Botanisches Institut, Universität Kiel, 24118 Kiel, Germany
The chloroplastic inner envelope protein of 110 kD (IEP110) is part of the protein import machinery in the pea. Different hybrid proteins were constructed to assess the import and sorting pathway of IEP110. The IEP110 precursor (pIEP110) uses the general import pathway into chloroplasts, as shown by the mutual exchange of presequences with the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSSU). Sorting information to the chloroplastic inner envelope is contained in an NH2-proximal part of mature IEP110 (110N). The NH2-terminus serves to anchor the protein into the membrane. Large COOH-terminal portions of this protein (80-90 kD) are exposed to the intermembrane space in situ. Successful sorting and integration of IEP110 and the derived constructs into the inner envelope are demonstrated by the inaccessability of processed mature protein to the protease thermolysin but accessibility to trypsin, i.e., the imported protein is exposed to the intermembrane space. A hybrid protein consisting of the transit sequence of SSU, the NH2-proximal part of mature IEP110, and mature SSU (tpSSU-110N-mSSU) is completely imported into the chloroplast stroma, from which it can be recovered as soluble, terminally processed 110NmSSU. The soluble 110N-mSSU then enters a reexport pathway, which results not only in the insertion of 110N-mSSU into the inner envelope membrane, but also in the extrusion of large portions of the protein into the intermembrane space. We conclude that chloroplasts possess a protein reexport machinery for IEPs in which soluble stromal components interact with a membrane-localized translocation machinery.
A central question in chloroplast biogenesis deals with
the mechanisms of intraorganellar sorting of nuclear-encoded plastid preproteins to the different
subcompartments. Chloroplasts are highly structured, and
six suborganellar locations can be distinguished; i.e., the
outer and inner envelope membranes (IEPs),1 the thylakoid membrane network, the intermembrane space between the two envelope membranes, the stroma, and the
thylakoid lumen. Nuclear-coded precursor proteins exist
for all these compartments, which use the general import
machineries that are present in both envelope membranes
(Tranel et al., 1995 The nuclear-coded chloroplastic IEPs identified to date
are synthesized in the cytosol with a stroma-targeting presequence (Flügge et al., 1989 We have recently identified the IEP of 110 kD (IEP110)
as a constituent of the chloroplastic protein import machinery (Kessler and Blobel, 1996 Chloroplast Protein Import Assays
Chloroplasts were isolated from the pea leaves of 10-12-d-old plants and
purified on silica sol gradients (Waegemann and Soll, 1991 Chloroplasts were treated with the protease thermolysin in 300 mM
sorbitol, 50 mM Hepes-KOH, pH 7.6, 0.5 mM CaCl2 for 20 min at 4°C at
200 µg protease/mg chl. The reaction was terminated by the addition of 10 mM EDTA, and chloroplasts were recovered by centrifugation through a
40% Percoll cushion and washed once (Joyard et al., 1983 After hypotonic lysis in 10 mM Hepes-KOH, pH 7.6, chloroplasts were
separated into a soluble stromal and a total membrane fraction by centrifugation for 10 min at 165,000 g. Soluble proteins were precipitated with
TCA. Import products were analyzed by SDS-PAGE (Laemmli, 1970 Construction of cDNAs Coding for IEP110 Hybrid
Proteins and Synthesis of Labeled Proteins
cDNAs coding for the different hybrid proteins were constructed by recombinant PCR (Higuchi, 1990 The role of IEP110 in protein import and its specific topology prompted us to study its import in some detail. The
protein sequence of IEP110 shows the potential to form a
hydrophobic
After import of the tp110-110N translation product into
intact pea chloroplasts under standard import conditions,
the terminally processed form was recovered almost exclusively in the envelope membrane fraction (Fig. 2 A). Processed 110N was resistant to extraction at pH 11.5 (0.1 M
Na2CO3), indicating a stable insertion into the membrane
similar to in situ (Lübeck et al., 1996
The COOH terminus of IEP110 that is exposed to the
intermembrane space in intact pea chloroplasts is inaccessible to the noninvasive protease thermolysin, but is degraded by trypsin, which proteolytically penetrates through
the outer envelope (Lübeck et al., 1996 The IEP110 Transit Peptide Contains Stromal
Targeting Information
Next, we wanted to know if the targeting information
