1 Department of Botany, Ludwig-Maximilian University Munich, 80368 Munich,
Germany
2 Department of Botany, University Kiel, 24098 Kiel, Germany
* Author for correspondence (e-mail: soll{at}uni-muenchen.de)
Accepted 25 November 2002
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Summary |
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Key words: Direct insertion, Protein-free liposomes, Chloroplast import
![]() |
Introduction |
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The OEP7 has a single transmembrane domain and an
Nin-Cout orientation
(Salomon et al., 1990;
Waegemann et al., 1992
). The
insertion of OEP7 is dependent on temperature, but independent of light and a
membrane potential (Salomon et al.,
1990
). The protein is able to bind to and insert into a
protein-free membrane. The topology of this protein is defined by positively
charged amino acids of the C-terminus flanking the transmembrane domain
(Lee et al., 2001
;
Schleiff et al., 2001
).
Additionally, it was shown that the topology of OEP7 is sensitive to the lipid
asymmetry of the outer envelope (Schleiff
et al., 2001
).
Toc34 also contains a single transmembrane domain but with a
Cin-Nout orientation
(Seedorf et al., 1995).
Insertion of Toc34 was found to be stimulated by ATP
(Li and Chen, 1997
;
Seedorf et al., 1995
;
Tsai et al., 1999
) and GTP
(Chen and Schnell, 1997
;
Tsai et al., 1999
). The
cytosolic region was suggested to influence the insertion given that partial
deletion resulted in the reduction of the insertion efficiency
(Li and Chen, 1997
). Two
positive charges flanking the transmembrane domain at the cytosolic site seem
to influence the orientation of Toc34 (May
and Soll, 1998
).
The outer envelope is a membrane with several unique and important
features. The outer envelope of chloroplasts contains a lower concentration of
phosphatidylcholine (PC) and a higher concentration of phosphatidylglycerol
(PG) in the inner than in the outer leaflet of the bilayer
(Dorne et al., 1985). Charged
lipids like PG were found to be important for association and insertion of
proteins into bilayers (van't Hof et al.,
1991
; van't Hof et al.,
1993
) because of electrostatic interaction with positively charged
amino acids or rejection of negatively charged amino acids. The outer envelope
is the only membrane facing the cytosol to contain the nonbilayer lipid
monogalactosyldiacylglyceride (MGDG)
(Bruce, 1998
). Nonbilayer
lipids are thought to play an important role in protein membrane interaction
and insertion. For example, MGDG stimulates the association of the transit
sequence of preferredoxin and pre-SSU (small subunit of rubisco) with lipid
surfaces (Chupin et al., 1994
;
Pilon et al., 1995
;
van't Hof et al., 1991
;
van't Hof et al., 1993
).
Phosphatidylethanolamine (PE), another nonbilayer lipid, was found to assist
protein folding of membrane proteins
(Bogdanov and Dowhan, 1998
;
Bogdanov et al., 1999
) and is
required for efficient protein transport across the plasma membrane of
Escherichia coli (Rietveld et
al., 1995
).
Toc34 is a subunit of a larger hetero-oligomeric translocation complex; therefore, binding and insertion into the membrane, as well as integration into the complex, probably represent distinct steps in the translocation pathway. To dissect this process we investigated insertional and topological constraints of Toc34 for translocation in chloroplasts and in a reconstituted protein-free liposome system. Our results show that Toc34 inserts into chloroplast outer envelopes even after inhibition of the translocation pore Toc75. Consistent with this observation is the ability of Toc34 to insert into liposomes. Interestingly, GTP also stimulates Toc34 insertion into protein-free liposomes. We suggest that the topology of Toc34 is partly determined and maintained by the size of the cytosolic domain. The positive-inside rule can be restored by deletion of the hydrophilic GTPase domain.
