Botanisches Institut, Christian-Albrechts-Universität Kiel, D-24118 Kiel, Germany
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Abstract |
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The chloroplastic outer envelope protein
Toc34 is inserted into the membrane by a COOH-terminal membrane anchor domain in the orientation
Ncyto-Cin. The insertion is independent of ATP and a
cleavable transit sequence. The cytosolic domain of
Toc34 does not influence the insertion process and can
be replaced by a different hydrophilic reporter peptide.
Inversion of the COOH-terminal, 45-residue segment,
including the membrane anchor domain (Toc34Cinv), resulted in an inverted topology of the protein, i.e., Nin-Ccyto. A mutual exchange of the charged amino acid
residues NH2- and COOH-proximal of the hydrophobic -helix indicates that a double-positive charge at
the cytosolic side of the transmembrane
-helix is the sole determinant for its topology. When the inverted
COOH-terminal segment was fused to the chloroplastic
precursor of the ribulose-1,5-bisphosphate carboxylase
small subunit (pS34Cinv), it engaged the transit sequence-dependent import pathway. The inverted peptide domain of Toc34 functions as a stop transfer signal
and is released out of the outer envelope protein translocation machinery into the lipid phase. Simultaneously, the NH2-terminal part of the hybrid precursor
remained engaged in the inner envelope protein translocon, which could be reversed by the removal of ATP,
demonstrating that only an energy-dependent force but
no further ionic interactions kept the precursor in the
import machinery.
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Introduction |
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THE majority of chloroplast proteins are encoded for
in the nucleus, synthesized in the cytosol, and posttranslationally imported into the organelle. The
standard import route is facilitated by two protein translocation machineries located in the chloroplastic outer and
inner envelope membranes, which act jointly during translocation (Schnell and Blobel, 1993; Alefsen et al., 1994
).
Proteins destined to be translocated into chloroplasts
carry NH2-terminal transit sequences, which are both necessary and sufficient for targeting and import. The transit
sequence recognition is mediated by a protease-sensitive
receptor polypeptide located in the outer envelope membrane (Cline et al., 1985
). A low ATP concentration, between 5-50 µM, is required for binding of the precursor
protein to the outer envelope translocon in a step not well
characterized yet, while high concentrations, between 50-
1,000 µM, are necessary for complete translocation into
the chloroplasts (Flügge and Hinz, 1986
; Schindler et al.,
1987
; Olsen et al., 1989
).
In contrast, proteins destined for the chloroplastic outer
envelope are generally synthesized without an NH2-terminal cleavable transit sequence but are targeted by internal
information (Salomon et al., 1990; Li et al., 1991
; Ko et al.,
1992
; Fischer et al., 1994
; Kessler et al., 1994
; Seedorf et al.,
1995
; Li and Chen, 1996
; Chen and Schnell, 1997
), except
for the 86- and 75-kD subunits of the translocon at the
chloroplastic outer envelope (Toc86,1 Hirsch et al., 1994
;
Kessler et al., 1994
; Toc75, Tranel et al., 1995
; Tranel and
Keegstra, 1996
). The Toc75 precursor uses the general import machinery during the initial phase of the import. It is
processed in the stroma to an intermediate-size form while the majority of the mature protein remains in the chloroplastic outer membrane by an unknown stop transfer
mechanism. In a second phase of translocation, the protein
is inserted into the membrane and terminally processed in
a poorly understood way. The transit sequence-independent targeting and insertion seem to require neither protease-sensitive polypeptide components of the outer envelope membrane nor the hydrolysis of ATP. Current
evidence suggests that none of the protein translocon components of the chloroplastic outer envelope is involved in
the presequence-independent insertion pathway (Salomon
et al., 1990
; Li et al., 1991
; Ko et al., 1992
; Fischer et al.,
1994
; Seedorf et al., 1995
; Li and Chen, 1996
; Chen and
Schnell, 1997
; for review see Soll et al., 1992
). This insertion mechanism is different from the presequence-independent integration of proteins into the mitochondrial
outer membrane, which uses subunits of the TOM machinery (for review see Lill and Neupert, 1996
).
An issue that remains to be solved is how these membrane proteins achieve their correct topology. (a) Does the
specific lipid composition influence targeting and topology
of the chloroplastic outer envelope proteins, and (b) what
is the role of the membrane spanning domains of these envelope polypeptides? Or, (c) do soluble segments of the
protein influence its insertion and topology? Toc34 has a
well-established topology, Ncyto-Cin with only one transmembrane segment. The -helical hydrophobic membrane anchor is located close to the COOH terminus of
the protein, which leaves only 3 kD of the polypeptide exposed to the intermembrane space. The major portion of
the protein, including its GTP-binding domain, is cytosolic. Insertion of Toc34 translation product into the chloroplastic outer envelope requires the presence of the hydrophobic transmembrane segment (Seedorf et al., 1995
;
Chen and Schnell, 1997
; Li and Chen, 1997
). This insertion
process renders Toc34 resistant to extraction at pH 11.5. Productive folding or assembly, which can be followed by
the appearance of an 8-kD protease-protected fragment,
seems to be stimulated by the presence of nucleoside-triphosphates and a protease-sensitive envelope membrane component (Seedorf et al., 1995
; Chen and Schnell,
1997
). Resistance to alkaline extraction together with the
appearance of the 8-kD proteolytic fragment can be used
to follow the productive insertion process of Toc34.