present in the presequence of IEP110 is essential for translocation into the organelle and insertion into the inner envelope. Therefore, the presequences between 110N and
SSU were mutually exchanged, resulting in the constructs
tpSSU-110N and tp110-mSSU (see Fig. 1). Import experiments into pea chloroplasts clearly show that the transit
peptide of IEP110 directs the SSU passenger protein into
the soluble stroma phase, where it is processed and resistant to thermolysin and to trypsin treatment (Fig. 3 A,
lanes 2-7). The import efficiency of tp110-mSSU, however, is less than 50% of the authentic pSSU. The transit
peptide of SSU also directed the 110N domain of IEP110
to chloroplasts in the reverse construct (Fig. 3 B, lanes 2 and 3). The processed 110N was recovered in a thermolysin-insensitive but trypsin-sensitive manner, indicating its correct insertion and orientation in the inner envelope
membrane. We conclude that tp110 contains only stromaltargeting envelope transfer information, and that the topogenic signal for envelope targeting to the inner envelope
membrane and correct insertion is present in the 110N domain of IEP110.
Further evidence for this conclusion results from studies
that demonstrate that tp110-110N import is competed for
by the addition of an excess pSSU (Fig. 4 A). A similar
competition for the import yield can be observed between
the 35S-labeled pSSU translation product and an excess of
E. coli-expressed pSSU (Fig. 4 B). E. coli-expressed
pSSU was introduced into the import reaction as a ureadenatured protein. While a competition at the level of
translocation was always observed, the binding of reticulocyte lysate-synthesized translation products to the outer
envelope membrane is not competed for to the same extent (Fig. 4, A vs. B). This might be caused by a certain
amount of nonspecific binding to the chloroplast surface
that cannot be competed for by denatured (i.e., unfolded)
pSSU, which might bypass the primary binding site and
thus compete only at the level of translocation. The results
(Fig. 4, A and B), however, clearly demonstrate that pSSU
and tp110-110N use common components of the chloroplast import machinery.
Translocation across the envelope membranes is followed by processing in the stroma. The stromal processing
protease is a metalloenzyme and can be specifically inhibited by o-phenanthroline (Abad et al., 1989 Inner Envelope Targeting of IEP110.
As shown above, the 110N region contained all the sequence determinants necessary for targeting and insertion
into the inner envelope membrane. To distinguish domains within this region that could be involved in either
targeting or insertion, 110N was subdivided into N1, which
contained the most NH2-proximal amino acids of 110N,
and N2, which contained the COOH-proximal portion of
the 110N region (details are outlined in Fig. 1 A). The sequence of the N2 subdomain was expressed in frame with
tp110 or tpSSU, resulting in the hybrid proteins tp110110N2 and tpSSU-110N2. When import reactions were
performed with either tp110-110N2 or tpSSU-110N2, processed 110N2 was recovered exclusively in the soluble
stromal phase (Fig. 5, A and B, lanes 2 and 3), as was also
indicated by its resistance to either thermolysin or trypsin
treatment (Fig. 5, A and B, lanes 4-7). The results suggested that the 110N2 domain did not contain sufficient
targeting information for the inner envelope membrane.
The 110N1 domain, which also comprises the putative
membrane-anchoring To test this notion, tpSSU-110N-mSSU, which contained both the N1 and N2 subdomains, was synthesized in
reticulocyte lysate and used in a standard import experiment. Interestingly, processed, mature 110N-mSSU was
largely recovered in the membrane fraction (Fig. 5 D).
110N-mSSU was resistant to thermolysin but accessible to
trypsin, demonstrating that the hybrid protein attained an in situ-like membrane orientation; i.e., the COOH-proximal portion of 110N-mSSU is exposed to the intermembrane space. Between 10 and 20% of processed, mature
110N-mSSU was recovered as a soluble protein in a
trypsin-inaccessible location, i.e., in the stromal phase. Up
to this point, the data indicate that 110N1 contains the information that anchors the protein into the membrane but functions with low efficiency, while 110N2 in combination
with 110N1 strongly increases the yield of membraneinserted 110N, maybe because 110N2 contains essential
targeting or sorting information that only works in a cooperative way with 110N1.