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Materials and Methods |
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|
Transcription and translation
Both coupled and uncoupled transcription and translation was used. The
uncoupled transcription translation is described elsewhere
(Schleiff et al., 2001). For
coupled transcription and translation the T7-TNT-Kit from Promega (Madison,
WI) was used. Proteins were synthesised in 50 µl containing 100 units
T7-polymerase, 25 µl reticulocyte-lysate, 2 µg DNA, 1 µl
RNase-inhibitor, 2 µl TNT-buffer and 2 µl amino acid mix without
methionine or leucine. The reaction mixture was supplemented with
[35S]-methionine (1000 Ci/mmol) or [3H]-leucine (148
Ci/mmol), respectively, and the reaction was carried out for 1 hour at
30°C. The translation mixture was centrifuged for 1 hour at 250,000
g at 4°C and the post ribosomal supernatant was used for
import.
Protein import into chloroplasts and mitochondria
Chloroplasts and mitochondria from garden pea were isolated by standard
procedures and further purified on Percoll gradients
(Schleiff et al., 2001;
Day et al., 1985
). Import into
mitochondria was carried out as described in
(Rudhe et al., 2002
). For
chloroplast use, chlorophyll concentration was determined to standardise
import results (Arnon, 1949
;
Mourioux and Douce, 1981
;
Schindler et al., 1987
).
Standard import into chloroplasts equivalent to 40 µg chlorophyll was
performed in 100 µl import buffer (10 mM methionine (or leucine), 20 mM
potassium gluconate, 10 mM NaHCO3, 3 mM MgSO4, 330 mM
sorbitol, 50 mM Hepes/KOH pH 7.6) containing 1-10% of in vitro translated
[35S]- or [3H]-labelled proteins. Import was initiated
by addition of organelles to import mixture and stopped after the times
indicated. Intact chloroplast were reisolated through a Percoll cushion (40%
Percoll in 330 mM sorbitol, 50 mM Hepes/KOH, pH 7.6), washed once in 330 mM
sorbitol, 50 mM Hepes/KOH, pH 7.6, 3 mM MgCl2, and used for further
treatments as described previously
(Schleiff et al., 2001
).
Liposome preparation and insertion experiments
Purified plant lipids were provided by Nutfield Nurseries (Surrey, UK).
Outer envelopes of chloroplasts from pea were purified as described
(Schleiff et al., 2001).
Liposomes with various lipid content (Table
1) were prepared as follows. The lipids were mixed in a glass tube
to yield a final concentration of 5 µmol total lipid content and dried
under N2-flow. Lipids were dissolved in 1 ml trichlormethane
followed by N2-drying and complete removal of the organic solvent
under vacuum for at least 3 hours. The created lipid film was either stored at
-80°C under argon or directly dissolved in buffer S (50 mM Hepes-KOH, pH
7.6, 0.2 M sucrose, degassed using N2) for synthesis of liposomes S
or in buffer N (50 mM Hepes-KOH, 125 mM NaCl, degassed) for the synthesis of
liposomes N. The solution was vortexed and freeze-thawed five times. The
multilamellar vesicles were extruded 21 times through a 100 nm pore
polycarbonate filter mounted in the mini-extruder (Liposofast, Armatis,
Mannheim, Germany) to give unilamellar liposomes
(MacDonald et al., 1991
). The
insertion of Toc34 and mutants into the liposomes was carried out as described
(Schleiff et al., 1999
;
Schleiff et al., 2001
).
|
Quantification and data presentation
The amount of imported or inserted protein was quantified by two different
methods. First, the SDS-page gel slice was dissolved in 30%
H2O2 and 60% HClO4 for 16 hours at 60°C
followed by cooling and 1:10 dilution into Rotiszint 22 eco scintillation
cocktail (Roth, Germany) and scintillation counting. Second, the radioactivity
was quantified using the Phospho-Image Reader FLA 5000 (Fuji-Film, Tokyo,
Japan) and quantified using Aida-Image Analyser (Raytest
Isotopenmessgeräte GmbH, Staubenhard, Germany). The radioactivity of the
proteins was normalised to the amount of labelled amino acids present in each
construct and in the 8 kDa fragment in order to normalise for the amount of
protein seen. Binding (B) and insertion (I) efficiency was quantified using
the results of one experiment as follows:
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![]() |
Results |
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Heterologously expressed proteins containing a typical transit sequence,
like preSSU, normally compete for translocation with other precursor proteins
that use the general translocation pathway
(Schleiff et al., 2001).
