(Received for publication, March 13, 1995; and in revised form, June 27, 1995)
From the
In order to get insight into the functioning of transit sequences in chloroplast protein transport, the import of the full-length transit peptide of ferredoxin (trfd) was investigated. trfd rapidly associated with chloroplasts under import conditions and becomes protected against externally added proteases. Import of radiolabeled trfd is inhibited equally efficiently by nonlabeled trfd as well as by the intact precursor of ferredoxin. This strongly suggests that trfd enters the general import pathway of proteins into chloroplasts. trfd import was stimulated by ATP, which is the first demonstration that ATP is involved in membrane translocation of a targeting signal. Imported trfd was membrane-associated but was also partially degraded by internal proteases, most likely present in the stroma, indicating that the membrane-associated fraction of trfd is en route to its functional localization. The degradation products are exported out of the organelle. In contrast to the import of the precursor of ferredoxin, the import of trfd was independent of protease-sensitive components on the chloroplast surface, indicating that the initial binding of precursor proteins may be facilitated by transit sequence-lipid interactions.
The majority of the chloroplastic proteins is encoded on the nuclear DNA and synthesized in the cytosol. These proteins contain an N-terminal extension(1) , the transit sequence that is necessary(2, 3) and sufficient (4) to import proteins post-translationally into chloroplasts (for review see (5) ). The import process is initiated by binding of precursor proteins to the chloroplast surface(6) . Maximal binding requires the utilization of low amounts of ATP (100 µM) in the intermembrane space (7) and the presence of protease-sensitive components on the chloroplast surface(8) . Binding can also be observed in the absence of ATP (9) and after protease treatment of chloroplasts(10) , indicating that different binding stages and modes can exist. The subsequent translocation of the precursor proteins across the chloroplast envelope membranes requires a 1 mM ATP concentration (11, 12) in the stroma. Imported proteins are processed by a specific stromal protease (1, 13, 14) routed to their final localization within the chloroplast and assembled into holoenzymes.
Analysis of transit sequences reveals that there is little similarity in amino acid sequences(15) . They are enriched in hydroxylated and small hydrophobic amino acids, have a positive charge, and lack acidic amino acids(16, 17) . Despite the poor homology in the primary structure of transit sequences(15) , they are able to perform their essential and specific functions in protein import processes (i.e. organelle-specific targeting, translocation across the envelope membranes, correct processing, and intraorganellar routing of precursor proteins).
This can be
illustrated with the precursor of ferredoxin (prefd), ()which follows the general import pathway(18) .
prefd is imported into the chloroplast stroma, where it is subsequently
processed(11, 19) . The apoprotein is converted into
the biologically active holoprotein by insertion of the 2Fe-2S
cofactor(20) . Import of the largely unfolded prefd is
independent of cytosolic factors(21) , indicating that prefd
itself contains all of the information for organelle-specific targeting
and for the productive interaction with the import machinery leading to
the translocation across the envelope membranes. Both processes require
the presence of a functional transit sequence, because mature proteins
do not bind to chloroplasts (22) and attachment of the
ferredoxin transit sequence is sufficient to direct a foreign protein
to the chloroplast stroma (23) . The prefd transit sequence is
also required for the interaction with the stromal processing enzyme,
because deletions in the C-terminal region of the transit sequence
strongly interfere with correct maturation of prefd(24) . Very
recently, it was demonstrated that prefd causes a transit
sequence-dependent reduction in electrochemical resistance of the
envelope in intact chloroplasts. The most likely interpretation of this
phenomenon was that the transit sequence opens protein-conducting
channels(25) . How transit sequences function is completely
unknown. However, it can be anticipated that they will exert specific
interactions with components of the envelope membranes such as
proteinaceous receptors (5) and envelope membrane
lipids(26) .
In order to get insight into the way transit sequences function, we studied the import of the transit peptide of ferredoxin (trfd) into chloroplasts. It is shown that trfd follows the ATP-dependent import pathway as is used by prefd. Import of trfd was independent of protease-sensitive components on the chloroplast surface. Imported trfd is rapidly degraded by internal chloroplast proteases followed by an efficient export of the degradation products.
[H]prefd was obtained by growing Escherichia coli BL21 cells (DE3) containing the vector pET11d
in 35 ml of SV medium (containing per liter 28 g of ammonium
ferrosulfate, 0.2 g of magnesium sulfate, 8 g of potassium dihydrogen
phosphate, and 30 g sodium hydrogen phosphate with a pH of 6.9) at 37
°C with the following additions: 0.4% (w/v) glucose, 5 mg/liter
thiamin, and 50 mg/liter ampicillin until the optical density was 0.6.