Since processed, mature 110N-mSSU was detectable in
the membrane as well as in the soluble phase, the following question arose: does 110N-mSSU integration into the
inner envelope occur via a soluble intermediate, or is a significant portion of 110N-mSSU missorted to the stroma?
Therefore, 35S-labeled tpSSU-110N-mSSU was incubated
with intact chloroplasts in the presence of 3 mM ATP for
5 min at 0°C to allow binding but no translocation. Chloroplasts were separated from unbound precursors by centrifugation. Organelles were resuspended in 25°C tempered
fresh import buffer in the presence of 3 mM ATP, and were incubated further at 25°C for various times (Fig. 6, A
and B). At the earliest time point during the chase period
(30 s), a significant portion (20%) of the labeled protein
was recovered as membrane-bound precursor, while about
one third was found as soluble processed mature form in
the stroma. About 50% of the protein had already reached
the inner envelope membrane. Most of the chloroplastbound precursor was imported during the chase period,
and only ~5% of the total labeled protein remained on the chloroplast surface. Furthermore, the proportion of
soluble 110N-mSSU intermediate declined steadily and
was at the brink of detectability after 10 min. Simultaneously, the membrane-recovered portion of 110N-mSSU
increased to more than 80% of the initially introduced labeled proteins. Because of the removal of unbound precursor before the beginning of the chase period, we analyzed only the import and insertion of prebound molecules;
i.e., the degradation of soluble 110N-mSSU can be excluded because, otherwise, we should not be able to detect
an almost constant amount of radioactivity during the entire chase period (Fig. 6 B). In the attempts to observe
more of the soluble 110N-mSSU protein in the stroma, the chase period was initiated by resuspending the chloroplasts in ice-cold import buffer, followed by a slow warming period. In general, the results were similar to those described in Fig. 6, except that at the early time points, 30 s
and 2 min, bound tpSSU-110N-mSSU made up more than
50% of the total labeled protein, while processed 110NmSSU showed a 1:1 distribution between the stroma and
the inner envelope (data not shown). The final result of
the chase period was identical to that described in Fig. 6.
These data clearly demonstrate that a soluble stroma localized intermediate can be chased to a membrane-bound
location with an orientation that most of the protein is exposed to the intermembrane space (see also Fig. 4).
Our data demonstrate that chloroplasts have the capability to reexport proteins into the inner envelope after their
complete import into and processing in the stroma. This is
not only a membrane insertion process, but the translocation machinery involved can export a large polypeptide
chain across the membrane into the intermembrane space.
The SSU reporter peptide in 110N-mSSU has a length of
140 amino acids, which is completely translocated across
the inner envelope into the intermembrane space, as indicated by its complete sensitivity to trypsin. The components of an export machinery are unknown at the present
time, and it remains to be seen if it shares common constituents with the inner envelope import machinery. The
identification of a chloroplast-coded, inner envelope-
localized protein (Sasaki et al., 1993 tpSSU-110N-mSSU is translocated into the inner envelope after processing in the stroma as the mature form; i.e.,
the export signal is not present in the transit sequence, but
internal targeting information is necessary. This topogenic
signal is localized in amino acids 38-269, most likely as a
bipartite signal. The NH2-proximal part (N1, amino acids
38-149), which also contains the putative membrane anchor region of IEP110, is sufficient for targeting to the inner membrane and proper insertion, albeit with low efficiency. The N2 region of IEP110 (amino acids 150-269)
cannot direct proteins to the inner envelope, but remains
in the stroma upon import. Compartimentation of N1 or
N2 upon import is most likely to be independent of the
stroma-targeting signal used, since both tpSSU and tp110
contain only envelope transfer stroma-targeting information. In combination with the N1 region, N2 directs the
protein quantitatively to the inner envelope. We propose
that N1 contacts the membrane surface very early and that
N2 subsequently initiates the translocation process, e.g., as
a start transfer signal.
