(Received for publication, March 27, 1995; and in revised form, May 2, 1995)
From the
The spinach triose phosphate/phosphate translocator and the
37-kDa protein are both integral components of the chloroplast inner
envelope membrane. They are synthesized in the cytosol with N-terminal
extensions, the transit peptides, that are different in structural
terms from those of imported stromal or thylakoid proteins. In order to
determine if these N-terminal extensions are essential for the correct
localization to the envelope membrane, they were linked to the mature
parts of thylakoid membrane proteins, the light-harvesting chlorophyll a/b binding protein and the CFII-subunit
of the thylakoid ATP synthase, respectively. In addition, the transit
peptide of the CF
II-subunit that contains signals for the
transport across both the envelope and the thylakoid membrane was fused
to the mature parts of both envelope membrane proteins. The chimeric
proteins were imported into isolated spinach chloroplasts, and the
intraorganellar routing of the proteins was analyzed. The results
obtained show that the N-terminal extensions of both envelope membrane
proteins possess a stroma-targeting function only and that the
information for the integration into the envelope membrane is contained
in the mature parts of the proteins. At least part of the integration
signal is provided by hydrophobic domains in the mature sequences since
the removal of such a hydrophobic segment from the 37-kDa protein leads
to missorting of the protein to the stroma and the thylakoid membrane.
The majority of the proteins found in higher plant chloroplasts are encoded in the nucleus, synthesized as higher molecular weight precursors in the cytosol, and post-translationally transported to their final destination (for recent reviews, see Flügge(1990), de Boer and Weisbeek(1991) and Soll and Alefsen(1993)). All chloroplast precursor proteins have to be bound to the organelle and, subsequently, either translocated across, or inserted into, the envelope membranes that surround the plastid. For the precursor proteins that are located to the interior of chloroplasts (i.e. stroma, thylakoid membrane, thylakoid lumen), it has been demonstrated that the information for targeting to the chloroplast and translocation across the envelope is present in N-terminal transit sequences (van den Broeck et al., 1985; Schreier et al., 1985). In some instances, they also contain information for the subsequent targeting to or across the thylakoid membrane (Hagemann et al., 1990; Clausmeyer et al., 1993; Ko and Cashmore, 1989).
Much less is known about the intraorganellar
sorting of proteins to the inner envelope membrane. In organello import studies on two prominent components of this compartment, i.e. the triose phosphate/phosphate translocator (TPT) ()and the 37-kDa protein, had revealed that the
post-translational import of these nuclear-encoded proteins involves,
similar to the import of stromal or thylakoid proteins, binding of
precursors to the outer envelope membrane, ATP-dependent translocation
into the organelle, and proteolytic removal of the transit peptide
(Flügge et al., 1989; Dreses-Werringloer et al., 1991; Willey et al., 1991).
According to
structure predictions, the transit sequences of the TPT and the 37-kDa
protein do, however, not possess the typical features of other
chloroplast proteins but tend to form a positively charged amphiphilic
-helix like mitochondrial presequences (Dreses-Werringloer et
al., 1991; Willey et al., 1991). It has been shown
recently that these preproteins have the ability to interact in
vitro with mitochondrial import receptors and to be imported into
mitochondria from fungi (Brink et al., 1994). Therefore, it
appears feasible that these transit peptides play a role in determining
the targeting to and integration into organellar membranes in general.
Alternatively, the transit peptides may function as
envelope-membrane-translocation signals, and the actual ``membrane
insertion'' signal may be contained in the respective mature
proteins as is the case for thylakoid integration of the
light-harvesting chlorophyll a/b binding protein
(Lamppa, 1988). It is evident that these signals must be different for
proteins of the envelope and the thylakoid membranes in order to ensure
correct intraorganellar sorting.
