shown). This interchangeability of the side of the addition was also observed with the other two peptides (data not shown), indicating identical interactions with both sides (in particular with the pore entrances) of the Toc75 channel.
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The second major effect of the addition of 1 µM SynB2 to the
trans compartment was a shift of the reversal potential to a less positive value (control, Erev = 48 mV; 1 µM SynB2, Erev = 41 ± 1 mV (n = 3)). One possible explanation is the
transport of the positively charged SynB2 but to account for the
measured reversal potential shift the permeability (calculated
according to Goldman-Hodgkin-Katz) would have to be
PSynB2 = 6700 (with unchanged
PK+/PCl = 14.3). But such a high permeability would be in total disagreement
with the observed block. Therefore, it seems more probable that SynB2
binds and alters the selectivity of the Toc75 channel for the permeant
ions K+ and Cl, a
reduction of the cation permeability to
PK+/PCl = 9.7 could easily account for the observed reversal potential shift.
The strength of the block was concentration-dependent, a micromolar concentration inducing a complete block (Fig. 3 B) (see above) but even with nanomolar concentrations the current was visibly reduced (Fig. 3 A). Moreover the salt concentration exerted a considerable influence. If the peptide was added to the high salt compartment the channel block did not set until a concentration of 5 µM SynB2 was reached (data not shown).
The situation was completely altered after lowering of the pH. At pH 4.0 even the addition of 10 µM SynB2 to each compartment (250 mM/20 mM KCl, 10 mM NAc/HAc, pH 4.0, cis/trans) did not induce a block (Fig. 3 C). However, after perfusion of both compartments with an electrolyte solution of pH 7.0, even the residue of SynB2 in the trans chamber (a single perfusion accounts for an ~100-fold dilution > 0.1 µM) caused a nearly complete block (Fig. 3 C), in accordance with the previous results (see above). Thus, the conclusion can be reached that the effect of SynB2 on the Toc75 channel at pH 7.0 is completely due to electrostatic interactions (see Discussion).
TrOE33
Essentially the effect of TrOE33 on the Toc75 channel was very similar to the effect of SynB2. The addition of nanomolar concentrations to the low salt compartment induced a voltage-dependent block (Fig. 4 A). This block was also concentration and salt dependent (data not shown). However, there are some significant differences between the results obtained with TrOE33 and those obtained with SynB2.
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First of all, the voltage-dependent block caused by TrOE33 was even at
a concentration as low as 20 nM nearly complete (Fig. 4 A),
indicating a stronger affinity of TrOE33 to the binding site(s) within
the pore as compared with SynB2. This notion is sustained by the fact
that the block was not immediately reversible. This is shown in Fig. 4
A. First, a voltage ramp from 0 mV to +120 mV was applied
and thereafter a voltage ramp from 0 mV to 80 mV. At higher positive
potentials the channels are increasingly blocked and apparently some of
them stay blocked hence causing the discontinuity of the two current
traces at 0 mV. During the second voltage ramp the current increased in
a stepwise fashion at approximately 40 to 50 mV, indicating that
some of the channels are apparently "cleared" of the obstructing
transit peptide(s). Hence, the affinity of TrOE33 to the binding
site(s) within the pore is obviously higher than that of SynB2 as the
current reduction is not completely reversible and a potential of the
opposite polarity is necessary to expel the TrOE33 molecules from the channel.
Second, even nanomolar concentrations of TrOE33 are sufficient to shift
the reversal potential to a less positive value (Fig. 4 A;
control, Erev = 47 mV; +20 nM TrOE33
trans, Erev = 42 mV), whereas higher concentrations of SynB2 (~1 µM) were necessary to
cause a comparable effect (see above, Fig. 3 B, Hinnah,
1999). Following the argumentation for SynB2, the genuine transit
peptide TrOE33 thus exhibits not only a higher affinity for the binding site(s) within the pore but also for the binding sites at the channel
mouths. As TrOE33 carries only four positive charges at pH 7.0 it is
unlikely that the higher affinity is based merely on electrostatic
interactions. This notion is corroborated by the results of experiments
performed at pH 4.0. Under these conditions the effect of TrOE33 on the
Toc75 channel is unaltered (Fig. 4 B), 5 nM TrOE33 added to
the low salt compartment caused the onset of current reduction and even
the reversal potential is slightly shifted (250 mM/20 mM KCl, 10 mM
NAc/HAc, pH 4.0, control, Erev = 11 mV; +5 nM TrOE33 trans,
Erev = 9 mV (n = 2)).