pIEP110 takes the general chloroplast import pathway,
as shown by the mutual exchange of transit sequences between pIEP110 and pSSU. tp110 seems to be less efficient
than tpSSU, which supports two- to threefold higher import yields. Simultaneously, more binding to the chloroplast surface is generally detected in constructs that contain tp110. The reason for this is unclear at the moment. Furthermore, the stromal processing peptidase seems to
also process pIEP110. Import into chloroplasts and integration into the inner envelope of pIEP110 and tpSSU110N are rapid processes, and a soluble intermediate was
beyond detectability. Only when we used tpSSU-110NmSSU was a soluble intermediate in the translocation
pathway clearly visible. While ~50% of mature 110NmSSU has already reached the inner envelope after a 30-s
chase period, the results in Fig. 5 clearly establish that soluble mature 110N-mSSU can be exported into and across
the membrane. Two reasons could explain these differences: (a) the passenger protein mSSU retards the integration and translocation process because its primary sequence or secondary structure are not well suited for the
export machinery; and (b) the mSSU part in 110N-mSSU
interacts transiently with its "natural" partners of the
pSSU import and assembly pathway, e.g., chaperonin 60 or the large subunit of ribulose-1,5-bisphosphate carboxylase. Independently of the reason for the kinetic differences in 110N-mSSU export, our results show very clearly
that the signal responsible for reexport to the inner envelope protein is present in 110N and is "stronger" than the
potential interaction of mSSU with stromal proteins. At
the moment, it cannot be excluded that a fraction of 110NmSSU is never released from the membrane before insertion,
indicating that parallel mechanisms that are used alternatively might exist, or that the currently available experimental approaches are not elaborate enough to resolve
this problem. Lowering the temperature during the critical
chase period did not result in a block of membrane insertion; only in a general slowdown of the entire process.
Therefore, specific inhibitors that block single steps in the
process are needed to describe the sequence of translocation and insertion in detail. However, our results demonstrate that a reexport machinery exists in the chloroplasts'
stroma that can cooperate faithfully with an inner envelope machinery. In the future, this soluble intermediate
will be used as a tool to search for the components of the
reexport pathway. The initial protein import experiments
of tpSSU-110N-mSSU in the presence of NaN3 or guanosine nucleotides did not show significant alterations in
the yield of inner envelope-localized 110N-mSSU (Lübeck,
J., and J. Soll, unpublished data).
Studies of sorting of proteins to the inner mitochondrial
membrane have suggested that the yeast cytochrome oxidase subunit Va uses a "stop-transfer" mechanism, i.e.,
one that never leaves the inner membrane, even during
processing by the matrix processing protease (Miller and
Cumsky, 1993; Brink et al., 1995
; Knight and Gray,
1995
; Hageman et al., 1990
; Grossmann et al., 1980
). An
envelope transfer stroma-targeting sequence as a cleavable NH2-terminal extension is the common denominator
for all of the preproteins (for review see Cline and Henry,
1996
; Lübeck et al., 1997
). Further transport into and
across the thylakoid membranes is accomplished by four
or five distinct pathways that involve (a) a chloroplast Sec
A homologue (Knott and Robinson, 1994
; Nohara et al.,
1995
; Berghöfer et al., 1995
; Robinson et al., 1994
; Yuan et
al., 1994
) and probably Sec Y and Sec E to complement the system (Laidler et al., 1995
); (b) a unique,
pH-dependent pathway whose components have not yet been identified (Cline et al., 1993
; Robinson et al., 1994
); (c) a SRP54
homologue-dependent integration of thylakoid proteins
(Franklin and Hoffman, 1993; Hoffman and Franklin,
1994; Yuan et al., 1993
); (d) a so-called spontaneous thylakoid insertion pathway (Michl et al., 1994
); and (e) a routing pathway that could involve a chloroplast homologue of
the NEM-sensitive factor (Huguency et al., 1995; Malhotra
et al., 1988
).