In this study, we demonstrate that a double-positive
charge at the cytosolic side of the -helicial membrane anchor dictates the topology of Toc34. Furthermore, this domain acts as a functional stop transfer signal for the outer
envelope protein translocon. The signal anchor sequence
is released laterally from the translocon into the lipid
phase of the membrane.
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Materials and Methods |
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Chloroplast Protein Import Assays
Chloroplasts were isolated from pea leaves of 10-12-d-old plants by standard procedures and purified further on silica-sol gradients (Waegemann
and Soll, 1991). Chlorophyll (chl) concentration was determined as described (Arnon, 1949
). Standard import assays contained chloroplasts
equivalent to 30 µg chl in 100 µl import buffer (330 mM sorbitol, 50 mM
Hepes-KOH, pH 7.6, 3 mM MgCl2, 10 mM methionine, 10 mM cysteine,
20 mM potassium gluconate, 10 mM NaHCO3, 2% [wt/vol] BSA) and
1-10% of reticulocyte lysate in vitro-synthesized 35S-labeled proteins.
Translocation reactions were initiated by the addition of organelles and
allowed to continue for 10 min at ambient temperature. Chloroplasts were
recovered from the import reaction by centrifugation (5,000 g, 5 min, 4°C)
through a Percoll cushion (40% [vol/vol] Percoll in 330 mM sorbitol, 50 mM Hepes-KOH, pH 7.6), washed once in Hepes-sorbitol (Waegemann
and Soll, 1991
), and used for further treatments.
Chloroplasts were treated with thermolysin before a translocation experiment with 750 µg protease/mg chl for 30 min on ice in 330 mM sorbitol, 50 mM Hepes-KOH, pH 7.6, 3 mM MgCl2, and 0.5 mM CaCl2. The reaction was terminated by the addition of 10 mM EDTA. Intact
chloroplasts were recovered on silica-sol gradients and washed twice as
described above. When chloroplasts were treated with thermolysin after
an import experiment, organelles were incubated at a final concentration
of 100 µg protease/mg chl for 15 min at 4°C. The reaction was stopped by
the addition of 10 mM EDTA, and the chloroplasts were recovered by
centrifugation and washed once (Joyard et al., 1983; Cline et al., 1984
).
When indicated, chloroplasts were treated with trypsin (Marshall et al.,
1990
; Lübeck et al., 1996
). Chloroplasts equivalent to 200 µg chl were incubated with 200 µg trypsin (10,700 Na-benzoyl-L-arginine ethyl ester U/mg
from bovine pancreas) for 60 min at 25°C in 50 mM Hepes-KOH, pH 8.0, 0.1 mM CaCl2, and various sorbitol concentrations (200 mM = hypotonic;
330 mM = isotonic; 600 mM = hypertonic conditions). The reaction was
stopped by the addition of 1 mM PMSF and a fivefold molar excess of soybean trypsin inhibitor. Intact chloroplasts were recovered by centrifugation through a Percoll cushion and washed twice under isotonic conditions
as described above. PMSF was present at all washing steps. Chlorophyll
concentration was determined, and equal amounts of organelles were
loaded onto the SDS-PAGE on a chl basis.
After hypotonic lysis in 10 mM Hepes-KOH, pH 7.6, chloroplasts were
separated into a soluble and a total membrane fraction by centrifugation
for 10 min at 165,000 g. Soluble proteins were precipitated by 10% TCA.
In some cases, lysed organelles were treated with 0.1 M Na2CO3, pH 11.5, for 10 min on ice. Insoluble material was recovered by centrifugation as
described before, and soluble proteins were precipitated by 10% TCA.
Import products were analyzed by SDS-PAGE (Laemmli, 1970) followed
by fluorography (Bonner and Laskey, 1974
).
Construction of cDNAs Coding for Toc34 Hybrid Proteins and Synthesis of Labeled Proteins
cDNAs coding for the different hybrid proteins, including single amino
acid exchanges, were constructed by recombinant PCR (Higuchi, 1990).
Toc34Cinv was constructed using two extra-long (105-bp) primers, which
coded for the inverted sequence of the Toc34 COOH terminus (see Fig.
1). Products were cloned into a vector suitable for in vitro transcription
and controlled by DNA sequencing (Sanger et al., 1977
). The original cDNAs
coding for Toc34 and the precursor form (pS) have been described (Klein
and Salvucci, 1992
; Seedorf et al., 1995
). In vitro transcription was done
using either T7 or SP6 RNA-polymerase, as outlined before (Salomon et
al., 1990
). Proteins were synthesized in a reticulocyte lysate system in the
presence of [35S]methionine and [35S]cysteine (1,175 Ci/mmol) for 1.5 h at
30°C. Overexpression of unlabeled or radioactively labeled proteins was
done in Escherichia coli BL21 (DE3) cells using the pET vector system
(Novagen Corp., Madison, WI) (Waegemann and Soll, 1995
). Proteins
were isolated from inclusion bodies and solubilized in 8 M urea. The final
urea concentration in the import assays never exceeded 80 mM.