In the work presented, the function of the transit peptides and the mature parts of the TPT and the 37-kDa protein were characterized by gene fusion experiments. We have produced a series of chimeric proteins in which the transit peptides and mature parts of proteins of the envelope and thylakoid membranes were reciprocally exchanged and compared the import and intraorganellar routing of these chimeras with that of the corresponding authentic proteins.
Figure 1:
The transit peptide of the TPT
functions as a stroma-targeting signal. In vitro-synthesized S-labeled precursor proteins (TPT, TPT-LHCP) were imported into intact chloroplasts as described
under ``Materials and Methods.'' Before or after thermolysin
treatment (100 µg/ml in the presence of 2 mM CaCl
for 30 min at 0 °C; protease -, +), EGTA was added
(final concentration, 5 mM) and the chloroplasts were
reisolated. Thylakoids (T) and envelope membranes (E)
were isolated from the import assays, and aliquots were analyzed by
SDS-PAGE (see ``Materials and Methods'') and fluorography. In lanes (t), 0.2 µl of the translation mixture were loaded.
The positions of the precursor proteins (p) and of the mature
proteins (mTPT, mLHCP) are indicated by arrows.
To further check the targeting
function of transit sequences of envelope membrane proteins, N-terminal
parts of the 37-kDa inner envelope membrane protein were fused to the
mature parts of LHCP and TPT, respectively. Previous studies suggested
that the cleavage site within the 37-kDa protein is located between
amino acid residues 21 and 22 of the precursor (Dreses-Werringloer et al., 1991). The N-terminal 21 amino acid residues were
therefore used for the construction of the fusion proteins,
37-LHCP and 37
-TPT, respectively. Import of
the chimeric proteins into isolated chloroplasts and subsequent
treatment of the chloroplasts with protease revealed that the proteins
were only bound to, but not imported into, chloroplasts. In contrast to
the authentic 37-kDa protein, they were neither inserted into the
envelope membrane (Fig. 2A) nor imported into the other
suborganellar fractions (not shown). The first 21 amino acid residues
obviously do not contain sufficient information for the import of
proteins into chloroplasts which suggests that the actual transit
peptide of the 37-kDa protein may comprise more than the first 21 amino
acid residues that were used in these chimeras. This assumption was
confirmed by the analysis of a truncated 37-kDa protein lacking the
N-terminal 21 amino acid residues (but containing an additional start
methionine), ATG-
37. Fig. 2B shows that
(i) this protein was indeed larger than the mature 37-kDa protein (lanes 1 and 4) and (ii) it was processed in
vitro by a partially purified stromal processing protease to a
product that was identical in size not only to the in vitro processing product obtained with the authentic 37-kDa precursor
protein (lanes 2 and 4) but also to the native 37-kDa
protein isolated from envelope membranes (not shown). The functional
transit peptide of the 37-kDa protein thus comprises more than 21 amino
acid residues as previously thought (Dreses-Werringloer et
al., 1991). Three N-terminal parts of increasing length (48, 54,
and 60 amino acid residues) of the 37-kDa precursor protein were
therefore used in subsequent experiments as ``transit
peptides'' and fused to the LHCP mature protein.
Figure 2:
A, the N-terminal 21 amino acid residues
of the 37-kDa protein are not sufficient for translocation into
chloroplasts. In vitro-synthesized S-labeled
precursor proteins (37-kDa protein (37), 31
-TPT, 31
-LHCP) were imported into intact
chloroplasts as described under ``Materials and Methods.''
Before or after thermolysin treatment (protease -, +), the
envelope membranes were isolated from the import assays and analyzed by
SDS-PAGE and fluorography. The positions of the precursor proteins (p) and of the mature proteins (m) are indicated by arrows. B, in vitro processing of the N-terminally truncated
37-kDa protein (ATG-
37) and of the
37-kDa precursor protein by the stromal processing protease. Processing
of the in vitro-synthesized
S-labeled proteins by
a partially purified stromal processing protease was carried out as
described under ``Materials and Methods.'' At the times
indicated, the reactions were terminated by the addition of SDS-sample
buffer and analyzed by SDS-PAGE and fluorography. The position of the
mature 37-kDa protein is indicated by an arrow. C, N-terminal
parts of the 37-kDa protein contain stroma-targeting information.