; Brink et al., 1995
; Knight
and Gray 1995
; for a review see Lübeck et al., 1997
). The
data indicate that the presequence of the inner envelope-
localized phosphate-triose-phosphate translocator is cleaved
off by the general stromal-processing peptidase (Brink et
al., 1995
; Knight and Gray, 1995
). The topogenic signal for
the integration into the inner envelope, however, seems to
be contained in the mature part of the protein. Since it was not possible to separate the translocation process of the
phosphate-triose-phosphate translocator from the inner
envelope insertion process, there remained the question of
how the insertion pathway took place, e.g., via a membrane-associated form or via a soluble stromal intermediate followed by a putative anchestral reexport pathway.
; Lübeck et al., 1996
).
IEP110 is anchored into the membrane by an NH2-terminal hydrophobic stretch of amino acids with the potential
to form an
helix. The topology of IEP110 can be assessed by treating intact chloroplasts with the protease trypsin. Trypsin penetrates the chloroplastic outer envelope and degrades proteins in the intermembrane space, as
well as those exposed on the outer surface of the inner envelope (Marshall et al., 1990
; Lübeck et al., 1996
), while
leaving the inner envelope membrane and the chloroplast
integrity intact. IEP110 is sensitive to trypsin, demonstrating that the majority of the protein, ~80-90 kD, is exposed
to the intermembrane space between the two envelopes (Lübeck et al., 1996
). This asymmetrical arrangement of
IEP110 might result in distinguishable import stages in
contrast to the phosphate-triose-phosphate translocator
that is deeply buried in the membrane (Flügge et al.,
1989
). The data presented in this paper reveal the existence of a reexport pathway that can direct proteins in a
posttranslocational manner from the stroma into the chloroplastic inner envelope independently of a cleavable NH2-terminal presequence. Furthermore, the export machinery is capable of translocating polypeptide domains
across the inner envelope into the intermembrane space.
Materials and Methods
). Chlorophyll
(chl) was determined as described (Arnon, 1949
). A standard import assay
contained chloroplasts equivalent to 30 µg chl in 100 µl import mix (330 mM sorbitol, 50 mM Hepes-KOH, pH 7.6, 3 mM MgSO4, 10 mM methionine, 10 mM cysteine, 20 mM potassium gluconate, 10 mM NaHCO3, 2%
(wt/vol) BSA, 3 mM ATP, and 1-10% of reticulocyte lysate in vitro-synthesized 35S-labeled precursor proteins. Import was carried out for 20 min at 25°C. Chloroplasts were reisolated from the import assay by centrifugation at 4°C through a Percoll cushion (40% [vol/vol] Percoll in 300 mM
sorbitol, 50 mM Hepes-KOH, pH 7.6), washed once in Hepes-sorbitol
(Waegemann and Soll, 1991
), and used for further treatments. Prebinding
of precursor proteins to chloroplasts was done under conditions identical
to those described above, except that all steps were carried out at 0°C.
; Cline et al.,
1984
). Chloroplasts (equivalent to 200 µg chl) were treated with 200 µg
trypsin (10700 N
-benzoyl-l-arginine ethyl estes U/mg from bovine pancrease) in 300 mM sorbitol, 50 mM Hepes-KOH, pH 8.0, and 0.1 mM
CaCl2 for 1 h at 20°C in a final volume of 200 µl. The reaction was stopped
by the addition of 1 mM PMSF and a fivefold excess of soybean trypsin inhibitor (Marshall et al., 1990
; Lübeck et al., 1996
). Chloroplasts were recovered and washed as described above. Protease inhibitors were present at all subsequent steps. Chlorophyll was determined (Arnon, 1949
), and
equal amounts of organelles were loaded onto the SDS-PAGE on a chl
basis.