Pelleted cells were resuspended in 5 ml of SV medium with 0.4% (w/v)
glucose, 5 mg/liter thiamin, 50 mg/liter ampicillin, and 1 mM
isopropyl-1-
-D-galactopyranoside. After 30 min, 1.0 mCi
of [
H]leucine (158 mCi/mmol) (Amersham Corp.) was
added, and the cells were allowed to grow for another 3 h.
prefd was purified as described(28) ,
except that the cells were lysed by sonication (Branson) and a smaller
gel filtration column (1.5
40 cm) was used. Protein
concentrations were determined according to Bradford (29) with
bovine serum albumin as reference.
Purified trfd was labeled by reductive methylation using
[C]formaldehyde(31) . In short, 2 mg of
trfd (0.47 µmol) dissolved in 500 µl of distilled water was
added to 500 µl of 20 mM Hepes, pH 7.6, containing 62.5
µmol of sodium cyanohydrogen boride (NaCNH
B) and 3.75
µmol of [
C]formaldehyde (59 mCi/mmol)
(Amersham Corp.) and was incubated for 1.5 h under nitrogen with
constant mixing. After the addition of another 62.5 µmol
NaCNH
B and further incubation of 1.5 h, trfd was
precipitated by 10% (w/v, final concentration) trichloric acid (TCA).
The trfd precipitate was washed 3 times with ice-cold acetone and,
after evaporation of the acetone, dissolved in 600 µl of distilled
water to a concentration of 2 mg/ml, divided into aliquots, and stored
under nitrogen at -20 °C. The
C-labeled transit
peptide of ferredoxin could be visualized as a single band by
Tricine/SDS-PAGE (32) followed by fluorography. It was shown
that all applied radioactivity was present in the peptide band. The
specific activity of [
C]trfd was 49 mCi/mmol,
and trfd contained 0.8 [
C]methyl group per
molecule.
Because chlorophyll interferes with the analysis of trfd by Tricine/SDS-PAGE(32) , the peptide was precipitated by 80% acetone, followed by centrifugation for 5 min at 14,000 rpm. The supernatant, which did not contain trfd as was verified by liquid scintillation counting, was removed, and traces of acetone were evaporated. The pellet was resuspended in 6 M urea, 10 mM Tris/HCl, pH 7.6, and 2 mM DTT by sonication for 15 min in a bath sonicator. Samples containing prefd were analyzed directly by SDS-PAGE according to Laemmli(35) . Protease pretreated chloroplasts were obtained by incubation of chloroplasts equivalent to 1 mg of chlorophyll with 250 µg of thermolysin for 20 min at 4 °C in the dark. Subsequently, the chloroplasts were reisolated by centrifugation through a preformed 50% Percoll gradient containing 2 mM EDTA in order to block the thermolysin activity. Chloroplast fractionation was performed by hypertonic lysis in 10 mM Hepes, pH 8.0, followed by a centrifugation for 30 min at 60,000 rpm in a Beckman TLA 100.3 rotor. The membrane pellet was resuspended for further analysis. In order to decrease the sample size of the supernatant fraction, the proteins were precipitated by 80% acetone. The pellet was resuspended in 6 M urea, 10 mM Tris/HCl, pH 7.6, and 2 mM DTT by sonication for 15 min in a bath sonicator.
The ability of [C]trfd to associate
with chloroplasts was investigated under conditions where prefd is
imported and correctly processed in the light at 25 °C in a buffer
containing 2 mM ATP(28) . Analysis by Tricine/SDS-PAGE
of reisolated and washed chloroplasts from the incubation mixtures
showed that in time increasing amounts of trfd became stable associated
with the organelle (Fig. 1A).
Figure 1:
Time course of import of
[C]trfd. A, total
chloroplast-associated trfd. B, protease-protected
chloroplast-associated trfd. C, quantitative representation of A and B.
, total associated trfd;
,
protease-protected trfd. 4 µg of trfd is used per incubation. In A and B, trfd is visualized by Tricine/SDS-PAGE and
fluorography.
To investigate whether chloroplast associated trfd was bound to the chloroplast surface, the incubation mixtures were treated with thermolysin, which is not able to enter the chloroplast intermembrane space and which can only digest proteins that are present on the chloroplast surface(34) . Interestingly, the majority of the associated trfd was not degraded (Fig. 1B). In control experiments, comparable amounts of trfd in the import buffer were digested within 30 s by identical amounts of thermolysin (data not shown). Furthermore, trfd associated to large unilamellar vesicles with a lipid composition comparable with the chloroplast outer envelope membrane was found to be completely digestable by thermolysin (data not shown). This indicated that binding to lipid surfaces does not result in protection against proteases. It can therefore be concluded that trfd had reached a protease-protected position, which we define as ``import.'' Quantification of this time course experiment (Fig. 1C) shows that association and import of trfd is a linear process in time.
