(Received for publication, August 22, 1994; and in revised form, December 12, 1994)
From the
In order to analyze the information content of a chloroplast transit sequence, we have constructed and analyzed by in vitro assays seven substitution and 20 deletion mutants of the ferredoxin transit sequence. The N-terminal part and the C-terminal part are important for targeting, and in addition the C-terminal region is required for processing. A third region is important for translocation but not for the initial interaction with the envelope. A fourth region is less essential for in vitro import. Purified precursors were tested for their ability to compete for the in vitro import of radiolabeled wild-type precursor, which confirmed the important role in chloroplast recognition of both the N- and the C-terminal domain of the transit sequence. Monolayer experiments showed that the N terminus was mainly involved in the insertion into mono-galactolipid-containing lipid surfaces whereas the C terminus mediates the recognition of negatively charged lipids. A sequence comparison to other transit sequences suggests that the domain structure of the ferredoxin transit sequence can be extended to these sequences and thus reveals a general structural design of transit sequences.
The majority of the chloroplast proteins are encoded in the nucleus. These proteins are synthesized as precursors in the cytosol and are subsequently imported. The molecular cloning of a large number of chloroplast precursors and gene fusion techniques, in combination with both in vitro and in vivo analysis of protein localization, have allowed delineation of organellar targeting from organellar routing signals (De Boer and Weisbeek, 1991). It is now established that proteins are post-translationally routed to the chloroplast stroma by virtue of a cleavable N-terminal transit sequence (Chua and Schmidt, 1979). During the chloroplast uptake process, precursors proceed through different stages in all of which the transit sequence fulfills a role. After recognition, precursors pass through an initial binding stage before they are translocated across the envelope (Cline et al., 1985) and finally are processed to their mature size in the stroma (Robinson and Ellis, 1984; Oblong and Lamppa, 1992). It can be envisaged that several components of the chloroplast import machinery that are involved in these steps serve as partners for putative recognition motifs present in the transit sequence. Several experimental approaches indicated the role of envelope proteins especially in the early stages of translocation (Cline et al., 1985; Theg et al., 1989; Olsen and Keegstra, 1992; Perry and Keegstra, 1994). On the other hand, the membrane lipids are thought to be involved in protein uptake by chloroplasts as well as other protein translocation processes (De Kruijff, 1994). It could be shown directly that an isolated peptide corresponding to the ferredoxin transit sequence inserts into a lipid surface, mimicking the outer membrane of the chloroplast (Van't Hof et al., 1993). The insertion affects the secondary structure of the targeting signal (Horniak et al., 1993).
At present, the vast number of transit sequences available (Von Heijne et al., 1991) has not given any clear ideas on how these peptides can serve as specific targeting signals enabling productive interaction of the precursor in all the steps of the import process. For this reason and also because of the limited experimental analysis aimed at this topic the motifs which serve as a basis for recognition are not yet understood, but several somewhat contradictory hypotheses have been put forward (Von Heijne and Nishikawa, 1991; Karlin-Neuman and Tobin, 1986; Keegstra, 1989). Despite the absence of an obvious common motif, chloroplast targeting is specific (De Boer and Weisbeek, 1991), and several competition studies have indicated the existence of one general pathway utilized by all of the precursors tested so far (Perry et al., 1991; Schnell et al., 1991; Cline et al., 1993). The lack of knowledge on the structure of transit sequences is contrasted by the notion that the topogenic sequences used in other protein translocation systems like secretory signal sequences (Gierasch, 1989) and mitochondrial presequences (Roise and Schatz, 1988) each have a structural design that allows specific motifs to be formed.
Thus
far, mainly the precursors for the small subunit of ribulose
bis-phosphate carboxylase (pSSU) ()and light-harvesting
chlorophyll a/b-binding protein (pLHCP) have been used to analyze the
information content of a transit sequence (de Boer and Weisbeek, 1991).
With respect to targeting, large deletions in both N and C terminus of
the pSSU transit sequence reduced the import efficiency (Reiss et
al., 1987, 1989). Alteration of specific residues near the
processing sites were shown to block processing while import was less
affected (Ostrem et al., 1989). Since only large deletions
were analyzed and since analysis was limited to the C terminus of the
transit sequence only, the full information content of transit
sequences is still quite obscure.