However, no sensitivity of Toc34 insertion in the presence of excess preSSU
could be observed as judged by the appearance of the 8 kDa fragment after
proteolysis, whereas translocation of preSSU was abolished
(Fig. 2A, lanes 4 and 5). We
also used spermine, a known inhibitor of the import channel Toc75
(Hinnah et al., 1997
). As
before, spermine inhibited import of preSSU
(Fig. 2A, lanes 2 and 6) but
had no influence on the insertion of Toc34 into the outer envelope of
chloroplasts, as judged by the appearance of the 8 kDa fragment after
thermolysin treatment (Fig. 2A,
lanes 6 and 7). A similar result was observed after treatment of the
chloroplasts with CuCl2 or spermidine (not shown), which both
inhibit the function of the Toc complex
(Hinnah et al., 1997
;
Seedorf and Soll, 1995
).
Together, the results suggest that pore forming and/or protease-sensitive
proteins (Seedorf et al.,
1995
) are not involved in the insertion process of Toc34. However,
from the current data it cannot be excluded that a protease-protected or
-resistant receptor for Toc34 might exist.
Toc34 does not insert into the outer membrane of mitochondria
When Toc34 was expressed in E. coli, insertion of the protein into
the inner membrane of the bacterium was observed (not shown). This raised the
question of whether Toc34 can insert into any available membrane or if
insertion is initiated by similarities of the lipid bilayers that is,
the existence of the nonbilayer lipid PE in the E. coli membrane. To
answer this question we used pea mitochondria, which do not contain nonbilayer
lipids, to study the insertion of Toc34. The import competence of the purified
mitochondria was supported by import and maturation of alternative oxidase
(AOX) (Fig. 2C, lane 2, lower
panel) and the protease resistance of the mature form
(Fig. 2C, lane3). When Toc34
was incubated with mitochondria, binding was observed
(Fig. 2C, lane 2, upper panel).
However, we did not observe insertion of Toc34 deduced from the absence of the
proteolytical 8 kDa fragment after thermolysin treatment of mitochondria
(Fig. 2C, lane 3). To confirm
this conclusion the mitochondria were incubated with sodium carbonate and
membranes were recovered. Toc34 was not observed in the pellet fraction
(Fig. 2C, lane 4), further
supporting our idea that Toc34 was not inserted into the membrane. Therefore,
we conclude that Toc34 does specifically insert into the outer envelope of
chloroplasts, but not into the outer membrane of mitochondria.
Toc34 inserts into protein-free membrane bilayer
Toc34 insertion seemed to be independent of the outer envelope
translocation machinery. Therefore, we wanted to determine whether Toc34 could
be inserted into a lipid bilayer directly. Liposomes with a lipid composition
comparable to the average composition of the outer envelope were incubated
with [3H]-labelled Toc34. Toc34 was inserted into protein-free
liposomes, as judged from the appearance of the 8 kDa fragment
(Fig. 3A, lanes 3 and 4). To
confirm that Toc34 was inserted into the bilayer, liposomes were extracted
with sodium carbonate before (Fig.
3A, lane 5) and after (Fig.
3, lane 6) thermolysin treatment. Both Toc34 and the 8 kDa
fragment were detectable in the membrane fraction, whereas the nonspecific 8
kDa product observed in the translation product was not. This is in line with
the notion that the 8 kDa product observed in the translation product did not
account for the 8 kDa observed after proteolysis. To prove that insertion of
Toc34 is specific and dependent on the transmembrane domain, liposomes were
incubated with Toc34 lacking the transmembrane region
(Fig. 3B, upper panel,
Toc34-252) and with Tic40 (Fig.