Import of the 37-kDa precursor protein (37) and of the
chimeric proteins (37
-LHCP, 37
-LHCP, 37
-LHCP) into chloroplasts was carried
out as described under ``Materials and Methods.'' After
treatment of the chloroplasts with thermolysin (100 µg/ml for 30
min), envelope membranes (E) and thylakoids (T) were
isolated from the import assays and analyzed by SDS-PAGE and
fluorography. The positions of the mature 37-kDa protein (m37)
and of the mature LHCP protein (LHCP) are indicated by arrows.
Incubation of
these chimeric precursor proteins (37-LHCP,
37
-LHCP, 37
-LHCP) with isolated chloroplasts
demonstrated that each of the N-terminal extensions was able to
translocate the passenger protein, LHCP, into the organelle. As shown
in Fig. 2C, in all three instances the proteins
accumulated as processing products in the thylakoids, but not in the
envelope membrane. These results indicate that the transit peptide of
the 37-kDa protein, like that of TPT, is a stroma-targeting import
signal which is not sufficient to target a protein into the envelope
membrane. The processing products obtained showed slightly increasing
sizes for the hybrid proteins 37
-LHCP,
37
-LHCP, and 37
-LHCP, respectively (Fig. 2C), indicating that in all three instances
processing occurred most likely at the same cleavage site within the
common N-terminal 48 residues that were derived from the 37-kDa
precursor protein. Initial evaluation of the putative processing site
of the 37-kDa precursor protein had been performed by radiosequencing
of the in vitro-synthesized and processed protein since the
N-terminal amino acid residue was found to be blocked
(Dreses-Werringloer et al., 1991). Maximum values for the
radioactivity released per cycle were obtained for cycles 2 and 15.
Reinspection of the amino acid sequence of the protein revealed that
these data were also compatible with a cleavage site between amino acid
residues 46 and 47 (-Asn-Ser-Arg
Asn-Leu-Arg) with leucine
residues at amino acid positions 48 and 61. In conjunction with the
experiments shown below (see Fig. 3), we suggest that the
processing site of the 37-kDa protein is located between amino acid
positions 46 and 47. Unfortunately, determination of the molecular mass
of the mature protein by MALDI-TOF-MS, a procedure that would lead to
an independent estimation of the cleavage site, was unsuccessful so
far.
Figure 3:
A, 37-CF
II
accumulates in the thylakoid membrane after import into isolated
chloroplasts. Import of the authentic CF
II protein
(CF
II) and the chimeric 37
-CF
II
protein into chloroplasts was carried out as described under
``Materials and Methods.'' The chloroplasts were subsequently
treated with thermolysin and fractionated into the thylakoids (T), envelope membranes (E), and the stroma (S). The different fractions were analyzed by SDS-PAGE and
fluorography. In lanes (t), 0.2 µl of the translation
mixture were loaded. The position of the precursor proteins (p) and of the processed proteins (mCF
II, mCF
II*) are indicated by arrows.
B, 37
-CF
II is processed by the stromal
processing protease. In vitro processing experiments were
carried out with in vitro-synthesized
S-labeled
precursor proteins (authentic CF
II precursor and
37
-CF
II protein) as described under
``Materials and Methods.'' The reactions were terminated at
the times indicated (in minutes) by the addition of SDS-sample buffer
and analyzed by SDS-PAGE and fluorography. C,
37
-CF
II is correctly integrated into the
thylakoid membrane. In vitro-synthesized
S-labeled precursor proteins (CF
II,
37
-CF
II) were imported into intact isolated
chloroplasts as described under ``Materials and Methods.''