As a model protein to analyze the information content of the transit sequence, we have chosen the precursor of the chloroplast stroma protein ferredoxin (preFd). This protein is imported efficiently in vitro via the general import route without a need for cytosolic factors and thus directly recognizes the chloroplast (Pilon et al., 1992a). Furthermore, the folding properties of this protein have been studied. This revealed that the transit sequence and the mature part behave structurally independently (Pilon et al., 1992b), which provides an explanation for the fact that the ferredoxin transit sequence is functionally dominant in gene fusions as it directs other chloroplast proteins as well as foreign proteins into chloroplasts (Smeekens et al., 1986, 1987; De Boer et al., 1991; Hageman et al., 1990). A previously performed deletion analysis (Smeekens et al., 1989) was limited to the C terminus of the transit sequence and indicated an important role of this part of the sequence in recognition. We have extended the analysis to the full transit sequence and performed a further detailed in vitro analysis of the effects of the mutations, which indicates the existence of distinct domains functional in targeting, translocation, and processing, respectively.
Stromal fractions of chloroplasts after import experiments were
obtained by osmotic lysis of chloroplasts, essentially as described by
Smeekens et al.(1986). Specifically, the chloroplasts that had
been protease treated and reisolated through Percoll gradients were
pelleted and then lysed by osmotic shock in 20 mM Tris-HCl, pH
8.0 (150 µl/30 µg chlorophyll). An equal volume of 100 mM HEPES/KOH pH 8.0, 660 mM Sorbitol was added, and the
sample was subjected to centrifugation for 10 min at 12,000
revolutions/min. The supernatant fluid was used as the stromal
fraction, the pellet was washed in 50 mM HEPES/KOH pH 8.0, 330
mM Sorbitol, pelleted again, and used as the thylakoid
fraction which includes some of the envelope membranes. For analysis by
SDS-PAGE the stromal fraction was recentrifuged at 12,000
revolutions/min in an Eppendorf centrifuge to remove residual
membranes, the proteins in the supernatant were then concentrated by
precipitation in 80% ice-cold acetone, and dissolved in SDS-containing
sample buffer. Stromal extract for in vitro processing was
obtained by osmotic lysis of chloroplasts in 20 mM Tris-HCl,
pH 8.0, at a chlorophyll concentration of 1 mg/ml. After 1 min an equal
volume of 100 mM HEPES/KOH pH 8.0, 660 mM Sorbitol
was added, and the sample was centrifuged for 10 min at 12,000 g. The supernatant was used as the stromal fraction. Standard
incubations for in vitro processing experiments contained 2.5
µl of translation mixture, 20 µl of stromal extract, and 200
µg/ml antipain. The volume was adjusted to 30 µl by the
addition of 50 mM HEPES/KOH pH 8.0, 330 mM Sorbitol.
Samples were incubated for 1 h at 25 °C. Then an equal volume of
SDS-containing sample buffer was added, and samples were heated for 5
min at 95 °C. In the control experiments, the stromal extract was
mixed with SDS-containing sample buffer and heated prior to the
addition of translation mixture.
In search of putative recognition motifs, we first theoretically analyzed a set of transit sequences with known cleavage sites. As transit sequences differ greatly in length, it is difficult to align them. However, within one precursor species a good alignment of homologous transit sequences is often possible (Keegstra and Olsen, 1989). Indeed, the transit sequences of five higher plant ferredoxins are aligned as shown in Fig. 1A. If we assume that these precursors are all derived from one ferredoxin precursor in a common ancestor of higher plants (Margulis, 1970; Raven 1970), one can envisage that due to natural mutation and selection functionally important primary sequences will remain conserved among plant species while less important sequences will be more variable.
Figure 1: Transit sequence alignments. Conserved (*) and semi-conserved (.) residues are indicated. Single-letter code is used. Proline residues are printed in boldface. Positively charged residues in the C-terminal region are printed in italics. Sequences corresponding to the F/W-G/P-K/R motif are double underlined; sequences that fit only loosely are underlined once. The sequences used in A are: ferredoxin transit sequences from A. thaliana (ara), spinach (spi), Silene pratensis (sil), pea (pea), and maize (may); in B, transit sequences from the A. thaliana chloroplast proteins: acyl carrier protein (acyl), TufA protein (tufa), rubisco activase (ract), EPSP synthase (epsp), ferredoxin (ferr), small subunit of rubisco (ssur), plastocyanin (plas); light harvesting complex type 11 protein (lhcp); in panel C, transit sequences from pea: ELIP (elip), ferredoxin-NADP reductase (fnre), ribosomal protein P5Cl18 (ri18), plastocyanin (plas), small subunit of rubisco (ssur), ferredoxin (ferr), superoxide dismutase (sodm), heat-shock protein 21 (hspt), LHCP-II (lhcp), ribosomal protein cl24 (ri24), glutamine synthase (glns), ribosomal protein cl9 (ri09), and ribosomal protein cl25 (ri25).