3B, lower panel). Both proteins were found to associate with the
membrane (Fig. 3B, lane 2);
however, after competition for nonspecific binding using N-liposomes
(Schleiff et al., 1999
;
Schleiff et al., 2001
), only a
small amount of Tic40 remained bound to the liposomes
(Fig. 3B, lane 3), and this was
rapidly degraded by the addition of thermolysin
(Fig. 3B, lane 4). We conclude
that neither Toc34 lacking the transmembrane domain nor Tic40 were inserted
into the membrane under the conditions used.
Quantification of the insertion (Materials and Methods) revealed that about 20% of the bound Toc34 was inserted (Fig. 3A, lanes 2 and 3; Fig. 4A, lanes 6 and 7; C3), as judged by the appearance of the 8 kDa fragment after protease treatment. This result was also achieved by using lipids purified from chloroplast outer envelopes (Fig. 4A, lanes 12 and 13). This 8 kDa transmembrane segment became protease accessible after membrane solubilisation (Fig. 4A, lane 14). The association or insertion was not altered when the concentration of the zwitterionic lipid PC was increased to 50 mol% (Fig. 4A, C4, lanes 8 and 9; Table 1). However, a decrease in the PC concentration to 16 mol% (Fig. 4A, C2, lanes 4 and 5) resulted in an increase of the association by about 25% and an increase of the insertion efficiency by twofold when compared with the association and insertion into liposomes of average lipid composition (Fig. 4A, C3, lanes 6 and 7). To test which of the other lipids most strongly influenced the association and insertion, liposomes containing a second nonbilayer lipid, namely PE, were used for insertion experiments (C1). Addition of 2 mol% (final concentration) of PE resulted in an increase of insertion of Toc34 (Fig. 4A, C1, lanes 2 and 3) comparable to the increase found using the lipid mixture C2. However, not only the nonbilayer lipid concentration was increased in C2, but also the content of anionic lipids. To verify that the insertion was dependent on the nonbilayer lipids, liposomes lacking PG were used to study Toc34 insertion (C5). Toc34 associated with PG-free liposomes with similar efficiency as with liposomes of average lipid composition (Fig. 4A, compare lanes 6 and 10), but the insertion efficiency increased by a factor of four (Fig. 4A, compare lanes 7 and 11) when compared with liposomes with the average lipid composition of the outer envelope.
|
From the results presented in Figs 2 and 4 we conclude that the insertion of Toc34 into the membrane occurs independently of channel proteins in vitro. Using synthetic protein-free liposomes we can clearly show that the insertion efficiency, but not the association of Toc34, is dependent on the presence of nonbilayer lipids (MGDG and PE) and on the concentration of anionic lipids (PG).
Insertion of Toc34 into protein-free liposomes is stimulated by
GTP
Insertion of Toc34 into the outer envelope of chloroplasts is stimulated by
ATP and GTP (Chen and Schnell,
1997; Tsai et al.,
1999
). This result was taken as indication for the existence of a
membrane-localised ATPase and GTPase involved in Toc34 insertion
(Tsai et al., 1999
). Because
our results show that Toc34 inserted into protein-free liposomes, we
investigated whether such a nucleotide effect also influences the insertion of
Toc34 into liposomes. Therefore, the insertion of Toc34 into protein-free
liposomes was carried out in the presence of different nucleotides. When GTP
was added before the addition of liposomes, the association of Toc34 with the
lipid surface increased, on average, by 60% compared with the association in
the absence of GTP (Fig. 4B,
lanes 1 and 3). The insertion efficiency in the presence of GTP increased by a
factor of four in comparison to the absence of GTP as determined by the
appearance of the 8 kDa fragment (Fig.