)
followed by fluorography (Bonner and Laskey, 1974
). Separation of envelope and thylakoid membranes was achieved after hypotonic lysis of chloroplasts (see above) by centrifugation for 30 s at 2,000 g. The resulting pellet contained thylakoid membranes. The supernatant was once again
centrifuged for 10 min at 165,000 g. Soluble proteins were precipitated
with TCA and treated as described above. The pellet fraction, containing
a mixture of envelope and remaining thylakoid membranes, was resuspended in 100 µl Hepes-KOH, pH 7.6, and layered over a 300-µl sucrose cushion (1 M sucrose, 10 mM Hepes-KOH, pH 7.6). After centrifugation for 10 min at 165,000 g, thylakoid membranes had pelleted through the
cushion and were pooled with the above thylakoid fraction. The supernatant, including the sucrose cushion, was diluted 1:5 with 10 mM HepesKOH, pH 7.6, and envelope membranes were recovered by centrifugation
for 10 min at 165,000 g.
). 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 pIEP110 and pSSU have
been described (Lübeck et al., 1996
; Klein and Salvucci, 1992
). In vitro
transcription was done using either T7- or SP6-RNA polymerase, as outlined before (Salomon et al., 1990
). Proteins were synthesized in reticulocyte lysate in the presence of 35S-labeled methionine and cysteine (1175Ci/
mmol) for 1.5 h at 30°C (Salomon et al., 1990
). Overexpression of pSSU
was done in Escherichia coli BL21 (DE3) cells using the pET vector system (Novagen, Madison, WI) (Waegemann and Soll, 1995
). pSSU was isolated from inclusion bodies and solubilized in 8 M urea. The final urea concentration in the assays never exceeded 80 mM.
Results
helix in the NH2 terminus (amino acids
100-140, Fig. 1 B), which seems to anchor the protein into
the inner envelope membrane (Lübeck et al., 1996
;
Kessler et al., 1996). Initial results had indicated that
pIEP110 uses the general import pathway into chloroplasts; however, the translation and import efficiencies of
full-length precursor of IEP110 (pIEP110) were rather
low, so we decided to use a truncated version of pIEP110,
namely the transit peptide of 110 and the NH2-proximal
part of mature IEP110 (tp110-110N; Fig. 1 A), which comprises amino acids 1-269, to study its import characteristics.
Fig. 1.
(A) Peptide domain combination of various hybrid precursor proteins used in this study and their nomenclature. (AA,
amino acids; tp, transit peptide; 110N, amino acids 39-269 of
pIEP110; 110N1, amino acids 39-149 of IEP110; 110N2, amino
acids 150-269 of pIEP110). (B) Detailed charge distribution and
hydropathy analysis of tp110-110N.
[View Larger Versions of these Images (34 + 28K GIF file)]
). No 110N was detectable in the soluble protein fraction of chloroplasts, and
only very little was detectable in the thylakoid membrane fraction (Fig. 2 A, lane 5). Under thylakoid isolation procedures such as those used here, these were shown to be
contaminated with envelope membranes (Keegstra and
Youssif, 1986
; Joyard et al., 1991
). Therefore, we conclude
that the low amount of 110N found in the thylakoid fraction results from contamination with envelope membranes.
Fig. 2.
Inner envelope-
targeting and insertion information is contained in the
NH2 terminus of IEP110. A
truncated IEP110 precursor, namely tp110-110N, comprising amino acids 1-269, was
used in standard import experiments with purified pea
chloroplasts (equivalent to
30 µg chl; A and B). (A)
Chloroplasts were separated
into different fractions containing either envelope membranes (Env), soluble stroma
(St), and thylakoid membranes (Thy). Membranes
were subsequently extracted
with Na2CO3 at pH 11.5 and
further separated into a pellet (P) and soluble fraction
(S). (B) Inserted, terminally processed 110N is sensitive
to the protease trypsin but
not to thermolysin. After completion of a standard import reaction, chloroplasts were either not treated (lanes 2 and 3) or
treated with protease (thermolysin [Th], lanes 4 and 5; trypsin
[Try], lanes 6 and 7). Organelles were reisolated through a 40%
(vol/vol) Percoll cushion washed once. Chlorophyll concentration was determined, and equal amounts of organelles were used
for further analysis. Organelles were separated into a soluble
stroma (St) and membrane (M) fraction. (C) pSSU is imported
into the chloroplast stroma, where the processed mature form is
resistant to both proteases, thermolysin and trypsin. All experimental conditions are as decribed in B. 35S-labeled translation
product (TL) is shown as an internal standard, 10% of which was
added to a standard import reaction. Fluorograms are shown.