The
addition of increasing amounts of labeled trfd to isolated intact
chloroplasts under import conditions led to an increased association of
trfd to chloroplasts (data not shown). Association and import of
[C]trfd is saturable. Moreover, association and
import are tightly coupled over a large range of transit peptide
concentrations. From these results it can be calculated that maximal 16
± 2
10
trfd molecules/minute/chloroplast are
imported assuming that 30 µg of chlorophyll corresponds to 4.5
10
chloroplasts(21) . The value of the V
of trfd import is close to the value of the V
of 22
10
molecules/minute/chloroplast reported for prefd
import(37) .
Quantification of the experiment shown in Fig. 1showed that nearly all chloroplast-associated trfd was
present in the trfd band (data not shown). Fractionation of reisolated
chloroplasts from incubation mixtures revealed that all
chloroplast-associated radioactivity is localized in the membrane
fraction (Fig. 2A). These observations do not exclude
the possibility that part of the transit peptide is degraded during or
after import. That this may be the case is suggested by the observation
that the transit sequence cleaved off from imported prefd could not be
detected in the membrane fraction nor in the soluble fraction, although
it contains 7 of the 13 [H]leucine residues (Fig. 2B). This demonstrates that the transit sequence
is rapidly digested after processing. It should be realized that
processing of prefd cannot be observed by Tricine/SDS-PAGE as used in Fig. 2B, because this gel system does not separate
prefd from holoprotein of ferredoxin. However, control experiments
using SDS-PAGE demonstrated that prefd was correctly processed under
the experimental conditions (data not shown).
Figure 2: Intrachloroplast localization of imported trfd and prefd. A, localization of trfd. Lane 1, total associated trfd; lane 2, imported trfd; lane 3, soluble fraction; lane 4, protease-treated soluble fraction; lane 5, membrane fraction; lane 6, protease-treated membrane fraction. B, localization of prefd. Lane 1, trfd standard; lane 2, total associated prefd; lane 3, imported prefd; lane 4, membrane fraction; lane 5, soluble fraction. Intensities of (poly)peptide bands cannot be directly compared because different lanes do not necessary contain the same amount of chloroplasts.
To get direct insight
into possible trfd degradation within chloroplasts, TCA precipitation
experiments were done (Fig. 3). Intact trfd in import buffer
without chloroplasts (Fig. 3, lane 1) or with
chloroplasts in conditions under which no import can take place (Fig. 3, lane 2) can be nearly quantitatively
precipitated by TCA. In contrast, a large fraction of trfd incubated
with lysed chloroplasts is not precipitable due to digestion by
proteases released from the chloroplasts (Fig. 3, lane
3). Under import conditions a substantial fraction (13 ±
1%) of the added C radioactivity is nonprecipitable (Fig. 3, lane 4), which demonstrates that indeed part
of the added trfd is degraded, like in case of transit sequence
liberated from the import precursor. This is most likely due to
digestion inside the chloroplasts, but in principle this could also, in
part be due to digestion by proteases liberated from chloroplasts
during the incubation. To get an estimate of the maximal contribution
of such released proteases, trfd was incubated in the supernatant of
chloroplasts preincubated under import conditions. This leads to
substantially less (7 ± 1%) degradation (Fig. 3, lane
5). Thus, it has to be concluded that at least 6% of the added
trfd is degraded by internal chloroplast proteases. This has to be
compared with 15% of the added trfd, which is associated as intact trfd
to chloroplasts under import conditions (Table 1).
Figure 3: Determination of the recovery of intact trfd after import into chloroplasts. After incubation for 20 min under import conditions, proteins are precipitated by TCA, and the percentage of precipitable and nonprecipitable counts, expressed as the percentage of the total amount of added trfd, is determined by liquid scintillation counting. For each experiment, 2.5 µg of trfd was used. Mean values and standard deviations of three independent measurements are shown. Lane 1, control experiment, trfd incubated in import buffer in the absence of chloroplasts; lane 2, incubation of trfd with chloroplasts in conditions under which no import take place (in the dark without ATP at 20 °C); lane 3, incubation of trfd in import buffer with lysed chloroplasts; lane 4, import experiment, trfd incubated in import buffer in the presence of chloroplasts under import conditions; lane 5, incubation of trfd in the supernatant of preincubated chloroplasts under import conditions. Open bars represent TCA-precipitable counts; closed bars TCA-nonprecipitable counts.