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Chloroplastic ATP Levels
Intact chloroplasts were incubated in import buffer in the presence of 2 mM
ATP and 2 µCi [-32P]ATP for 10 min at 25°C. An aliquot was removed,
and chloroplasts were separated from the aqueous medium by centrifugation (15 s, 9,000 rpm) through a silicon oil layer (Wirtz et al., 1980
). Residual chloroplasts were recovered by centrifugation and resuspended in import buffer in the absence of ATP. Aliquots were removed from the
organelle suspension at different time intervals and recovered by centrifugation through silicon oil. Chloroplasts were extracted with 80% dimethyl-keton. Nucleotides were separated by thin layer chromatography on glass
plates coated with polyethyleneimine cellulose using 0.5 M KH2PO4 as
solvent. ATP-containing spots were localized in UV light and scraped off
the plates, and radioactivity was determined after addition of scintillation
cocktail in a scintillation counter.
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Results |
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Toc34 has an established topology, i.e., Ncyto-Cin with a single hydrophobic -helical region at the COOH terminus
acting as a membrane anchor domain. Our aim was to
study the role of the membrane anchor region in the topology of Toc34 and its influence on the protein import pathway. Therefore, the hybrid protein Toc34Cinv was constructed containing 45 amino acids of the COOH terminus, including the membrane anchor domain, but in an inverted sequence (for details see Fig. 1). Accurate targeting
and insertion of Toc34 into the chloroplastic outer envelope can be proven by a combination of two experimental
approaches: (a) Toc34 is largely resistant to extraction at
pH 11.5 in situ (Seedorf et al., 1995
); and (b) Toc34 is sensitive to thermolysin in situ, except for an 8-kD COOH-terminal fragment (Seedorf et al., 1995
). When Toc34Cinv translation product was incubated with intact chloroplasts,
the protein integrated into the outer envelope membrane
in a largely protease-resistant form (Fig. 2 A, bottom, lane
2), in contrast to the wild-type Toc34 (Fig. 2 A, top, lane
2). Protease resistance of inserted hybrid protein is compatible with the sequence of Toc34Cinv because the protein contains only three amino acids COOH-terminal of
the inverted membrane anchor (see Fig. 1). The appearance of proteolytic fragments, which are formed from a
subpopulation of Toc34Cinv, was most likely due to an incomplete transfer of the hydrophilic domain across the
outer envelope membrane. Toc34Cinv inserted into the
membrane in a way that renders it largely resistant to extraction at pH 11.5, i.e., it behaves as an integral membrane protein like the wild-type Toc34 (Fig. 2 A, lanes 3 and 4). The proteolytic fragments generated from inserted
Toc34Cinv were also resistant to alkaline extraction, demonstrating that the membrane anchor region had integrated into the lipid bilayer even in those cases where
translocation was not complete. Insertion of Toc34 translation products into chloroplasts yielded the expected results (Seedorf et al., 1995
; Chen and Schnell, 1997
), i.e., the
integrated protein was sensitive to thermolysin but resistant to alkaline extraction (Fig. 2 A, top, lanes 3-6).
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Next we wanted to know if the hydrophilic NH2-terminal part of Toc34 influences the insertion reaction. It was replaced by the mature form of the Rubisco small subunit (mS), which is a soluble stroma-localized protein. Both hybrid proteins, mS34C and mS34Cinv, inserted indistinguishably in the chloroplastic outer envelope to the analogue "wild-type" Toc34's (Fig. 2, A and B), i.e., inserted mS34C and mS34Cinv were resistant to extraction at pH 11.5 (Fig. 2 A, lanes 9 and 10), and mS34C was susceptible to a thermolysin treatment while mS34Cinv proved to be largely resistant (Fig. 2 A, lanes 7 and 8). The proteolytic fragments of inserted mS34Cinv are most likely due to incomplete translocation of the NH2-terminal polypeptide chain across the outer envelope membranes. Solubilization of chloroplast membranes by detergent before protease treatment resulted in complete proteolysis (not shown).