[View Larger Version of this Image (30K GIF file)]
). Imported mature
110N was recovered in a thermolysin-inaccessible but trypsin-accessible manner (Fig. 2 B), giving further evidence that tp110-110N is correctly translocated, processed, and
inserted into the inner envelope membrane. Control import experiments (Fig. 2 C) using the precursor of the
small subunit of ribulose-1,5-bisphosphate carboxylase
(pSSU), a protein localized in the stroma, show that imported mature SSU is inaccessible to trypsin. These results indicate that the chloroplastic inner envelope was not penetrated by trypsin under these experimental conditions,
corroborating earlier results that used the latency of the
Hill reaction to measure chloroplast intactness after
trypsin treatment (Lübeck et al., 1996
). We conclude that
tp110-110N can be used as a bona fide substrate to study
the import of pIEP110 into pea chloroplasts, since both
full-length and truncated precursors are targeted to the inner envelope membrane and exhibit identical behavior to extraction at pH 11.5 and sensitivity to trypsin.
Fig. 3.
The presequence
of IEP110 contains only
stroma-targeting information. The presequence of
IEP110 and SSU were mutually exchanged, and the resulting hybrids, tp110-mSSU
and tpSSU-110N, were used under standard chloroplast
import conditions. (A) Chloroplasts were separated into
a soluble stroma phase and a
total membrane fraction either before or after treatment with thermolysin or trypsin. (B) The stroma-directing presequence of SSU (tpSSU) cannot deviate 110N to the stroma.
Import and subfractions of chloroplasts are as decribed in A. All
further experimental conditions and abbreviations are as decribed in Fig. 2.
[View Larger Version of this Image (35K GIF file)]
Fig. 4.
IEP110 uses the
general import and processing components of chloroplasts. (A) tp110-110N import into pea chloroplasts
was assayed in the absence or
presence of increasing pSSU
concentration. pSSUex was
synthesized in E. coli and
added as a urea-denatured protein to the import assay
(final urea concentration 80 mM). Import experiments
were initiated by the addition
of organelles (equivalent to
30 µg chl). (B) E. coli-synthesized pSSUex competes the import of reticulocyte lysate made 35S-labeled pSSU
with similar efficiency as
tp110-110N. Experimental conditions are as desribed in
A. (C) Tp110-110N and
pSSU are both processed in vitro by a stromal extract. A stromal processing extract was incubated for either 0 min (lanes 1 and 4) or 90 min (lanes 2, 3 and 5, 6) with the respective translation product in the absence (lane 2 and 5) or presence (lane 3 and 6) of 10 mM 1,10 o-phenanthroline.
[View Larger Version of this Image (37K GIF file)]
). In vitro processing of pSSU and tp110-110N by a stromal extract demonstrated that both precursor proteins could be converted
to their respective mature forms (Fig. 4 C, lanes 1, 2, and 4,
5). Furthermore, the processing of both preproteins was
inhibited by o-phenanthroline (Fig. 4 C, lanes 3 and 6). Together, the data presented in Fig. 4, A-C, strongly indicate
that pIEP110 uses the general import machinery and processing system of the majority of chloroplast-targeted preproteins.
Fig. 5.
The N1 and N2
subdomains of IEP110 are
both necessary for efficient
envelope targeting and insertion. (A and B) amino acids 150-269 of IEP110 were
fused to the presequence of
either IEP110 or SSU, resulting in tp110-110N2 and
tpSSU-110N2, respectively. 35S-labeled translation product was incubated with intact
pea chloroplasts (equivalent
to 30 µg chl), and the localization of processed, mature
110N2 was tested by chloroplast subfractionation before (lanes 2 and 3) or after treatment with the proteases thermolysin (lanes 4 and 5) or
trypsin (lanes 6 and 7). All
other experimental conditions are as decribed in Fig. 2. (C) The tp110-110N1-mSSU translation product is imported into pea chloroplasts. Chloroplasts were subfractionated into a soluble stroma phase (lanes 2, 4, and 6) and a membrane fraction (lanes 3, 5, and 7) either before (lanes 2 and 3) or after treatment of the organelles with the proteases thermolysin (lanes 4 and 5) or trypsin (lanes 6 and 7).