The three regions that have been described by von Heijne et al.(1989) are obvious for the Silene pratensis preFd. At the N terminus the consensus sequence MA is present. The N-terminal region of 15 amino acids that is uncharged and the C-terminal region of the 10 residues in which several positively charged amino acids are present are easily located. A fit to the semiconserved processing site motif Ile/Val-X-Ala/Cys (Gavel and von Heijne, 1990) can be found at residues 44 and 46 in the S. pratensis sequence and in the corresponding sequence of the Arabidopsis thaliana protein. The C- and N-terminal regions are connected by the middle region which according to Von Heijne et al.(1989) is highly variable. However, next to the above mentioned regions, more motifs of primary sequence conservation can also be recognized among the ferredoxin transit sequences. In the middle region the helix-breaking residue proline is conserved at several positions in the sequence, and toward the end of the middle region a conserved consensus sequence, FGLK, is found in four out of five sequences. The latter motif is similar to a proposed framework block that was described previously by Karlin-Neuman and Tobin(1986) for the pSSU and pLHCP proteins.
We next checked whether the motifs which are quite obvious in the ferredoxin transit sequence are also found in other transit sequences (see Fig. 1, B and C). We chose to analyze the transit sequences from two plant species: the model plant A. thaliana and Pisum sativum which is the plant from which we isolate chloroplasts for in vitro studies. From both plant species many sequences with known cleavage site are available. Due to the differences in length the alignment is (as expected) very poor but still the now more loosely conserved motifs which were obvious in the preFd transit sequence can again be recognized in most of the sequences. Proline residues are present in the central region as is a sequence of 3-7 amino acids similar to the FGLK found in the ferredoxin precursors. The latter sequence can be described as containing the loose motif: aromatic residue, turn promoting residue, positive charge often spaced by 1 or 2 other residues similar to the block proposed by Karlin-Neuman and Tobin (1986).
In order to
analyze possible functions of the putative domains of the ferredoxin
transit sequence, we introduced new restriction sites for enzymes that
produce blunt ends. Together with these new restriction sites we
introduced some changes in the primary structure, thus generating seven
new substitution mutants. In our efforts to introduce new sites, we
were limited by the sequence as we allowed only similar amino acids to
be introduced. We made an exception for positions 57 and 58 in the
sequence (which is in the mature region of the protein) where 2 charged
residues were replaced by uncharged residues. The new restriction sites
were subsequently used to create 18 new deletion mutants covering the
full-length of the ferredoxin transit sequence region (see Fig. 2). The new mutants as well as two made previously by
Smeekens et al.(1989), t36 (12-47) and t7
(
41-47), were brought under the control of the T7 promoter
in vector pET-11d or in case of the two most N-terminal deletions
pBluescript (SK). These plasmids were subsequently used as templates
for in vitro transcription using T7 polymerase. The high
activity of the T7 polymerase allows the synthesis of large quantities
of capped mRNA and thus should allow a reproducible in vitro translation in a wheat-germ lysate in the presence of
[
H]leucine to generate radioactive precursor
proteins. For all the clones the translation of mRNA was efficient and
with comparable efficiency, except for two mutants
21-25 and
45-57 which were expressed poorly in vitro for
unknown reasons.
Figure 2: Substitution and deletion mutants of precursor ferredoxin. Single-letter code is used. In the substitution mutants the residues identical to the wild-type sequence are indicated by dots. Deletions are indicated by dashes. The arrow indicates the position of the processing site in the wild-type protein.
As some deletion mutants also carry a substitution, we first analyzed the effects of these amino acid changes on the import into isolated pea chloroplasts. All substitution mutants were correctly processed and were localized in the stroma (not shown) suggesting that none of the amino acids altered fulfills an essential role in these processes. As import of the wild-type precursor usually is complete within 20 min, we chose the 20 min time point to compare the import efficiencies of the substitution mutants quantitatively (see Fig. 3). Except for two mutants all the substitutions in the transit sequence did not significantly alter the import efficiency. Remarkably, mutant 33G/F34W/G35P was imported with a significantly better efficiency compared to wild-type preFd. The substitution mutant in the C terminus of the transit sequence G42S/V44W/T45C reduced the import efficiency to one-fourth of the wild-type level. Interestingly, this mutant does not match to the consensus sequence for cleavage sites any more (Gavel and von Heijne, 1990), but it is still processed correctly. The lower import efficiency of the latter substitution mutant thus has to be taken into account when analyzing the effects of deletions in these regions on the import efficiency.
Figure 3: In vitro import efficiency of wild-type and substitution mutants of prefd into isolated pea chloroplasts (20 min incubation time). Each bar is the average of three experiments with standard error.