To obtain further experimental evidence for the putative topology of the different Toc34 proteins, they were synthesized in a reticulocyte lysate system in the presence of [3H]leucine. The 8-kD proteolysis-resistant fragment (see above) can only be detected in experiments using [3H]leucine-labeled Toc34 because the COOH terminus does not contain any methionine or cysteine residues. The appearance of the 8-kD fragment either from [3H]Toc34 or [3H]mS34C translation product shows that both polypeptides obtained a topology Ncyto-Cin, i.e., indistinguishable to Toc34 in situ (Fig. 2 B, lanes 1 and 2, and 5 and 6). Thermolysin treatment of inserted [3H]Toc34Cinv yielded no 8-kD fragment, but did yield a fragmentation pattern similar to the 35S-labeled Toc34Cinv (Fig. 2 B, lanes 3 and 4). mS does not interact with chloroplasts to a detectable amount (Fig. 2 B, lane 7). From these data, we conclude that the Toc34 COOH-terminal 45 amino acids seem to be the only determinant for chloroplastic targeting, and they simultaneously dictate the topology of the inserted protein. The inverted COOH terminus of Toc34 can function as a bona fide membrane anchor in the chloroplastic outer membrane but simultaneously inverts the topology of the protein into Nin-Ccyto.
As demonstrated above, the COOH-terminal region of Toc34 contains an outer envelope targeting domain regardless of its orientation. In light of this, we asked if the stroma-targeting envelope transfer signal present in the transit sequence of pS is able to override the membrane anchoring information present in the COOH terminus of Toc34. We therefore compared the import and binding of preSSU with its derived hybrid proteins pS34C and pS34Cinv. Under conditions that allow import to occur at optimal rates (25°C and 2 mM ATP), pS is efficiently translocated into the chloroplasts and processed to the mature form (Fig. 3 A, lanes 1 and 2). The mature form mS is recovered as a protease-protected soluble protein (Fig. 3 A, lanes 3-6). The ratio of radioactivity recovered in pS and processed mature mS was 1:19, as determined by laser densitometry of exposed x-ray films (n = 5). pS34C is imported into intact chloroplasts with a yield similar to preSSU. It is processed like the wild-type protein to yield mS34C (Fig. 3 B, lanes 1 and 2). The ratio of chloroplast-bound precursor and mature mS34C was 2:8 (n = 5). Upon subfractionation of the chloroplasts, an equal distribution of mS34C is observed between the total membrane and the soluble protein fraction (Fig. 3 B, lanes 3 and 4). However, mS34C only adheres to the membranes since it is completely extractable at pH 11.5 (Fig. 3 B, lanes 5 and 6). In comparison to preSSU, the binding of pS34C is only slightly increased, indicating that most but not all of the hybrid precursor might enter the standard import route. Translocation still takes place when pS34Cinv translation product was incubated with intact chloroplasts under standard import conditions. However, the processing resulted primarily in a product of intermediate size (iS34Cinv) and only in a very small amount of mature mS34Cinv (Fig. 3 C, lanes 1 and 2). The ratio between pS34Cinv, iS34Cinv, and mS34Cinv was 6:3:1 (n = 5). iS34Cinv was completely recovered in the total membrane fraction (Fig. 3 C, lanes 3 and 4). Upon alkaline extraction, 80% of iS34Cinv was recovered in the pellet fraction while 20% could be extracted (Fig. 3 C, lanes 5 and 6). These latter results indicated that most of iS34Cinv had inserted into the lipid bilayer of the outer or inner envelope while a smaller portion of iS34Cinv remained in a proteinaceous environment, e.g., the protein translocation pore. When the outer and inner envelope membranes were separated from chloroplasts after pS34Cinv translocation, iS34Cinv was recovered in the outer envelope (Fig. 3 E). The translocation efficiency of pS34Cinv is less than for pS and pS34C because more pS34Cinv remained bound to the chloroplast surface in a protease-accessible manner (Fig. 3 B, lanes 1 and 2). On the other hand, insertion of mS34Cinv translation product into the outer envelope attains a largely protease-protected topology (Fig. 2, lanes 7 and 8). We do not think that these results are contradictory because pS34Cinv can enter two chloroplastic translocation pathways: a receptor-dependent pathway as well as a receptor-independent pathway, depending on the experimental conditions. This might result in a protein being fixed with both the NH2 terminus and the COOH terminus at the surface of the organelle. In contrast, mS34Cinv has only one opportunity, namely to enter the receptor-independent pathway. This is also corroborated by further findings presented below.
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Furthermore, when we used urea-denatured, overexpressed pS34Cinv in a standard import reaction, iS34Cinv
was again the major reaction product (Fig. 3 D, lanes 1 and
2). Processed, mature mS34Cinv was below 10% of the total. Only very little binding of urea-denatured pS34Cinv
was detected. This might be due to the missing secondary
structure of the unfolded precursor, which could prevent
(a) the interaction with the membrane due to the absence
of the -helical conformation of the COOH terminus and (b) the accumulation of the precursor at receptor sites on
the organellar surface.