(D) The tpSSU-110N-mSSU translation product is imported into the chloroplasts under standard conditions. Chloroplasts are fractionated either before or after protease treatment, as outlined above. All experimental conditions and abbreviations are as outlined in Fig. 2.
[View Larger Version of this Image (29K GIF file)]
helix, could therefore be solely responsible for membrane targeting. A hybrid protein was
synthesized which consisted of tp110 fused to the N1 domain, which contained SSU as a COOH-terminal extension and putative reporter for intermembrane space location. When the tp110-110N1-mSSU translation product
was imported into chloroplasts, 110N1-mSSU was again
largely recovered in the soluble stromal phase. About 10%
of 110N1-mSSU was found in the membrane fraction (Fig.
5 C, lanes 2 and 3). Soluble mature 110N1-mSSU was resistant to the proteases thermolysin and trypsin. However, membrane-bound 110N1-mSSU was resistant to thermolysin but accessible to trypsin, i.e., 110N1 functions as the
correct topogenic signal, albeit with low efficiency. The efficiency of membrane targeting and insertion might be
strongly enhanced by the 110N2 domain.
Fig. 6.
110N-mSSU can be translocated into the inner envelope membrane via a soluble stromal intermediate. (A) 35S-labeled
tpSSU-110N-mSSU translation was incubated with intact chloroplasts (equivalent to 200 µg chl in 500 µl reaction volume) at 0°C
to allow binding but not import. After 5 min, the chloroplasts were reisolated through a 40% Percoll cushion, washed, and resuspended in fresh 25°C tempered import buffer. Aliquots
(equivalent to 30 µg chl) were taken at the time intervals indicated, and the translocation reaction was halted by the addition
of ice-cold import buffer. Chloroplasts were separated into soluble and total membrane fractions. Products were analysed by
SDS-PAGE and fluorography. (B) Quantification of the fluorogram shown in A. Different exposures of the x-ray film were
quantified using a laser densitometer. The sum of radioactivity
found in either the soluble or membrane fraction at 0.5 min was
set to 100%.
[View Larger Version of this Image (45K GIF file)]
Discussion
) had indicated previously that an independent transport pathway should exist for this membrane. No data exist as to whether this protein
translocates co- or posttranslationally into the inner envelope.
; see also Glick et al., 1992
). Sorting of subunit 9 of Fo-ATPase to the inner membrane of Neurospora mitochondria, however, was proposed to occur after complete translocation into the organelle via a conservative
sorting mechanism (Rojo et al., 1993). The soluble matrix
localization of processed mature subunit 9 of Fo-ATPase
was not analyzed (Rojo et al., 1993; see also Stuart and
Neupert, 1996
). The soluble intermediate we describe in
this study for the translocation of 110N-mSSU into the inner envelope might involve a sorting system that is not
present in mitochondria, e.g., a Sec, NSF, or SRP homologue-dependent pathway. More work is required to answer these questions related to chloroplastic biogenesis.
Received for publication 7 February 1997 and in revised form 24 March 1997.
Address all correspondence to Professor Jürgen Soll, Botanisches Institut, Christian-Albrechts-Universität, D-24118 Kiel, Germany. Tel.: (49) 431880-4210. Fax: (49) 431-880-1527. E-mail: jsoll{at}bot.uni-kiel.deThis work was supported by the Deutsche Forschungsgemeinschaft.
chl, chlorophyll; IEP, inner envelope protein; IEP110, chloroplastic IEP of 110 kD; m, mature form of; p, precursor of; SSU, small subunit of ribulose-1,5-bisphosphate carboxylase; tp, transit peptide of; 110N, NH2-proximal part of mature IEP110.
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