We next
analyzed the chloroplast import characteristics and localization of the
deletion mutants in comparison with the wild-type precursor. For most
deletion mutants some import or association to the chloroplasts could
be detected (see Fig. 4for a selected set of proteins). To
compare the kinetics of import of the deletion mutants, we performed
time course experiments. Quantitative data on the time courses of
import of a set of deletion mutants selected from each region of the
transit sequence are shown in Fig. 5. It can be seen that the
reduction in import efficiency relative to the wild-type precursor
results from a reduced initial import rate for all of the deletions
investigated. For all the proteins, except one, the import rate is
initially fast and then levels off. Due to the similar shapes of the
curves the import efficiencies relative to the wild-type preFd are the
same whether the early time points or the 20-min time point are
considered. Only for mutant 43-45 the import seems to be
more linear with time. The latter observation might reflect a subtle
difference in the mechanism by which mutants in this particular region
are imported. As the comparison of the time courses of import indicated
that the 20-min time point is a proper incubation time to compare the
efficiency of import of most deletion mutants, we decided to use the
20-min time point to quantitatively compare the import efficiencies of
all the mutants (see Fig. 6for quantitative data). As none of
the deletion mutants can be imported with the wild-type efficiency, we
conclude that all the regions of the transit sequence are involved in
determining the overall efficiency of the import reaction. However,
depending on the position of the deletions in the sequence several
additional consequences can be observed as a result of the deletions.
We will next describe these effects as observed for each region of the
transit sequence.
Figure 4:
Chloroplast import and fractionation
experiments with deletion mutants. TM, translation mixtures:
20% of the amount added to each of lanes 1-4. Lanes
1, whole chloroplasts. Lanes 2, whole chloroplasts after
protease treatment. Lanes 3, stromal fraction. Lanes
4, membrane fractions. Samples were analyzed by SDS-PAGE and
fluorography. The exposure time was 3 days except for mutant
15-25 and
6-10, where the exposure time was 6
days.
Figure 5:
Time
course of import of several deletion mutants: quantitative data. Open squares, wild-type; open circles,
43-45; open triangles,
39-42; closed
squares,
35-38; closed circles,
26-34; closed triangles,
11-14. Data are
expressed as the amount of added precursor that was imported in the
indicated incubation time.
Figure 6: Import efficiencies relative to wild-type prefd of deletion mutants. Quantitative data of 20-min incubations. Each bar represents the average of at least three independent experiments with standard error.
Some deletions cause a severe defect such that no
import or association is observed (Fig. 6). Among these are the
two very long deletions 12-47 and
26-45 which
cover more than one putative region of the transit sequence.
The uncharged N terminus is especially important in determining the efficiency (Fig. 6). Two deletions in this region result in a complete loss of import. The first 5 amino acids cannot be missed. Two small deletions in the region from residue 6 to 14 still allow import into the stroma and correct processing (see Fig. 4), but the 9 amino acid deletion in this region completely blocks the import.
Deletion mutant 15-25 in the central region is a special
case. Under conditions that allow efficient import of the wild-type
precursor, about 4% of the added mutant precursor is found associated
to the outside of the chloroplast and can be degraded by thermolysin
indicating that it is only bound to the outer surface of the envelope.
Although this protein thus has a residual capacity to recognize the
chloroplast, it can apparently not be translocated. As the deletion of
residues 15-25 is quite large, we also created the smaller
deletion
15-21. This clone was constructed by duplicating
amino acids 11-14(ASLW) present in mutant
15-25. The
duplicated sequence is similar to residue 22-25 (ASSW) present in
substitution mutant L25W which behaves identical to the wild-type
preFd. The partial filling in of the gap resulted in a clone that was
poorly (approximately five times less efficient than wild-type preFd)
translated. With this protein a partial restoration of the import could
be observed as some protein became protected against protease (Fig. 6). However, a significant amount of precursor-sized
protein remained associated to the outer surface of the chloroplasts
(not shown). Deletions in the region from residue 15-25 thus seem
to effect the translocation across the envelope more than recognition.
Two relatively large deletion mutants in the second part of the
middle region, 26-32 and
26-34, are imported with
relatively good efficiency. Compared to these two larger deletions the
small deletion mutant
33-34 has a relatively large effect on
the import efficiency especially when compared to the parent
substitution mutant which imports with an efficiency better than
wild-type. The mutants with a deletion in this region partly disturb
the FGLK motif (see Fig. 2), but all are processed correctly
upon import (see Fig. 4for
26-34). Whereas for the
N-terminal half of the transit sequence a correlation is found between
the size of the deletion and the import efficiency, such a correlation
is not directly obvious for the more C-terminal half.