The general import pathway into chloroplasts by the Toc and Tic machinery requires protease-sensitive receptor components at the chloroplastic surface, which recognize the NH2-terminal transit sequences. In contrast, the insertion of chloroplastic outer envelope proteins, which do not contain a presequence, is independent of a protease-sensitive surface component. To investigate further if and to what extent pS34C and pS34Cinv used the standard import pathway, chloroplast surface-exposed precursor receptors were removed by treatment with the protease thermolysin. The binding of pS to protease-shaved chloroplasts was reduced to 10% in comparison to untreated organelles (Fig. 4, C, lanes 1 and 3; and D). In contrast, the amount of binding of pS34C or pS34Cinv was not significantly influenced by a protease pretreatment, indicating that both proteins inserted largely via the COOH-terminal membrane anchor region under conditions that discourage the Toc pathway. Under import conditions, (2 mM ATP and 25°C), thermolysin-shaved chloroplasts were also blocked in complete translocation of pS (Fig. 4 C, lanes 7-10). The residual import yield varied between 10 and 15% (n = 6; Fig. 4 D) for protease-pretreated chloroplasts. In parallel experiments, the efficiencies of pS34C import into thermolysin-shaved chloroplasts as measured by the appearance of processed mS34C dropped to 15- 20% of control imports (Fig. 4, A, lanes 7-10; and D). The import yield was even less for the translocation of pS34Cinv into protease-pretreated chloroplasts (Fig. 4 B, lanes 7-10). Only 5% iS34Cinv was still detected (Fig. 4 D). The precursor proteins pS34C and pS34Cinv, which were bound to protease-shaved chloroplasts, were resistant to alkaline extraction, indicating that they inserted by the COOH terminus into the outer envelope membrane.
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To distinguish in vitro between presequence-dependent
and -independent interactions with the organellar surface,
i.e., via the COOH-terminal -helix, chloroplasts were incubated with pS, pS34C, and pS34Cinv, respectively, under binding conditions (50 µM ATP and 4°C) for 10 min,
followed by a chase period under translocation conditions
(2 mM ATP and 25°C). pS bound to the chloroplastic surface and translocation intermediates (TIM 3/4), which are
well characterized for the pS import pathway (Waegemann and Soll, 1991
, 1996
), were detected upon thermolysin treatment (Fig. 4 C, lanes 1 and 2, and 13 and 14). The
bound pS could be chased into the chloroplasts upon the
addition of ATP and raising the temperature to 25°C,
demonstrating that the binding state was productive (Fig. 4 C, lane 15). pS34C and pS34Cinv also bound to the chloroplast surface with a yield similar to pS (Fig. 4, A and B,
lanes 1 and 13). Upon thermolysin treatment, the translocation intermediates (TIM 3/4) could be detected (Fig. 4,
B and C, lanes 2 and 14). However, chloroplast-bound hybrid proteins did not translocate into chloroplasts to any
significant extent upon establishing import conditions
(Fig. 4, A and B, lane 15), probably because they had also
inserted during the course of the incubation period into
the chloroplastic outer envelope with the COOH-terminal
-helical region. This is supported by the observation that
the surface-bound pS34C and pSCinv were resistant to alkaline extraction (not shown). These results indicate that
conditions that do not allow import, i.e., low ATP and low
temperature, favor the insertion of Toc34 via the
-helical
membrane anchor region.
From the data presented in Fig. 4, we conclude that pS34C and pS34Cinv use preferentially the Toc and Tic complex-dependent pathway under experimental conditions that favor this pathway (2 mM ATP, 25°C) and functional receptor polypeptides. Whenever we manipulate the optimal requirements by lowering the temperature or the ATP concentration or by removing precursor recognition sites, the membrane insertion route via the COOH-terminal membrane anchor becomes more prominent.
pS34Cinv is imported into chloroplasts at 2 mM ATP
and processed to an intermediate-size form (see Fig. 4 B),
most likely by the stromal-processing protease. Therefore,
it should also engage the Tic complex during this process.
Furthermore, iS34Cinv behaves as an integral membrane
protein that cofractionates with the chloroplastic outer envelope (Fig. 3 E) when the inner and outer membrane are
separated by shearing forces (Keegstra and Youssif, 1986).
To test if iS34Cinv is in contact with the Tic complex, we
compared the sensitivity of the imported and processed forms of pS, pS34C and pS34Cinv, to thermolysin and
trypsin, respectively. Under strictly controlled conditions,
thermolysin only shaves proteins off the chloroplastic surface, while trypsin penetrates the outer envelope membrane and gains access to the intermembrane space and to
proteins exposed on the surface of the inner membrane (Marshall et al., 1990
; Lübeck et al., 1996
; see also Fig. 5 C). mS and mS34C were resistant to both thermolysin and
trypsin (Fig. 5 A, lanes 1-6), indicating that they had
reached the stroma. In contrast, iS34Cinv was resistant to
thermolysin but sensitive to trypsin (Fig. 5 A, lanes 7-9). A
proteolytic fragment was generated by trypsin (iS
C; Fig.