The chloroplasts were osmotically lysed, and the thylakoid membranes
obtained were incubated without or in the presence of thermolysin (100
µg/ml for 30 min; protease -, +). All samples were
subsequently analyzed by SDS-PAGE (10-20% polyacrylamide) and
fluorography. The positions of the precursor proteins (p) and
mature proteins (m), as well as the specific
protease-protected degradation products (m*), are indicated by arrows.
To test if the chimeric protein was
properly integrated into the membrane, thylakoids were treated with
protease after the import reaction. In the case of the authentic
CFII protein, this treatment led to a degradation of the
stroma-exposed part and resulted in the appearance of a specific and
indicative product of
3 kDa that represents the membrane span and
the lumen-located N terminus of the protein (m*, Fig. 3C).
After import of the chimeric
37
-CF
II protein, protease treatment of the
thylakoids yielded a degradation product of
4.5 kDa (m*, Fig. 3C). The size of this degradation product
correlates well with the
3-kDa product observed for the native
CF
II protein plus the additional 14 amino acid residues
from the 37-kDa protein remaining at the N terminus of CF
II
after processing suggesting that the chimera was correctly integrated
into the thylakoid membrane.
The correct integration of
37-CF
II into the thylakoid membrane is
remarkable in two respects. First, it confirms that the N-terminal
region of the 37-kDa precursor protein is capable of importing
hydrophobic passenger proteins into the chloroplast, and, second, it
shows that it can functionally replace the bipartite transit peptide of
the CF
II protein. Since this N-terminal segment does not
have the typical structure of a bipartite transit peptide (von Heijne et al., 1989), it appears unlikely that it has
thylakoid-targeting properties. Instead, we assume that the residual 14
residues lead, for as yet unknown reasons, to the suspension of the
otherwise strict requirement for a transient hydrophobic domain during
membrane integration of CF
II.
This phenomenon
is currently under investigation.
Incubation of
isolated, intact spinach chloroplasts with CFII-TPT showed
that the protein accumulated exclusively as the unprocessed precursor
in the organelles (Fig. 4A). This indicates that
CF
II-TPT has probably not reached the thylakoids, because
the transit peptide of CF
II can only be removed by
thylakoid processing protease (Michl et al., 1994). This was
confirmed by fractionation of the chloroplasts after the import
reaction which showed that the hybrid protein was found in exactly the
same fractions as the authentic TPT which was analyzed in parallel.
Both proteins were apparently quantitatively integrated into the
envelope membrane, in contrast to CF
II which was found
exclusively in the thylakoids after import (Fig. 4A).
Thus, in spite of the thylakoid-targeting transit peptide at its N
terminus, CF
II-TPT was efficiently integrated into the
envelope membrane, proving that mature TPT carries all the information
necessary for its envelope membrane-specific insertion.
Figure 4:
A, the mature TPT contains information for
membrane integration. In vitro-synthesized S-labeled precursor proteins (CF
II, CF
II-TPT, TPT) were imported
into intact chloroplasts as described under ``Materials and
Methods.'' After the import reaction, the chloroplasts were
treated with thermolysin (100 µg/ml) for 30 min at 0 °C. Stroma (S), thylakoids (T), and envelope membranes (E) were isolated from the import assays and analyzed by
SDS-PAGE and fluorography. The positions of the precursor protein (pCF
II-TPT) and of the mature proteins (mCF
II, mTPT) are indicated by arrows. B, The mature 37-kDa protein contains information for
membrane integration. Import of the authentic 37-kDa protein (37) and of the chimeric CF
II-
37
protein into chloroplasts was carried out as described under
``Materials and Methods.'' After treatment of the
chloroplasts with thermolysin (100 µg/ml for 30 min), the
chloroplasts were fractionated into thylakoids (T), envelope
membranes (E), and the stroma (S), and the different
fractions were analyzed by SDS-PAGE and fluorography. In lanes
(t), 0.2 µl of the translation mixture were loaded. The
position of the mature 37-kDa protein (m37) is indicated by an arrow. C, CF
II-
37 is integrated into
the envelope membrane. In vitro-synthesized
S-labeled precursor proteins (37-kDa protein (37), CF
II-
37) were
imported in intact chloroplasts as described under ``Materials and
Methods.'' The chloroplasts were subsequently treated with
thermolysin (100 µg/ml for 30 min). Envelope membranes were
isolated from the import assays and were treated either with 100 mM Na
CO
(pH 11.5) or 100 mM NaOH as
described under ``Materials and Methods.'' The membrane
pellets (p) and the supernatants (s) were
subsequently analyzed by SDS-PAGE and fluorography. D,
CF
II-
37 is processed by the stromal processing
protease. In vitro-synthesized
S-labeled
precursor proteins (CF
II, CF
II-
37, TPT) were imported into intact chloroplasts as described under
``Materials and Methods'' in the absence(-) or presence
(+) of 2 mM EDTA. After the import reaction, chloroplasts
were treated with thermolysin (100 µg/ml) for 30 min at 0 °C.