In order to determine the localization of the degradation products of trfd, intact chloroplasts were isolated by centrifugation after the incubation. In both the resuspended pellet and the supernatant, the percentage of intact and degraded trfd was determined by TCA precipitation. Table 1demonstrates that under import conditions virtually all chloroplast-associated trfd is intact (TCA-precipitable) and that the degradation products (TCA-nonprecipitable) are present in the chloroplast supernatant. It thus has to be concluded that the trfd degradation products generated by internal chloroplast proteases are rapidly exported.
trfd competes for import of prefd(27) . Fig. 4shows that the reverse is also true. Unlabeled trfd and
prefd equally efficiently inhibit the import of
[C]trfd, indicating that prefd and trfd compete
for the same limiting import step. Competition is a specific process
depending on the transit sequence because apofd is not able to inhibit
trfd import (Fig. 4).
Figure 4:
Inhibition of
[C]trfd import by trfd, prefd, and apofd.
Competition by trfd (
), prefd (
), and apofd (
). 1
µg of [
C]trfd is used per incubation mixture
of 150 µl containing 30 µg of
chlorophyll.
Binding and import of precursor
proteins into chloroplasts require ATP as energy source(5) .
ATP also affects chloroplast association and import of
[C]trfd (Fig. 5). In the absence of
exogenous ATP, trfd already displays some association and import into
chloroplasts. However, increasing the ATP concentration strongly
stimulates both trfd association and import, indicating that
ATP-consuming proteinaceous components are involved in trfd import.
trfd association and import is maximal around 1-2 mM ATP, which is very similar to the ATP concentration of 1 mM at which prefd import is maximal(21) .
Figure 5: ATP dependence of trfd import. Open bars, total associated trfd; closed bars, imported trfd. 4 µg of trfd is used per incubation. Incubation mixtures contained 200 nM nigericine in order to prevent intrachloroplast ATP production. The import experiment is performed at 4 °C under green light. Chloroplasts are incubated for 30 min at 0 °C in the presence of 200 nM nigericine in the dark to deplete them of ATP.
Digestion of proteinaceous components localized on the chloroplast surface by thermolysin reduces the import of precursor proteins into chloroplasts(33) , as is shown for prefd in Fig. 6. In contrast, the import of trfd is hardly affected by protease pretreatment.
Figure 6:
Effect
of chloroplast protease pretreatment on trfd and prefd import. Closed bars, control chloroplast; open bars,
protease-pretreated chloroplasts. Mean values and the standard
deviation of three individual experiments are shown. 1 µg of
[C]trfd (0.2 µmol) and 2.34 µg of
[
H]leucine-prefd (0.16 µmol) are used per
incubation mixture of 150 µl containing 30 µg of
chlorophyll.
The aim of this study was to investigate the functioning of transit sequences in chloroplast protein import. The approach was to study the import of the full-length transit peptide of ferredoxin.
It was shown that trfd enters the general import pathway of proteins
into chloroplasts. This conclusion is based on the following
observations. First, associated trfd is largely protected against
externally added protease, indicating that trfd has reached an internal
chloroplast localization. Second, [C]trfd import
is equally efficiently inhibited by nonlabeled trfd and prefd. Third,
trfd inhibits the import of prefd into chloroplasts(27) .
Fourth, trfd import is saturable and occurs with a maximal velocity
close to the value of the V
of prefd
import(37) . Finally, trfd import is stimulated by ATP.
This
is the first example of a targeting signal for which translocation
across a membrane is stimulated by ATP. This differs strikingly from
the situation in mitochondria where the translocation step of the
presequence is driven by the membrane potential () across
the inner membrane(38, 39, 40, 41) .
This difference in the energy required for protein translocation into
chloroplasts and mitochondria demonstrates that the mechanisms of
protein import into both organelles are fundamentally different. The
stimulation of trfd import by ATP can, for instance, be due to
interactions of trfd with membrane-associated chaperonines and to the
fact that ATP is consumed in transit sequence-chaperone binding/release
steps. From a comparison of transit sequences, it was postulated that
they are unstructured, which makes them prone to interact with
chaperonines(42) .