5 A, lane 9), which was recovered in the total soluble protein fraction of chloroplasts (Fig. 5 B, lane 4). These data
indicate that trypsin removed the membrane anchor of
iS34Cinv, so that translocation into the stroma could proceed through the inner envelope translocon. The predicted
localization of iS34Cinv, i.e., anchored in the outer envelope membrane while simultaneously engaging the Tic
complex, was further established by carrying out the
trypsin treatment under different osmotic conditions. The
rationale behind this approach is that at hypertonic conditions (0.6 M sorbitol), a retraction of the inner envelope membranes from the outer and a widening of the intermembrane space occurs because H2O is extruded from the
stroma into the surrounding medium (Block et al., 1983
;
see Fig. 5 C). Under these conditions, iS34Cinv should become trypsin sensitive. On the other hand, isotonic or
slightly hypotonic conditions do not result in a membrane
rearrangement, and trypsin treatment should result in the
appearance of iS
C. This was found to be the case (Fig.
5 B). Chloroplast-inserted iS34Cinv was completely degraded by trypsin treatment under hypertonic conditions
(Fig. 5 B, lanes 9 and 10). At isotonic and hypotonic conditions, iS
C remained protease protected (Fig. 5 B, lanes 3 and 4, and 7 and 8). iS
C is not protease resistant per se, as demonstrated by treating the soluble iS
C with trypsin
(Fig. 5 B, lane 6), which results in complete proteolysis.
We conclude that the inverted COOH-terminal segment
of Toc34 can serve as a stop transfer signal for the Toc machinery. The stop transfer signal is recognized by the outer
envelope translocon and laterally released into the plane
of the membrane. Simultaneously, the iS part of the protein continuously interacts with the inner envelope translocon.
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The latter prediction was analyzed using the following
experimental procedures. Intact chloroplasts were preincubated either in the absence or presence of E. coli expressed pS (Fig. 6 A), pS34Cinv (Fig. 6 B), or Toc34 (Fig. 6
C) under import conditions for 5 min. Organelles were recovered by centrifugation and washed once in import
buffer in the presence or absence of 2 mM ATP. A second
round of protein translocation was started by the addition of 35S-labeled pS reticulocyte lysate translation product.
Import of 35S-labeled pS was only slightly diminished
when chloroplasts were preincubated in the presence of
overexpressed pS, in comparison to a mock preincubation
in the absence of overexpressed pS (Fig. 6 A, lanes 1 and 2,
and 5 and 6). When 35S-labeled pS reticulocyte translocation was added simultaneously with E. coli expressed unlabeled pS, binding and translocation of 35S-labeled pS was
almost completely abolished (Fig. 6 A, lanes 3 and 4). This
demonstrates that the heterologously expressed precursor
could compete with the reticulocyte lysate-synthesized precursor. In contrast, 35S-labeled pS import into chloroplasts that were preincubated with pS34Cinv was very
strongly inhibited (Fig. 6 B, lanes 1 and 6), independent of
the presence of pS34Cinv during the second round of
translocation. Binding of pS to the chloroplast surface was not influenced significantly (Fig. 6 B, lane 5), indicating
that inhibition occurred in a later step of the translocation
reaction, e.g., at the level of the Tic complex. This is corroborated by the observation (Fig. 6 B, lanes 7 and 8) that
the inhibitory effect of pS34Cinv preincubation was completely reverted when chloroplasts were reisolated and
washed in the absence of ATP before the second round of
translocation. The ATP concentration in chloroplasts
drops to below 10 µM ATP (Fig. 6 D) during the reisolation and washing procedure. This low level of ATP is not sufficient to support translocation (Olsen et al., 1989). We
conclude that an ATP-dependent pulling force in the
stroma, e.g., exerted by hsp70 or hsp100, looses its grip on
the partly translocated protein, upon which the precursor
retracts from the inner envelope translocon. The Tic complex then becomes available for new rounds of translocation.
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Heterologously expressed pS34Cinv competes effectively with 35S-labeled pS reticulocyte lysate translation product when added simultaneously to chloroplasts (Fig. 6 B, lanes 3 and 4). This further shows that pS34Cinv enters the standard import pathway. Chemical amounts of Toc34 can neither compete for pS nor for pS34Cinv import (Fig. 6 C), corroborating our earlier notion that under optimal import conditions (2 mM ATP, 25°C), the presequence-dependent standard import route is preferred to the membrane insertion pathway.
Our data so far indicated two findings: (a) Inversion of
the COOH-terminal 45 amino acids of Toc34 causes a reorientation of the membrane topology; and (b) the inverted COOH-terminal segment functions as a stop transfer signal for the Toc machinery. However, it remained to
be established if the orientation determinants reside in the
transmembrane or in the flanking sequences. As can be
seen in Fig. 7, two positively charged amino acids are
placed in direct vicinity (position-1 and -3) of the cytosolic
side of the Toc34 transmembrane segment, while in the
case that two positively charged amino acids are closely
spaced in the lumenal segment of Toc34, they are directly
accompanied by a negatively charged residue. This double-positive charge might represent a determinant for
Toc34 orientation. Therefore, a mutual charge exchange
was performed (see Fig. 7 A), which resulted in a
Toc34+
and Toc34Cinv+
. Toc34+
contains the
membrane segment in a wild-type orientation, while the
two positive charges at the NH2-proximal site of the transmembrane segment were replaced by two negatively
charged amino acids. At the COOH-proximal end, one
negatively charged amino acid was replaced by a positively
charged amino acid resulting in a double-positive charge
close to the COOH-proximal site of the transmembrane
segment. The similar amino acid exchanges were also
placed in Toc34Cinv, resulting in Toc34Cinv+
.