Thylakoids (T) or envelope membranes (E),
respectively, were subsequently isolated from the import assays and
analyzed by SDS-PAGE and fluorography. The positions of the precursor
proteins (p) and mature proteins (m) are indicated by arrows.
Analogously,
we studied the translocation of CFII-
37 into
chloroplasts. As shown in Fig. 4B,
CF
II-
37 was processed to the 37-kDa mature
protein and, like the authentic 37-kDa precursor protein, targeted
exclusively to the envelope membrane. Thus, similar to
CF
II-TPT, the imported protein reached its correct
destination even if the import was mediated by the thylakoid-targeting
transit peptide of CF
II. The protein was apparently
correctly integrated into the envelope membrane, because it showed the
same characteristics upon treatment of the membranes with alkaline
reagents like the 37-kDa protein that was obtained from import of the
authentic precursor. Both proteins were almost unaffected by 0.1 M carbonate treatment (pH 11.5) and were partially (
70%)
extracted by 0.1 M NaOH (Fig. 4C). Such
treatments are known to extract soluble and peripheral proteins only,
while integral membrane proteins remain associated with the membrane
sheets (Fujiki et al., 1982). In contrast to the chimera
CF
II-TPT, CF
II-
37 was correctly
processed to the size of the mature polypeptide. According to the
results shown in Fig. 3B, this processing was probably
performed by stromal processing protease. This was confirmed by import
experiments in the presence of EDTA. Under these conditions, the
maturation of the authentic CF
II precursor protein (that is
cleaved by thylakoid processing protease only) was not affected due to
the cation independence of the protease (Musgrove et al.,
1989) (Fig. 4D). On the other hand, the processing of
both the TPT precursor protein and CF
II-
37 was
inhibited by EDTA to a similar extent, confirming that maturation of
CF
II-
37 by stromal processing protease occurs
at a processing site derived from the 37-kDa protein. Additional
support for this conclusion comes from the observation that the size of
this processed product was identical to that obtained with the
authentic 37-kDa precursor protein (see Fig. 4B).
Figure 5:
A, the C-terminal hydrophobic region of
the 37-kDa protein is required for targeting to the inner envelope
membrane. Import of the 37-kDa precursor protein (37) and of
the C-terminally truncated 37-kDa protein (37) into chloroplasts was carried out as
described under ``Materials and Methods.'' The chloroplasts
were subsequently treated with thermolysin (100 µg/ml for 30 min)
and fractionated into the stroma (S), the envelope membranes (E), and the thylakoids (T). The different fractions
were analyzed by SDS-PAGE and fluorography. B, the C-terminal
hydrophobic region of the 37-kDa protein anchors the protein in the
envelope membrane. Envelope membranes containing the imported 37-kDa
protein (37) or the C-terminally truncated 37-kDa protein
(37
) (see A) were treated either with 100
mM Na
CO
(pH 11.5) or 100 mM NaOH as described under ``Materials and Methods.'' The
membrane pellets (p) and the supernatants (s) were
subsequently analyzed by SDS-PAGE and
fluorography.