After import, protease-protected intact trfd could be identified solely in the membrane fraction. The absence of trfd in the soluble fraction, which mainly consists of the stroma, could indicate that trfd import is halted at the level of the envelope membranes. This would suggest that interaction of the mature region of precursor proteins with the import machinery is required to complete the import into the stroma. The observation that fusion proteins, for instance consisting of the ferredoxin transit sequence and the yeast mitochondrial manganese superoxide dismutase(23) , are correctly imported into the stroma suggests that this presumed interaction should be rather aspecific. Alternatively, and more likely, the absence of trfd in the stroma could be due to digestion of trfd during or after import. In agreement with this proposal, it was observed that part of the chloroplast-associated trfd was digested by internal chloroplast proteases. This protease activity was not associated to the chloroplast surface because incubation of trfd with chloroplasts, under the condition that no import could take place, resulted in only a marginal trfd degradation. Instead, the degradation was shown to be largely due to internal proteases. This protease activity could be present in the stroma, because the cleaved transit sequence after import of prefd into the stroma could not be detected in the membrane nor in the soluble fraction. The proposed degradation of trfd in the stroma indicates that the membrane-associated protease-protected trfd is en route to its functional localization.
The degradation of transit sequences after import may be required to prevent the accumulation of large amounts of transit sequence, which may very well have a poisoning effect. For instance, the surface-active and membrane-seeking properties of transit sequences (26) could lead to membrane insertion of large amounts of transit sequences, affecting membrane functioning. Furthermore, this result strongly argues against a second long-lived function of transit sequences in chloroplasts.
Surprisingly, although trfd was degraded by internal protease, the degradation products were almost entirely present in the external chloroplast medium. Therefore, the degradation products should be exported out of the chloroplast by a so far unknown mechanism. This transport process could enable the reuse of transit sequence degradation products in the cytosolic protein synthesis.
trfd is imported into chloroplasts along the general import pathway of the precursor protein; therefore differences in import characteristics of trfd and prefd may be related to differences in import requirements of transit sequences and mature part of precursor proteins. One striking difference between trfd and prefd import was the independence of trfd import to protease-sensitive components on the chloroplast surface. Also, the import of outer envelope membrane proteins was shown to be independent of protease-sensitive components on the chloroplast surface(44, 45, 46) , but these proteins likely follow an alternative pathway(44) . Therefore, the protease-sensitive components of the chloroplast surface seem not to be involved in trfd binding and import. This suggests that trfd initially binds to the chloroplast surface by interactions with the membrane lipids. This hypothesis is supported by the observation that prefd inserts, via its transit sequence, efficiently and specifically in lipid monolayers composed of a lipid extract of its target membrane (26) and binds to lipid vesicles(36) . Transit sequence-lipid interactions may result in the insertion of the transit sequence in lipid domains(36) , enabling the diffusion of precursors to the import machinery in a two-dimensional way, which will be more efficient than via three-dimensional diffusion through the aqueous phase. Besides this, transit sequence-lipid interactions result in the induction of secondary structures in the otherwise unstructured transit peptide, which may function as recognition motive for the import machinery (47) Furthermore, these interactions can result in reorientation of lipid molecules(48) . This change in lipid organization can directly be involved in protein import (49) or be required for the activation of the import machinery.
Comparison of the dissociation constants reveals that trfd binds with a 30-fold lower affinity to lipid vesicles than a precursor protein to chloroplasts(22, 36) . This suggests that the initial binding to the lipids is followed by an interaction with proteinaceous components of the import machinery.
Recent studies (50, 51, 52, 53) have identified several of these proteinaceous components. Schnell et al.(52) and Kessler et al.(53) identified six envelope membrane proteins associated to a translocation intermediate. Two of these proteins, of 34 and 86 kDa, are both integral outer envelope membrane proteins and are supposed to be exposed to the cytosol, due to their sensitivity to externally added proteases. Because trfd import is independent of protease-sensitive components on the chloroplast surface, it is unlikely that the 34- and 86-kDa proteins directly interact with the transit sequence and are involved in precursor protein targeting. Subsequently, it is unlikely that these proteins function as proposed by Kessler et al.(53) in the regulation of the presentation of transit sequences to the import machinery or by regulating the opening of the translocation channel. The 34- and 86-kDa proteins are most likely required for the import of the mature region of the precursor, and can perform the following functions. First, by interacting with the mature part region of the precursor, they could stabilize the binding of precursors to the chloroplast surface. Second, they may be required for a productive interaction of the precursor with other components of the import machinery. Finally, they may be required to bring the precursor in an import-competent conformation, for instance by reduction or by unfolding of mature regions of precursor proteins. In case of prefd, they could act as reductases because prefd import is stimulated by DTT (21) , whereas trfd import was independent of the DTT concentration (data not shown). Guérra et al.(43) observed the unfolding of a precursor protein when incubated with outer envelope membrane vesicles, but whether this activity was protein- or lipid-mediated is not known.