At pH 4.0, TrOE33 carries six positive charges as opposed to five for
SynB2, so the effect might at least partially be due to enhanced
electrostatic interactions. However, TrOE33 already exhibited a higher
affinity than SynB2 to Toc75 at pH 7.0, where it only carries four
positive charges as opposed to 4.8 for SynB2. Besides, the Toc75
channel is only weakly cation selective at pH 4.0 (PK+/PCl = 1.9) due to the presumed protonation of otherwise negatively charged
amino acids. These considerations render it rather improbable that the
effect is solely due to intensified electrostatic interactions. The
implications of these findings will be discussed in detail later.
CoxIV
The genuine mitochondrial presequence CoxIV also induces a voltage-dependent block of the Toc75 channel. But as visible in Fig. 5, A and B higher concentrations than with TrOE33 and also with SynB2 were necessary. CoxIV (17 µM) (Fig. 5 A) was added to the trans compartment under asymmetric conditions (250 mM/20 mM KCl, 10 mM MOPS/Tris, pH 7.0, cis/trans) only induced an incomplete block. However, in Fig. 5 A the bilayer contains only three Toc75 channels, this experiment can therefore not serve for quantification of the effect, see below (Fig. 5 B for depiction of a more representative result). But in return a low number of channels in the bilayer allows a higher degree of resolution and it becomes obvious that the block is caused by an increased frequency of closures (corresponding to the main conductance value) as already perceived with SynB2 and TrOE33 (Figs. 3, A and C, and 4 B). Thus, the same underlying mechanism can be assumed, that is plugging of the pore by the peptide. The block was salt dependent, addition of the same amount of CoxIV (17 µM) to the high salt compartment (cis) caused only a slight flickering of the channels toward the closed state (Fig. 5 A).
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The strength of the block is better represented in Fig. 5 B, the dark gray current trace is markedly reduced after addition of 3.33 µM CoxIV to the trans side of a bilayer containing ~26 Toc75 channels. But the block is still incomplete, whereas 20 nM TrOE33 (Fig. 4 A) was able to induce a block comparable with this one caused by 3.33 µM CoxIV, and 1 µM SynB2 have been sufficient to bring about a complete block (Fig. 3 B).
As visible in Fig. 5 A the reversal potential was also slightly shifted to a less positive value (from 47-45mV), but the effect was even less pronounced than with 1 µM SynB2. As the amount of available CoxIV was restricted the effect was not quantified by assay of higher concentrations.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Conductance
The main conductance of a channel is primarily a valuable
identifying feature. The main conductance of Toc75 of 1.32 nS in 1 M
KCl is identical to the main conductance of synToc75 (1.3 nS in 1 M KCl
(Bölter et al., 1998b)), stressing the homology of the two
proteins on a functional level. Toc75 shows two additional subconductance levels at 66% and 33% of the main conductance. As
indicated by the dissimilarity of the subconductance levels and the
direct transitions between the fully closed and fully open state (Laver
and Gage, 1997), these are most likely subconductances of a single-pore
channel. They may either be caused by long-lived conformational states
as, e.g., described for the "allosteric" cyclic-nucleotide gated
channels (Miller, 1997) or they may be due to slight alterations in the
electrostatic properties in the channel's entrance vestibules (Dani
and Fox, 1991). The two subconductances of Toc75 are in good accordance
with the two previously determined values (Hinnah et al., 1997) and
furthermore with the two subconductances of 965pS and 334pS observed
with synToc75 (Bölter et al., 1998b).
The main conductance of Toc75 corresponds well to that of porins
(-monomers) e.g., OmpF, OmpE, ScrY show a value of 1.4 nS in 1M KCl
(Benz et al., 1985; Schülein et al., 1991), and that of Tom40,
which shows under identical conditions (bilayer measurements, 1 M KCl
symmetrical solution) a main conductance of 900 pS with one
subconductance of 375 pS (Hill et al., 1998).