The analysis
of mutant 35-38,
35-42, and
39-42 is
not that straightforward, since the deletions are derived from
substitution mutant G42S/V44W/T45C that is itself imported at 25% of
the wild-type efficiency (Fig. 3). In view of the latter fact,
the deletion of residues 35-38 only seems to have a limited
effect on the import efficiency as it is still imported at a rate
comparable to the parent substitution mutant (compare Fig. 3and Fig. 6). The processing of
35-38 upon import,
however, is incomplete (see Fig. 4). The other deletions in the
positively charged C-terminal region effect the import efficiency and
especially processing. Interestingly,
43-45 is imported with
an efficiency which is intermediate between the wild-type level and
that of the substitution mutant from which it is derived. In contrast
to the substitution mutant, however, this deletion is not correctly
processed but instead is partially processed at a site which produces a
smaller than mature sized protein (see Fig. 4). The other
C-terminal deletion mutants
35-42,
39-42, and
41-47 are imported with a strongly reduced efficiency. They
become localized in the stroma but are not processed (see Fig. 4). The observed import of
41-47 is in contrast
to previously published data (Smeekens et al., 1989). We
explain this apparent discrepancy by the more optimized conditions
under which we perform our import experiments (Pilon et al.,
1992a). The import protocols used by Smeekens et al.(1989)
yielded lower import efficiencies too low to detect the import rate of
this mutant.
Import of mutant 43-57 which includes a
deletion of the last 5 amino acids of the transit sequence and the
first 10 amino acids of the mature sequence was barely detectable (Fig. 6).
46-57, which lacks only 2 amino acids of
the transit sequence, was also imported with a reduced efficiency.
During import this protein is also not processed (not shown).
Protein import into chloroplasts depends on protease-sensitive components (Cline et al., 1985). We found that relative to a mock incubation without protease the wild-type preFd import was reduced 5-10-fold by pretreatment (Cline et al., 1985) of chloroplasts with 200 µg/ml thermolysin for 30 min at 4 °C. The import efficiency of the imported deletion mutants was also reduced at least 5-fold by this treatment indicating that the imported deletion mutants use a similar import pathway (not shown).
As was shown in Fig. 4, several imported proteins were not or were incorrectly
processed. In order to be able to directly compare the sizes of the
imported proteins, we ran samples from different import reactions next
to each other on SDS-PAGE (not shown). This showed that the sizes of
the imported C-terminal deletion mutants correspond to their precursor
size. The lower band of 43-45 corresponds to a protein which
is smaller in size than the wild-type mature ferredoxin. The imported
N-terminal and middle region mutants are indistinguishable in size from
the wild-type mature protein.
Several deletion mutants were not
imported by the pea chloroplasts but still might posses an intact
processing enzyme recognition site. We therefore investigated whether
the processing site itself still can be recognized in an in vitro processing assay (see Fig. 7). The wild-type and T49S
substitution mutants were both processed specifically to the mature
size by the processing peptidase present in the stromal extract.
Exactly as observed in the import assays all the C-terminal deletion
mutants with a deletion closer to the processing site than residue 38
are not or (43-45) incorrectly processed. Interestingly, all
other deletion mutants are processed correctly, to the mature size, in
this in vitro assay. This also includes the N-terminal mutants
which are not imported. Since these proteins can be recognized by the
processing enzyme, we conclude that processing and import are not
strictly coupled. Only with mutant
15-21 processing seems
less efficient, but this might be due to the need to add five times
more translation mixture to this reaction because of the inefficient
translation of this protein.
Figure 7: In vitro processing experiment. Lanes 1, control samples; lanes 2, processing incubations. For the controls the translation mixtures of each mutant were added to preheated stromal extract mixed with SDS sample buffer and heated immediately at 95 °C for 5 min. For the processing incubations the translation mixtures were mixed with untreated stromal extract and incubated for 60 min at 25 °C after which SDS buffer was added, and samples were heated for 5 min. Radioactive protein bands are visualized by SDS-PAGE and fluorography. The arrows indicate the position of mature ferredoxin from S. pratensis (Pilon et al., 1992b).
The analysis of in vitro import so far has revealed several characteristic regions of the transit sequence involved in processing and the full import process. We next wanted to investigate whether the initial stages of import are affected by the mutations. It is now generally accepted that as a first step in import the formation of an early translocation intermediate (binding to the envelope surface) takes place (Cline et al., 1985). It has been shown that at 4 °C in darkness translocation intermediates are formed (Leheny and Theg, 1994). For the wild-type protein, the conditions we used in the binding assay (30 min at 4 °C in the dark) enable the reproducible formation of translocation intermediates. These proteins are fully susceptible to thermolysin degradation and have the precursor size (see Fig. 8, upper panel). Approximately 400 molecules are bound per chloroplast. The bound wild-type precursor can be chased to an imported and processed form (see Fig. 8, upper panel, lane 4). Binding is most reduced for the N- and C-terminal deletion mutants, whereas the two deletion mutants in the central part of the transit sequence are less affected. Deletion mutant 43-45 is relatively much more limited in its binding efficiency than in the overall import. This is rather surprising and suggests an important role of this region in early recognition whereas translocation is less effected.