4B, lanes 2 and 4). The increase of insertion is dependent on GTP
binding but not on GTP hydrolysis, because addition of the non-hydrolysable
GMP-PNP (guanosine 5'[imido]triphosphate) also increased the insertion
of Toc34 (Fig. 4B, lanes 5 and
6). By contrast, the addition of GDP or ATP did not alter the association or
insertion of Toc34 significantly (Fig.
4B, lanes 7-10). We conclude that the stimulation by GTP on the
insertion of Toc34 is an intrinsic effect, e.g. accessibility of the
hydrophobic membrane anchor due to a GTP-dependent conformational change
rather than due to the presence of additional proteinaceous components in the
envelope membrane.
The hydrophilic domain imposes a second constraint on membrane
topology of Toc34
After establishing that Toc34 insertion does not require proteinaceous
components, we wanted to investigate the constraints on the topology of Toc34.
Previous work had established that charges flanking the transmembrane regions
form one constraint for the topology of outer envelope proteins
(Schleiff et al., 2001). We
therefore incubated [3H]-labelled Toc34 with an inverted charge
distribution flanking the transmembrane domain (Toc34C++) with chloroplasts.
We observed an 8 kDa fragment after thermolysin treatment, suggesting that
Toc34C++ is inserted with Cin-Nout orientation
(Fig. 2B, lanes 5 and 6). This
result seems to contradict our notion of the charge dependency for Toc34
topology. However, the topology could be dependent on the size of the
hydrophilic domain. Therefore, we created deletion mutants as shown in
Fig. 1. One polypeptide,
(2-230)Toc34, contained a large deletion of the N-terminal hydrophilic
domain. The second polypeptide,
(2-230)Toc34C++, contained the same
deletion and, in addition, a membrane domain with inverted charges (see
Fig. 1). Both proteins labelled
either with [3H] or [35S] were imported into
chloroplasts. Both mutants contain five leucines in the transmembrane domain
and a further four leucines in the adjacent parts (see
Fig. 1), but they contain only
an N-terminal methionine. This asymmetric labelling allows the orientation of
the inserted polypeptides to be determined
(Schleiff et al., 2001
). The
quantification of association and insertion of the [3H]-labelled
proteins revealed that the association efficiency of Toc34 and Toc34C++
(Fig. 5A, upper panel, binding)
was comparable. In addition, the insertion efficiency did not differ
significantly (Fig. 5A, upper
panel, insertion). When the truncated forms of Toc34 were used, we observed an
increased association of
(2-230)Toc34 to the chloroplast surface
(Fig. 5A, lower panel, binding
wt; Fig. 5B, upper panel, lane
2); however, the insertion yield remained similar
(Fig. 5A, lower panel,
insertion wt; Fig. 5B, upper
panel, lane 3) when compared with full-length Toc34. Although
(2-230)Toc34C++ revealed similar association with the chloroplast
surface as the full-length protein (Fig.
5A, lower panel, binding C++;
Fig. 5B, lower panel, lane 2),
the insertion efficiency was drastically reduced, as judged by the appearance
of the 8 kDa fragment (Fig. 5A, lower panel, insertion C++; Fig.
5B, lower panel, lane 3). Analysis of the
[35S]-labelled protein revealed that
(2-230)Toc34 was
sensitive to thermolysin treatment and therefore inserted into chloroplasts
with an Nout-Cin orientation
(Fig. 5B, lane 6). We did not
observe a significantly smaller proteolytic fragment for the
[3H]-labelled proteins, which might be due to the resolution
capacity of the gel system used. Therefore, we conclude that the size of the
hydrophilic region is one of the main constraining influences on the topology
of Toc34.
|
The topology of Toc34 and mutants after insertion into protein free
liposomes
To test whether the size of the hydrophilic region also influences the
topology of Toc34 within protein-free liposomes, the mutants described
(Fig. 1) were used for
insertion experiments. Toc34C++ had a reduced association compared with
wild-type Toc34 when liposomes with an average lipid composition of the outer
envelope were used (Fig. 6A,
upper panel, binding). However, all of the bound Toc34C++ was inserted as
determined by the appearance of the 8 kDa fragment after protease treatment
(Fig. 6A, upper panel,
insertion C++). By contrast, only one quarter of the associated Toc34 was
inserted into the bilayer (Fig.