Open probability
When previously assayed by patch clamp experiments Toc75 displayed
a rather unusual voltage dependence of the open probability (Hinnah et
al., 1997). Only after preactivation at voltages above 100 mV and
subsequent voltage jumps to potentials above +100 mV channel activity
could be induced. Because the protein import of chloroplasts is not
dependent on a voltage gradient (Douwe de Boer and Weisbeek, 1991), we
already suspected at that time that the reason for this in vitro
requirement might be incomplete reconstitution. Indeed, circular
dichroism-spectra indicated a higher degree of refolding after
preincubation with Mega9 (Hinnah et al., 1997). The voltage-gradient
may have been necessary to effect a conformational change by
interaction with charged protein parts of the incompletely
reconstituted Toc75 thereby inducing a transport-competent form. A
similar kind of pore-formation has been described for Colicin
(Lazdunski, 1995). This concept is now substantiated by the fact that
after reconstitution with Mega9 or Triton X-100 Toc75 reveals the
typical, porin-like voltage dependence that has meanwhile been proposed
to be an intrinsic feature of all -barrel pores (Bainbridge et al.,
1998). Secondary structure prediction, circular
dichroism-spectroscopy measurements and topology studies indicate a
putative structure for Toc75 consisting of 16 transmembrane -sheets
(Hinnah et al., 1997; Sveshnikova et al., 2000). These would allow
folding into a -barrel, the typical structure of a multitude of
pores, e.g., bacterial porins (Jap and Walian, 1996; Kreusch et al.,
1994), the mitochondrial protein import pore Tom40 (Mannella et al.,
1996), and some pore-forming toxins like aerolysin (Bainbridge et al.,
1998). These pores show no significant sequence homology but apart from
their structure they share a strikingly similar voltage-dependence of
their open probability. Thus, the observed voltage-dependence
corroborates the hypothesis that after reconstitution with detergents
Toc75 refolds into a -barrel structure. In addition, the
voltage-dependence significantly resembles that of synToc75 when
measured under identical conditions (Bölter et al., 1998b) and of
Tom40. However, as already mentioned above this voltage dependence is
considered to be an in vitro effect without significance for regulation
in vivo (Klebba and Newton, 1998). According to the new concept of
regulated solute exchange across the outer chloroplast envelope
(Neuhaus and Wagner, 2000), the opening of the Toc75 channel should be
regulated to avoid an uncontrolled flux of ions across the outer envelope.
The block of ionic current by preproteins as presented in this study suggests a way by which Toc75 could be indirectly controlled. Possibly the negatively charged receptor Toc160 (155 negative charges at pH 7.0) attracts the positively charged preproteins and augments their concentration in direct proximity of the pore entrance. Together with the high affinity of Toc75 for preproteins this would ensure the constant occupation of the pore, which prevents any uncontrolled ionic current. Thus, it is plausible that the preproteins close the channel as they are transported whereas other Toc components keep the pore closed when no transport occurs.
Selectivity and pH effect
The pronounced cation-selectivity of Toc75 at pH 7.0 (PK+/PCl = 14.3) is identical to that of synToc75
(PK+/PCl = 13.3), once more confirming their similarity. Such a high selectivity is rarely found with large pores, e.g., most porins exhibit only a weak
selectivity (Jap and Walian, 1996; Cowan et al., 1992). However, the
mitochondrial protein import pore Tom40 is also fairly cation-selective
(PK+/PCl = 7.7) (Hill et al., 1998). Generally the ability to differentiate
between cations and anions can be ascribed primarily to electrostatic
interactions between ions and charged and/or polar groups of the
channel (Laio and Torre, 1999). These groups may be localized within
the pore and/or at the pore mouth(s) (the latter performing a
preliminary screening of the potential solutes) as, e.g., demonstrated
for porins (Karshikoff et al., 1994; Jap and Walian, 1996; Cowan et
al., 1992). In the case of Toc75 the cation-selectivity can be ascribed
to the presence of negatively charged groups, because at low pH (5.0 and 4.0) protonation of these groups rendered the channel progressively unselective. The localization of these groups remains to be elucidated, however, our data (namely the measurements with peptides) strongly suggest that they are not only situated in the constriction zone but
also at both channel entrances (see below). The cation-selectivity can
be reconciled with the natural substrates of the channel, the transit
peptides, which carry an overall positive charge (von Heijne et al.,
1989). But as demonstrated in the present study the recognition is not
solely based on the positive charge (see results with TrOE33), so the
electrostatic interactions between the transit peptide and the negative
binding site(s) of the channel could have different and/or additional
functions. According to the acid chain hypothesis of protein import
into mitochondria (Komiya et al., 1998; Schatz, 1997), the transport of
the positively charged presequences is actually driven by their
interaction with negatively charged binding sites (among others binding
sites at the channel Tom40) of increasing affinity. It is tempting to
imagine a similar scenario between transit peptides and binding sites of Toc160, Toc75, and perhaps additional components of the inner envelope.