Figure 8: Formation of early translocation intermediate (binding analysis). Upper panel, binding assay using wild-type prefd. TM, translation mixture: 10% of the amount added to each of lanes 1-4. Lanes 1 and 3, chloroplast pellets recovered from binding assays. Lane 2, same experiment as lane 1, but the recovered chloroplasts received thermolysin treatment prior to analysis by SDS-PAGE. Lane 4, same experiment as lane 3, but the recovered chloroplasts were incubated for 10 min under import conditions to chase the intermediate. Samples were analyzed by SDS-PAGE and fluorography. Lower panel, quantitative data of binding efficiencies relative to wild-type prefd of a set of deletion mutants. Each bar is the average of two experiments.
The in vitro import assays described so far define four distinct regions of the transit sequence. The first region consists of residue 1-14, and mutations in this region strongly effect the import efficiency, probably due to inefficient binding. The second region consists of residues 15-25 and is required for full translocation. The third region roughly consists of residues 26-38. Deletions in this region have a low effect on the in vitro import. Finally, the C-terminal residues are required for processing and binding although large deletions in this region still allow import to occur with low efficiency.
To further investigate
the role of each of these regions in the initial interaction with the
chloroplast, we made use of the possibility of overexpressing the
precursors in an E. coli strain which carries a copy of the T7
polymerase under control of an
isopropyl-1-thio--D-galactopyranoside-inducible promoter.
The proteins were expressed to different levels in E. coli,
but nevertheless all the deletion mutants accumulated in inclusion
bodies (not shown). The purification scheme worked out for the full
precursor could be used to purify all the deletion mutants to near
homogeneity, better than 95% pure, as judged from SDS-PAGE (see Fig. 9). Only in the case of mutant
26-34 a minor
contamination of an apparent molecular mass of 17 kDa remained present
throughout the purification procedure. We did not succeed in removing
this contamination.
Figure 9:
SDS-PAGE of purified precursor proteins. 2
µg of each protein was applied. The gels were stained with
Coomassie Brilliant Blue. The molecular masses of marker proteins are
indicated in the margin. The lane marked 35-43 should be marked
35-42.
The capability of the isolated precursors to
enter the import machinery was tested by an import competition assay
(see Fig. 10). These assays have been used successfully to
analyze the import pathway of several precursors (Cline et
al., 1993; Schnell et al., 1991; Perry and Keegstra,
1991). As a precursor enters the import machinery it can at a certain
stage occupy limiting components of the import machinery which at that
stage are not available for interaction with the radiolabeled wild-type
precursor obtained by in vitro translation in a wheat-germ
lysate. The addition of 0.5 µM of the purified full
precursor already severely reduced the import of the radiolabeled
precursor synthesized in vitro as evidenced by the much lower
intensity of the band seen on the auto radiograph (see Fig. 10A). Higher concentrations decreased the import
even further (see Fig. 10B for quantitative data). In
contrast, the presence of deletion mutants 6-14 and
41-47 even at the much higher concentrations of 2 and 4
µM had only a limited effect on the import of the in
vitro synthesized wild-type protein (see Fig. 10, A and B). This result suggests that these two mutant
proteins are no longer able to occupy limiting components of the import
machinery. In contrast the purified deletion mutants
15-25,
26-34, and
35-45 were nearly as efficient
competitors as the full precursor (see Fig. 10B)
suggesting that these proteins are capable of entering the import
machinery. Interestingly,
15-25 was itself not imported
although low amounts of the protein associated to the outside of the
chloroplast in import experiments (Fig. 4). The observed
competition thus suggests that this mutant lacks the information to
enter a later stage of the translocation pathway. In addition these
results suggest that the competition occurs at an early stage of the
import reaction.
Figure 10:
Import competition assay. The indicated
amount of unlabeled precursor was added to the import buffer, then 5
µl of translation mixture containing H-labeled prefd
(200,000 distintegrations/min obtained by in vitro translation) was added prior to the addition of isolated
chloroplasts (30 µg of chlorophyll in a final volume of 150
µl). Panel A, autoradiograph of a typical competition
experiment using the wild-type precursor and two deletion mutants which
were present in the amount (in µM) indicated above the
lanes. TM, 20% of the amount of precursor added to each of the
incubations. Panel B, quantitative data of import competition
experiments with the different precursors. Data are expressed relative
to control samples to which no competitor was added. Each data point
represents the average of two independent experiments. The precursor
proteins used for competition were: open circles, wild-type; closed circles,
6-14; open squares,
15-25; closed squares,
26-34; open
triangles,
35-42; closed triangles,
41-47.