6A, upper panel, insertion wt). The length deletion mutant with
the original charge distribution showed a slightly reduced association
(Fig. 6A, binding wt) but a
higher insertion efficiency (Fig.
6, insertion wt) when compared with the full-length protein. The
association of the length deletion with inverted charges was not reduced
compared with Toc34wt (Fig. 6A,
lower part, binding C++; Fig.
6B, lower panel, lane 2), but almost no insertion could be
observed (Fig. 6A, lower part,
insertion C++; Fig. 6B, lower
panel, lane 3). In addition, using [35S]-labelled mutant
polypeptides, we could not detect any proteolytically resistant fragment when
the truncated form of Toc34C++ was used
(Fig. 6B, lane 6). This
suggests that the insertion of this protein into liposomes of average lipid
composition occurs with Nout-Cin topology, unlike the
results observed using chloroplasts (Fig.
5B, lane 6).
|
The asymmetric distribution of PG between both leaflets of the outer
envelope (Dorne et al., 1985)
seems to be one of the most important determinants for the topology of OEP7
(Schleiff et al., 2001
).
Therefore, we tested whether the concentration of PG has an influence on the
insertion efficiency of Toc34 into protein-free membranes. The association and
insertion of Toc34C++ with liposomes lacking PG
(Fig. 7A, upper panel, binding
C++) was reduced compared with the association with liposomes containing PG
(Fig. 6A, upper panel, binding
C++). Toc34 inserted with higher efficiency than Toc34C++ into the liposomes
not containing PG (Fig. 7A,
upper panel, binding), which was comparable to the results seen for
chloroplasts (Fig. 5A). Both
truncated forms of Toc34,
(2-230)Toc34 and
(2-230)Toc34C++,
showed a reduced association and insertion efficiency compared with Toc34 when
liposomes lacking PG were used (Fig.
7A, lower panel; Fig.
7B, upper panel, lanes 2 and 3). However, the insertion of the
length deletion of Toc34C++ into liposomes without PG
(Fig. 7A, lower panel,
insertion C++; Fig. 7B, lower
panel, lane 2) increased compared with the insertion into liposomes of average
composition (Fig. 6A, lower
panel, insertion C++). Analysis of the translocation of the
[35S]-labelled proteins into liposomes not containing PG revealed
the same result as seen using chloroplasts. Only for the truncated version of
Toc34C++ was a protease-resistant form observed
(Fig. 7B, lane 6), indicating
that at least some of the protein had inserted in an
Nin-Cout orientation.
|
Together, our results indicate that three different factors influence the membrane topology of Toc34: first, the lipid asymmetry present between outer and inner leaflet of the outer envelope; second, the size of the cytosolic domain of Toc34; and third, the charge distribution flanking the transmembrane domain.
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Discussion |
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In line with earlier observations for isolated chloroplasts
(Chen and Schnell, 1997;
Tsai et al., 1999
), GTP
binding was also found to stimulate insertion into protein-free liposomes
(Fig. 4). This result supports
the hypothesis (Chen and Schnell,
1997
) that binding of GTP by Toc34 evokes a conformational change
rendering Toc34 more capable of insertion for example, by exposing the
hydrophobic transmembrane domain. ATP had no effect on the insertion of Toc34
into protein-free liposomes when the post-ribosomal supernatant was used
(Fig. 4). Therefore, we suggest
that the previously observed ATP effect is partly due to chaperones present in
the translation mixture and in the chloroplast preparation or to a conversion
of ATP to GTP by nucleoside diphosphate kinase present in chloroplasts
(Lübeck and Soll,
1995
).
In summary, our data suggest that proteinaceous components are not essential for the insertion of the transmembrane domain into the lipid bilayer of the outer envelope.