Pore size
The recalculation of the pore diameter under consideration of the
correction factor for the resistivity of the solution within the pore
resulted in a value of d = 15.4 Å for the constriction zone. However, the calculation is based on another assumption, the
unknown length of the constriction zone (here set as 5 Å, according to
Hille, 1992), thus still yielding only to an approximate value. Yet,
the notion that the pore diameter has to be considerably larger than
the previously calculated 8 to 9 Å is substantiated by the nearly
unrestricted passage of TBA+ (see Results). But
the method of probing the pore size with charged molecules is
disputable because the effective radius of a pore is not only
determined by its geometrical dimensions but also by charges and the
electric field within the pore (Eisenberg, 1996; Chen and Eisenberg,
1993), which may interact with the charged probes and distort the
result. To avoid this problem we used nonelectrolytes (PEG) of
different radius to estimate the pore size according to the polymer
exclusion method of Krasilnikov et al. (1992) and Bezrukov and
Kasianowicz (1997). The fit of the data according to Bezrukov and
Kasianowicz (1997) yields a value of d 22 Å as the
characteristic diameter of the channel. But, as pointed out by Smart et
al. (1997), this interpretation is based on the assumption that the
pore is a cylindrical conductor having one single characteristic
radius. But in reality most channel pores have a more complex internal
geometry see, e.g., the hourglass-shaped structure of porins (Cowan et
al., 1992). The assumption that Toc75 possesses a comparable geometry,
namely that the pore of Toc75 has a constriction zone with d
14 Å (thus, confirming the results of the calculation of the pore
size) flanked by two wide vestibules with an opening of
d 28 Å, is corroborated by the data obtained with
transit peptides, which are not intelligible without assuming this
internal structure of the Toc75 pore (see below). The diameter of ~14
Å of the constriction zone lies well within the range of the values of
other protein conducting pores, e.g., Tom40 with ~20 to 26 Å (Hill
et al., 1998; Kunkele et al., 1998) and the central pore of the Sec61p
complex has a diameter of 20 Å (Hanein et al., 1996). Studies with the
preprotein prOE17, which was imported into chloroplasts despite being
covalently linked to a tightly folded, protease-resistent BPTI-domain
of ~23 Å in diameter (Clark and Theg, 1997) suggests an even larger pore radius for Toc75. Conversely, this result may also indicate a
flexible structure of the pore in vivo, able to expand during import as
observed for the Sec61p-complex (Hamman et al., 1997).
Transit peptides
In brief, all of the three peptides caused two major effects: a voltage-dependent block and a shift of the reversal potential.
According to Woodhull (1973) the voltage-dependence of the block
demonstrates that the channel is plugged by binding of a peptide within
the electrical field (=within the pore). So the distinct closures in
the current trace can be attributed to the presence of the peptide
within the pore thus blocking the ionic current. Therefore, the
voltage-dependent block demonstrates that Toc75 contains a specific
binding site within the pore region. The specificity of this binding
site is highest for TrOE33 as indicated by the partially
irreversible block already caused by nanomolar concentrations.
SynB2 is also effectively bound at nanomolar concentrations, but the
block was immediately relieved in the absence of a voltage gradient
indicating that the attraction was essentially based on the overall
positive charge. CoxIV only induced a voltage-dependent block at
micromolar concentrations and the block was readily reversible. Thus,
the Toc75 channel displays a preference for the genuine transit peptide.
The question of a permeation of the peptides through the Toc75 channel cannot be decided. On account of the electrophysiological data it is impossible to discriminate between a peptide that is stuck in the pore (and consequently retreats) or one that is slowly translocated as both cause a transient block. To demonstrate permeation bi-ionic measurements would be necessary, but this is not feasible because sufficiently high amounts of peptides are not available.