It has been shown previously that the transit sequence can mediate the specific interaction of the precursor with lipids found in the chloroplast envelope membranes. Both negatively charged lipids like DOPG and the chloroplast-specific sulfolipid as well as the chloroplast-specific neutral mono-galactolipid were indicated to be important for this interaction (Van't Hof et al., 1993). In order to localize the regions of the transit sequence responsible for lipid recognition, we compared the insertion of the deletion mutants and wild-type precursor into lipid monolayers composed of different lipids. In such an experiment, small amounts of lipid molecules dissolved in chloroform-methanol are applied onto the surface of a buffer solution in a Teflon trough. Upon the evaporation of the organic solvents, the lipids will spontaneously orient at the air-water interface with the acyl chains oriented away from the buffer solution and the more polar head groups facing the aqueous subphase. In this way a mono-molecular lipid film mimicking one leaflet of a bilayer is formed. The lateral pressure is directly related to the packing density of molecules in the film and is measured on line. Different initial surface pressures can be obtained by controlling the amount of lipids spread. When proteins that are added to the subphase insert into the lipid layer this is measured as an increase in the surface pressure. At the limiting surface pressure the polypeptides will not be able to penetrate any more, and therefore the change in surface pressure will be zero.
The surface pressure increases ()
upon injection of protein underneath monolayers composed of lipids in
the total lipid extract of the chloroplast outer membrane were measured
at different initial surface pressures (
i) in the range of
18-36 mN/m for the precursors mentioned in Fig. 9. For all
the proteins the
decreased with increasing
i values in
a linear fashion. The data obtained for the insertion of the wild-type
precursor indicated a limiting pressure around 35 mN/m and a pressure
increase of 9.5 mN/m at an initial pressure of 18 mN/m. These data are
in agreement with previously published data (Van't Hof et
al., 1993). In order to compare the efficiency of insertion of all
the proteins, we determined from the
i/
curves the
pressure increase at an initial surface pressure of 28 mN/m. The
results are plotted in Fig. 11(solid bars). Relative
to the wild-type protein three deletion mutants insert less efficiently
into the total lipid extract. These are
6-14,
35-42, and
41-47. The loss of insertion is not
simply due to the shortening of the transit sequence by the deletion
because mutant
26-34 inserts with an efficiency comparable
to the wild-type protein. An even stronger interaction is seen with
15-25 which is the largest deletion studied here.
15-25 has a limiting pressure around 38 mN/m and causes a
pressure increase of 11.5 mN/m at an initial pressure of 18 mN/m (not
shown). In contrast, for the deletion mutants
6-14 and
41-47 a much lower limiting pressure of around 30 mN/m was
determined. This low limiting pressure means that they would not be
able to penetrate into a biological membrane in which surface pressures
between 30 and 35 mN/m are thought to exist (Demel et al.,
1975).
Figure 11:
Surface pressure increases caused by the
injection of the indicated different precursors underneath monolayers
composed of lipids present in the total lipid extract of the
chloroplast outer membrane, MGDG and DOPG. The surface pressure
increases at an initial surface pressure of 28 mN/m were determined
from i/
curves.
It has been shown previously that both the
chloroplast-specific neutral mono-galactolipid as well as lipids with a
negatively charged head group are important for the insertion of the
transit sequence (Van't Hof et al., 1993). We therefore
investigated whether the differences observed for the mutant proteins
can be contributed to alterations in the interactions with these
individual lipids with either a neutral sugar head group or a
negatively charged head group. To this aim we used chemically
synthesized MGDG and a DOPG species which are identical in their acyl
chain composition. The pressure increase due to insertion of the full
precursor in the chemically prepared MGDG corresponded nicely to the
pressure increases reported for the insertion of this protein into MGDG
isolated from pea chloroplasts (Van't Hof et al., 1993).
An interesting lipid specificity is seen for the deletion mutants (Fig. 11). The N-terminal deletion mutant 6-14 is
especially blocked in the ability to penetrate into the MGDG monolayer.
The most C-terminal deletion mutant
41-47 is mainly
disturbed in its capacity to interact with the negatively charged DOPG.
Mutant
35-42 has a reduced capacity to interact with both
lipids.
One way to establish whether electrostatic interactions or
hydrophobic interactions are dominant in determining the insertion into
the total lipid extract is by analyzing the effects of the inclusion of
high salt (400 mM NaCl) in the subphase. As the salt will mask
the negative charge of the anionic lipids, a reduced effect of
electrostatic interactions is expected. Indeed the insertion of both
wild-type and 41-47 into a monolayer of a negatively charged
lipid was observed (results not shown). In contrast to this, the
insertion of neither the wild-type precursor nor any of the mutant
proteins into a monolayer of the total lipid extract of the chloroplast
outer membrane was reduced by high salt. This result suggests that
hydrophobic interactions are dominant forces in determining the
insertion. A similar conclusion was reached for the interaction with
outer membrane lipids of peptides corresponding to different regions of
the transit sequence of the small subunit of ribulose bis-phosphate
carboxylase (pSSU) by Van't Hof et al.(1991).