In this paper, we address the question of how two
nuclear-encoded inner envelope membrane proteins, the TPT and the
37-kDa protein, are targeted to the chloroplasts and which parts of the
precursors, transit peptides or mature polypeptides, carry the signals
that are responsible for the correct targeting to and into the inner
envelope membrane. The results presented show unambiguously that the
transit sequences of both envelope membrane proteins are able to import
LHCP as a passenger protein into the chloroplast stroma from where it
subsequently integrates into the thylakoid membrane. These
stroma-targeting properties of the two transit peptides are
particularly remarkable, because they do not possess the typical
features of transit sequences of higher plant proteins which are
destined to the stroma or the thylakoid membrane (von Heijne et
al., 1989), but instead show the potential to form an amphiphilic
-helix like is frequently found in mitochondrial presequences. We
have therefore proposed earlier that this structural element may serve
as a ``membrane targeting domain'' (Dreses-Werringloer et
al., 1991) and, indeed, both preproteins can efficiently be
targeted to, and inserted into, the inner membrane of mitochondria from
fungi in vitro (Brink et al., 1994). Although these
results support a role of amphiphilic
-helices in recognition of
preproteins by mitochondrial surface receptors, the chloroplast import
experiments presented here show clearly that, in spite of their
structural similarity to mitochondrial import sequences, the two
transit peptides only serve to target proteins to the chloroplast
stroma but not to the envelope membrane. It should be noted in this
context that not all transit peptides of chloroplast envelope proteins
necessarily have a structure that is similar to mitochondrial
presequences. The precursor of the 2-oxoglutarate/malate translocator,
an integral protein of the chloroplast envelope that is even more
hydrophobic than the TPT, is synthesized with a transit peptide that
closely resembles those of soluble chloroplast proteins (without an
amphiphilic
-helix) but is nevertheless correctly targeted to the
chloroplast envelope (Weber et al., 1995).
The second major
outcome of the experiments presented is the finding that the
information for the targeting of TPT and the 37-kDa protein to and into
the envelope membrane is located in the respective mature parts of the
two precursor proteins. Despite the presence of a thylakoid-targeting
presequence of CFII that had been attached to the mature
proteins, both proteins were exclusively targeted to the envelope
membrane. Thus, analogous to the findings for most integral thylakoid
proteins, these envelope proteins also integrate apparently independent
of their transit peptides via uncleaved targeting signals. A similar
conclusion has been drawn from experiments with the maize Bt1
protein, a putative metabolite translocator protein from maize
endosperm (Li et al., 1992). This protein can be imported into
chloroplasts in vitro, and LHCP, when fused to the transit
peptide of the Bt1 protein, was found associated with the
thylakoids. Likewise, after fusion to the stroma-targeting transit
peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase,
the mature Bt1 protein was found after import in the envelope
membrane. Although in this instance a putative envelope membrane
protein from non-green plastids was analyzed with chloroplasts, the
results obtained are in line with our observations that transit
peptides of envelope membrane proteins often have a stroma-targeting
function and that signals for integration of proteins into the inner
envelope membrane of chloroplasts reside in the mature parts of these
proteins.