|
In contrast to wild-type Toc34, Toc34+
now inserts
into the chloroplastic outer envelope in a way that renders
it mostly protease resistant (Fig. 7 B, lanes 3 and 4), reminiscent of the insertion behavior of Toc34Cinv (compare
Fig. 7 B, lanes 5 and 6). Toc34Cinv+
instead now inserts
in a protease-sensitive manner (Fig. 7 B, lanes 7 and 8),
reminiscent of wild-type Toc34 (compare Fig. 7 B, lanes 1 and 2). These results indicate that the charge distribution
flanking the transmembrane segment is the prime determinant for the orientation of Toc34.
As shown above (Fig. 3), pS34C can be imported into
chloroplasts, where it is processed and recovered as soluble protein in the stroma. When we tested the binding and
import properties of pS34C+
, we found that it was no
longer able to translocate fully into chloroplasts (Fig. 7 C,
lanes 5 and 6). Instead, under import condition it was processed to iS34C+
and recovered in the membrane fraction, resistant to extraction at pH 11.5 (Fig. 7 C, lanes 7 and 8). Under binding conditions, the translocation intermediates TIM 3/4 indicated that pS34C+
had entered
the standard import pathway (Fig. 7 C, lanes 1 and 2). We
conclude that charge distribution around the transmembrane segment represents a stronger sorting signal than
orientation and hydrophobicity of the membrane domain.
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Discussion |
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---|
Toc34 is anchored in the chloroplastic outer envelope by a
single transmembrane hydrophobic membrane anchor in
an orientation Ncyto-Cin. If we compare the region of 28 amino acids NH2-terminal preceding and COOH-terminal
following the membrane anchor, a charge difference of 4:7
positive charges can be observed. Hence, it is not obvious
that posttranslational insertion of Toc34 obeys the positive
inside rule (von Heijne and Gavel, 1988; von Heijne, 1989
). Charge differences as sole determinant for the orientation of a membrane anchor region is difficult to reconcile because it would most likely require an intact electrochemical potential across the chloroplastic outer envelope
(Andersson and von Heijne, 1994
; Rapoport et al., 1996
).
At present, the evidence suggests that the outer envelope
lacks a significant electrochemical potential (Joyard et al.,
1991
). However, the specific lipid composition of the chloroplastic outer envelope results in a strongly negatively charged membrane surface (Joyard et al., 1991
). Positively
charged amino acids selectively positioned to a transmembrane segment might be prominent determinants for the
membrane orientation of a polypeptide. Our results establish that two positive charges in close proximity to the
transmembrane segment dictate the topology of Toc34.
The interconversion of the positive charge "motif" (Fig. 7)
NH2- and COOH-proximal to the transmembrane segment resulted in the interconversion of the Toc34 topology. From these observations we conclude that although
the hydrophobic
-helix of Toc34 is necessary for its insertion (Seedorf et al., 1995
), positively charged amino acids
flanking this region are the major determinants for its orientation. Characterization of the insertion behavior of
other single membrane spanning outer envelope proteins
will be necessary to validate and generalize this conclusion.
The general protein import machinery of the chloroplastic outer envelope can translocate proteins containing integral membrane components of either the inner envelope
or the thylakoid membrane. Consequently, amino acid sequences with the potential to form hydrophobic -helices
pass freely through the outer envelope translocon. The
2-oxoglutarate-malate translocator, a 12-helix-motif transporter of the inner envelope, uses the general import machinery as well as the three
-helices containing thylakoid
localized light harvesting chlorophyll a/b binding protein
(Hobe et al., 1994
; Weber et al., 1995
). The presence of a
stop transfer signal functional at the outer envelope protein translocon would obviously be deleterious to organelle biogenesis. Chloroplast preproteins localized in the thylakoid lumen contain a bipartite transit sequence.
The NH2-proximal part functions as an envelope transfer
domain, while the COOH-proximal part functions as thylakoid transfer domain and dictates which translocation
route it follows, i.e., a Sec-dependent or
pH-dependent
route (for review see Robinson and Mant, 1997
). Positive
charges placed around a hydrophobic domain determines the translocation pathway to be used. The hydrophobic
domain in thylakoid lumen presequences is much shorter,
10-13 amino acids, than in Toc34. Positively charged
amino acids in combination with a critical length of a hydrophobic transmembrane segment might represent a necessary combination of signals that is recognized by the Toc
machinery. The unique import pathway of preToc75 into the outer envelope membrane seems to involve a stop
transfer signal that is present in the COOH-proximal part
of the cleavable transit sequence (Tranel and Keegstra,
1996
). However, the nature of this signal is not clear, nor is
it clear whether it is deciphered by the Toc or the Tic machinery. Hydrophobic stop transfer regions from mouse
immunoglobulin M or vesicular stomatitis virus glycoprotein, which are both necessary and sufficient to halt translocation of proteins across the endoplasmatic reticulum,
do not halt translocation of proteins into chloroplasts
(Lubben et al., 1987
).