Lipid dependence of association and insertion of Toc34
The effect of lipids on the association and insertion of outer envelope
proteins has only recently received attention
(Schleiff et al., 2001;
Tu and Li, 2000
).
Investigation of insertion of OEP14 suggested that MGDG is not essential for
the insertion of outer envelope proteins
(Tu and Li, 2000
). However,
the result was obtained indirectly by treatment of chloroplasts with
duramycin. Duramycin induces aggregation of membrane vesicles containing PE or
MGDG (Navarro et al., 1985
)
and also induces artificial pore formation
(Sheth et al., 1992
). The mode
of interaction between duramycin and nonbilayer lipids is not clear yet and
the results are therefore rather difficult to interpret
(Tu and Li, 2000
). A more
direct analysis of OEP7 insertion, using a protein-free liposome system,
showed that the interaction is driven by the hydrophobicity of the
transmembrane domain and possibly by galactosyldiacylglycerides like MGDG
or/and sulfoquinovosyl-diacylgycerol
(Schleiff et al., 2001
). Here,
we show that the nonbilayer lipids MGDG and PE stimulate the association of
Toc34 to protein-free liposomes (Fig.
4). The insertion efficiency was enhanced when the nonbilayer
lipid content was increased (Fig.
4) or 2 mol% of PE was added to the lipid mixture. From that we
conclude that Toc34, like OEP7, associates with the surface of the organelle
by hydrophobic interaction. This is also consistent with the observation that
the free energy resulting from an association of the transmembrane segment
with the lipid surface is in the range of the energy found for OEP7
(Schleiff and Klösgen,
2001
). Furthermore, the insertion of Toc34, as well as of OEP7,
into protein-free liposomes was largely stimulated after depletion of PG
(Fig. 3). This supports our
hypothesis that the insertion of outer envelope proteins is dependent on the
lipid asymmetry present in the outer envelope
(Dorne et al., 1985
;
Schleiff et al., 2001
).
Constrains for the insertion efficiency and the topology of the outer
envelope protein Toc34
The charge distribution flanking the transmembrane domain has been shown,
using OEP7 as a model protein, to be one determinant of the topology of
proteins in the outer envelope of chloroplasts
(Schleiff et al., 2001). By
contrast, Toc34 inserted into chloroplasts and liposomes with an
Nout-Cin orientation, even after the reversal of the
charges flanking the transmembrane domain (Figs
2,
6 and
7). Only the complete deletion
of the cytosolic region resulted in a charge-sensitive topology (see Figs
5 and
7), which, in combination with
the lipid composition of the liposomes or the lipid asymmetry of the outer
envelope, results in the predicted topology. But the large hydrophilic domain
of Toc34 seems to represent an obstacle for the orientation of the
transmembrane anchor in such a way that it is energetically unfavourable to
translocate it across the lipid membrane. This suggests that the size of the
hydrophilic region represents a retention force, which overrules the
positive-inside rule.
Comparing the insertion of Toc34C++ into chloroplasts or liposomes without PG with its insertion into liposomes with PG clearly shows this. In all cases, the hydrophobic transmembrane domain inserts into the membrane with a Nout-Cin orientation. In the case of vesicles containing PG, the hydrophilic domain is exclusively retained on the membrane surface most probably because of electrostatic interaction with the charged polar lipids. In the case of chloroplasts or liposomes without PG, this electrostatic interaction is less strong and allows the translocation of the hydrophilic domain across the membrane, although only to a small extent.
In summary, we conclude that early steps in the targeting and insertion process of the chloroplast outer envelope protein Toc34 can be faithfully studied in a reconstituted system. The liposome system shows that all the determinants for targeting, insertion and topology are present in the primary sequence and tertiary structure of Toc34, as well as in the lipid composition of the target membrane. Whether any of these steps are facilitated or accelerated by outer envelope proteins remains to be seen.
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Acknowledgments |
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