Reversal potential
As already mentioned the observed shift in reversal potential cannot be explained in terms of permeation of the peptides. The required high permeability ratio is incompatible with the observed block. The only possible explanation is an alteration of the channel selectivity resembling that observed at low pH. The peptides bind to the selectivity filter(s) thus screening their negative charge and diminishing the cation selectivity. The selectivity filter(s) in question cannot be situated within the pore, because then binding would also cause block of the ionic current and furthermore be voltage dependent, but both is not the case. Therefore, the selectivity filter(s) have to be localized at the wider pore mouths, so that binding of peptides does not obstruct the passage of ions. Thus, the concept of an hourglass-shaped structure of the Toc75 channel with wide vestibules on either side of a constriction zone, as already indicated by the results of the PEG-method, is further corroborated.
The potency of the peptides to cause a shift of the reversal potential
corresponds well with their efficiency in inducing a block. TrOE33 is
already effective at a concentration of 20 nM, whereas 1 µM of SynB2
is necessary to induce a comparable result and 17 µM CoxIV is even
less effective and causes only a slighter shift. So the binding sites
at the pore entrances are likewise capable to distinguish between the
chloroplastic transit peptide and the mitochondrial presequence and its
derivative. Because TrOE33 is at pH 7.0 even less positively charged
than the other two peptides electrostatic interactions seem not to be
the only decisive factor in recognition. This observation is further
corroborated by the results of experiments at pH 4.0. At this low pH
the binding sites in the Toc75 channel are mostly protonated, and it
has lost its affinity for SynB2 and CoxIV. TrOE33 on the other hand is
now, as before, able to induce a voltage-dependent block and a shift of
the reversal potential suggesting that its affinity to all binding
sites is nearly unaltered. Furthermore it has already been demonstrated
by experiments with deletion mutant Ferredoxin precursors that the
uncharged N-terminal region of the transit peptide is essential for
import (Rensink et al., 1998; Pilon et al., 1995).
Thus, the specific recognition also seems to depend on the two other
possible forms of interaction, namely hydrogen bonds and/or van der
Waals interactions. Hydroxylated amino acids are specifically suited to
serve as H-donor and -acceptor, and as transit peptides can be
distinguished from mitochondrial presequences solely on the basis of
their high content in hydroxylated amino acids (von Heijne et al.,
1989) it is reasonable to assume an important function in recognition.
But hydrogen bonds and van der Waals interactions are relatively weak
interactions that can only secure specificity when conformational
compatibility exists. This would require a defined secondary structure
of transit peptides contrasting the conception that they form perfect
random coils (von Heijne and Nishikawa, 1991). Indeed our secondary
structure prediction of TrOE33 (see diagram in Material and Methods)
indicated -helical regions in the stromal targeting domain. It has
also been demonstrated that conditions that presumably mimic the
membrane environment or the contact with lipid surfaces in vitro induce a significant degree of -helical structure in several transit peptides (Endo et al., 1992; Horniak et al., 1993; Wienk et al., 1999;
Krimm et al., 1999). Thus, the necessary secondary structure may in
some of the transit peptides not be induced until they reach the direct
proximity of the chloroplast envelope. The presumed defined secondary
structure seems to consist of -helical elements, but it has to
differ from the amphiphilic -helix of mitochondrial presequences
because CoxIV is not recognized. Interestingly even the synToc75
channel (the presumed predecessor of Toc75) shows a lower
affinity to CoxIV than to SynB2, suggesting a highly conserved principle of recognition (Bölter et al., 1998b). In sum our data indicate that the Toc75 channel can recognize preproteins on the basis
of charge and conformation.
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ACKNOWLEDGMENTS |
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This work was supported by Human Frontier Science Program grants (to R.W. and J.S.) and a Deutsche Forschungsgemeinschaft grant SFB 431, P16 (to R.W.).
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FOOTNOTES |
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Address reprint requests to Prof. Dr.-Ing. R. Wagner, Universität Osnabrück, Biophysik, Fachbereich Biologie/Chemie, Barbarastrasse 11, Postfach 4469, D-49076 Osnabrück, Germany. Tel.: 49-541-969-2851; Fax: 49-541-969-2243; E-mail: wagner{at}uos.de.
Submitted October 29, 2001, and accepted for publication April 1, 2002.
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REFERENCES |
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|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
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-barrel membrane channels.
FEBS Lett.
431:305-308[Medline].
Erev = 5.1 ± 1.5 mV
(n = 5)) the current is reduced at a positive
voltage. At first some channels (three) stay closed during the
consequently applied voltage ramp from 0 mV to 