Our results (Figs. 3-6) clearly demonstrate that pS34Cinv is jointly imported by the Toc and the Tic complexes
and not via a Toc-independent pathway. The recognition
of the stop transfer signal must therefore occur within the
Toc complex. A fraction of processed iS34Cinv is still in a
proteinaceous environment, maybe in the pore component
Toc75. Most of the protein is resistant to extraction at pH
11.5, which is indicative for the insertion and interaction
with the lipid moiety of the membrane. Our results and
those obtained for preToc75 (Tranel and Keegstra, 1996)
indicate that the Tic complex harboring the precursor
polypeptide translocation intermediate does not influence
whether or not the Toc machinery can release a transmembrane segment into the lipid bilayer. Translocation arrest leaves the hydrophobic transmembrane segment in
the pore of the Toc complex, where it might have sufficient time to "sense" the lipid environment, which induces
the release into the bilayer. The mechanism of release of the
protein into the plane of the membrane is not known but
could be analogous to that proposed by Martoglio et al.
(1995)
and Liao et al. (1997)
(for review see Siegel, 1997
).
The partially processed polypeptide iS34Cinv remains
locked in the Tic complex in the presence of ATP while
the membrane anchor has already inserted into the lipid
bilayer of the outer membrane. Simultaneously, the Toc
complex becomes available for a new round of precursor
recognition and translocation initiation (Fig. 6). This indicates that in intact chloroplasts, the Toc complex can function independently of the Tic machinery, corroborating
earlier observations (Waegemann and Soll, 1991) that isolated purified outer envelope vesicles are also functional
in specific precursor recognition and partial translocation.
The ATP-dependent lock that keeps iS34Cinv in the Tic
complex could be represented by the action of hsp100,
which interacts on the stromal site with the Tic complex
(Akita et al., 1997; Nielsen et al., 1997
). The engagement
of the Tic complex by iS34Cinv leads to a block of these
translocation sites and to the inhibition of further import.
By the removal of ATP, the inhibition is released because
iS34Cinv retracts from the Tic complex and becomes exposed to the intermembrane space, indicating that no further ionic interactions keep the protein in the Tic complex.
The chloroplastic inner envelope contains an active ATP/
ADP carrier that might be responsible for the rapid drop
in chloroplastic ATP levels (Neuhaus et al., 1997
). iS34Cinv can probably not reengage the Tic machinery because it possesses only an incomplete import signal.
Intermediate processing of the presequence of SSU can
occur if the proper site is not available to the stromal-processing peptidase (Archer and Keegstra, 1993). About 120 amino acids are spaced between the authentic processing
site and the beginning of the membrane anchor domain.
This should suffice to guarantee faithful processing if both
the outer and inner envelope translocons were in close
proximity to each other during the entire import event.
Our data indicate that upon the release of the transmembrane segment into the plane of the membrane, the joint
translocation sites dissociate and clear away from each
other. This scenario would require a much longer spacing
between the outer envelope and the Tic complex, which
could explain our results (see also Fig. 5 C). It seems also
unlikely that iS34Cinv is inserted into the outer membrane
in a loop structure for two reasons: (a) iSSU34Cinv is resistant to the protease thermolysin, and (b) a loop structure would require a topology Ncyto-Cin, but we have demonstrated (Fig. 2) that mS34Cinv inserts in a topology Nin-Ccyto.
In the constructs pS34C and pS34Cinv, competition exists between two targeting signals, which could lead the polypeptides on alternative translocation pathways. Under experimental conditions that are optimized for import (2 mM ATP and 25°C), pS34C and pS34Cinv use preferentially the standard import pathway via the Toc and Tic complex. Removal of the presequence receptors from the chloroplast surface shifts pS34C and pS34Cinv to the outer envelope insertion pathway via the COOH-terminal hydrophobic region. Similarly, preincubation of chloroplasts under binding conditions (50 µM ATP at 4°C) for 10 min also results in a shift to the Toc-independent insertion pathway. While both pathways may operate simultaneously, our data establish that transport conditions can be selected that favor one over the other.
![]() |
Footnotes |
---|
Received for publication 6 August 1997 and in revised form 2 March 1998.
Address all correspondence to Prof. Dr. Jürgen Soll, Botanisches Institut, Am Botanischen Garten 1-9, Christian-Albrechts-Universität, D-24118 Kiel, Germany. Tel.: (049)-431-8804210. Fax: (049)-431-8804222. E-mail: jsoll{at}bot.uni-kiel.deThis work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
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Abbreviations used in this paper |
---|
chl, chlorophyll; inv, inverse; mS, small subunit of ribulose-1,5-bisphosphate carboxylase; pS, precursor form; Tic, translocon at the inner envelope membrane of chloroplasts; TIM, translocation intermediates; Toc, translocon at the outer envelope membrane of chloroplasts.
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