The question arises by what mechanism the insertion of
these proteins into the inner envelope membrane is achieved. For
mitochondrial proteins, two models have been proposed, a
``conservative sorting pathway,'' i.e. the proteins
are first completely imported into the organelle and then redirected
into or across the membrane, and a ``nonconservative
(stop-transfer) pathway'' in which the proteins are arrested in
the membrane during the translocation process. A clear example for the
conservative pathway is the Rieske Fe-S protein of complex III of the
mitochondrial respiratory chain, a protein located on the outer surface
of the inner mitochondrial membrane. The precursor is imported into
mitochondria, processed to its mature-sized form which is then
re-translocated across the inner membrane (Hartl et al.,
1986). It is still a matter of controversy to what extent this
mechanism also applies for intermembrane sorting to other proteins,
cytochrome b and cytochrome c
. The hydrophobic segments contained in the
bipartite transit sequences of these proteins were supposed to serve as
stop-transfer signals resulting in the arrest of the translocated
protein in the membrane and its subsequent release (van Loon and
Schatz, 1987; Glick et al., 1992). Alternatively, these
sequences could act as topogenic signals for redirecting the (not
necessarily completely) imported protein across the inner mitochondrial
membrane (Hartl et al., 1986, 1987; Gruhler et al.,
1995).
The 37-kDa protein of the chloroplast envelope membrane
structurally resembles the subunit Va of the cytochrome c oxidase of the inner mitochondrial membrane. Both proteins contain
an organelle targeting presequence and a hydrophobic sorting sequence
at the C terminus. It has been proposed that, during import, a yet
unidentified component of the inner mitochondrial membrane binds to the
subunit Va of the cytochrome c oxidase sorting sequence to direct it to
the inner membrane (Gärtner et al., 1995).
We have removed the C-terminal hydrophobic segment of the 37-kDa
protein and analyzed the sorting of the truncated protein
(37). It turned out that the truncated protein was
indeed partially missorted to the stroma and the thylakoids, suggesting
an important role of this hydrophobic segment in the envelope-targeting
process. A fraction of the truncated protein was still found associated
with the envelope membranes after import indicating that removal of the
hydrophobic domain obviously did not cause the protein to be generally
soluble. However, alkaline treatment of the membranes showed that the
truncated protein was not correctly integrated into the envelope
membrane. It can be concluded that the terminal hydrophobic region of
the 37-kDa protein plays a crucial role also in its anchoring in the
membrane although additional signals within the mature protein appear
to be required for specific targeting to the envelope membrane.
Analogous to the mitochondrial subunit Va of the cytochrome c oxidase protein, we assume a direct insertion mechanism for the
chloroplast 37-kDa protein although the conservative sorting pathway
can not be excluded by our experiments.
Hydrophobic proteins of the inner mitochondrial membrane, e.g. the ADP/ATP translocator, appear to be inserted into the inner membrane by a ``stop-transfer''-type of import mechanism. Complete translocation of the protein is prevented due to interaction of sorting signals with the components of the import apparatus, and the arrested protein leaves the import site by lateral diffusion without passing the matrix (Hartl and Neupert, 1990; Wachter et al., 1992; Glick et al., 1992). This mechanism might also hold true for the envelope membrane integration of hydrophobic chloroplast proteins like the TPT. The question then arises how specific sorting of nuclear-encoded hydrophobic proteins to either the envelope or the thylakoid membrane is achieved. It appears from hydrophobicity analyses (Kyte and Doolittle, 1982) of plastidial membrane proteins that, in general, the most hydrophobic thylakoid membrane proteins are coded for in the plastom (e.g. subunits I, III, and IV of the thylakoid ATPase; Hennig and Herrmann(1986)), suggesting that a high hydrophobicity might inhibit envelope translocation. On the other hand, the 22-kDa protein of photosystem II possesses at least three hydrophobic membrane-spanning segments (Wedel et al., 1992) and shows a higher hydrophobicity compared with that of the 37-kDa envelope membrane protein, but is nevertheless transported across the envelope membrane. Thus, although highly hydrophobic membrane proteins such as the TPT or the 2-oxoglutarate/malate translocator might not easily be transported across the envelope membrane, the degree in hydrophobicity cannot be the only determinant for the correct sorting to the different membranes in the chloroplast. Work is now in progress to elucidate the additional signals within the mature proteins that are involved in the membrane specificity of protein targeting within chloroplasts.
This work is dedicated to Prof. Dr. Johannes Willenbrink on the occasion of his 65th birthday.