As
penetration into a monolayer might involve the insertion of hydrophobic
domains, we analyzed the hydrophobicity profiles of the wild-type
precursor and of the five deletion mutants that have been used in the
monolayer studies (see Fig. 12). The mutant proteins were
aligned to the wild-type sequence at the processing site. Four
hydrophobic regions named A, C, D, and F and two hydrophillic regions B
and E are observed in the transit sequence of the full precursor. A
previously performed secondary structure prediction, according to Chou
and Fasman(1978), indicated the presence of five possible secondary
structure elements (Pilon et al., 1992b). A -strand was
found as the most likely structure for residue 3-14, coinciding
with hydrophobic region A. Two
-helices are predicted, residues
20-25 and 29-38, coinciding with hydrophobic regions C and
D. A
-turn with a high probability is predicted for residues
39-43 and coincides with the hydrophillic region E. Finally, the
processing region and beginning of the mature region was predicted as
-strand.
Figure 12: Hydrophobicity profiles of the transit sequences and first part of the mature protein of wild-type and mutant precursor ferredoxin proteins. The normalized consensus scale of Eisenberg et al.(1984) was used with a window length of 5 residues. The arrow indicates the position corresponding to the processing site.
Relative to the full precursor some structural changes
occur as a result of the deletions. In 6-14, which lacks the
capacity to recognize the chloroplast, the possible
-strand and
hydrophobic region A are removed. In
15-25, which is blocked
in its ability to fully translocate, hydrophillic region B is removed,
connecting regions A and C and thus resulting in a large N-terminal
hydrophobic domain that is predicted to be a
-strand. In
26-34, a deletion with reduced effects both import and on
lipid affinity, hydrophillic region D is removed. The secondary
structure prediction of the other regions is not affected by this
deletion. In
35-42, which is translocated with low
efficiency only but is not processed, both the hydrophobic region D and
a large part of the hydrophillic region E are removed. As a consequence
the potential turn corresponding to residue 39-43 of the
wild-type sequence is lost. The latter secondary structure element is
also lost for
41-47. In this mutant, which is particularly
blocked in its ability to recognize the chloroplast and is also not
processed, the hydrophillic region E was removed.
In view of the large variation in length of transit sequences in general, it might be assumed that these sequences contain several largely redundant sequence parts. However, our mutational analysis of the ferredoxin transit sequence, which with its length of 47 amino acids is quite a typical transit sequence, reveals a surprisingly high information content with respect to in vitro import. The whole sequence is important as most deletions interfere with the overall efficiency of import. In addition, the deletion analysis indicates the presence within the transit sequence of four distinct functional domains which each are more involved in a specific aspect of the import reaction. As will be discussed below, the functional domains coincide with four distinguishable parts of the primary sequence. A sequence comparison to transit sequences of other proteins suggests that the domain structure of the ferredoxin transit sequence can be extended to these sequences and thus reveals a general structural design of transit sequences.
The first domain of the preFd transit sequence comprises the N-terminal 14 amino acids. Deletions in this region strongly effect the import and initial binding but have no effect on processing. The N-terminal region thus seems to contain a special function in providing targeting to the correct membrane. The lack of competition observed using peptide analogs of the N-terminal region of either the ferredoxin or pSSU transit sequence seemed to indicate little function for the N-terminal region (Schnell et al., 1991; Perry et al., 1991). In contrast, the data presented in this paper clearly indicate a function for the N terminus. We assume that the N terminus although it is important cannot mediate recognition on its own; in other words, other parts of the transit sequence are required for functioning of the N terminus. The N-terminal region of the preFd transit sequence contains no residues with charged side chains. All transit sequences have such an uncharged N terminus which is enriched in amino acids with hydroxylated side chains (Von Heijne et al., 1989). Only the sequence of the first amino acids, MA, is well conserved among homologous ferredoxins and is thought to signal the removal of the initiator methionine in the cytosol (Flinta et al., 1986). This sequence is restored after deletion of residue 1-5 which resulted in a complete loss of import. The rest of the N terminus, especially from residue 4-14, is highly variable (Fig. 1). Several substitutions of residues by amino acids with similar properties: T to A, L to W, and S to C in this region have no effect on import (Fig. 3). In addition, the two smaller deletions in this region still allow a low efficiency of import. These data suggest that there is not a specific primary structure motif in the region from residues 4 to 14 required for function. Rather, it seems that recognition is fulfilled by the overall properties of the amino acids, perhaps as organized in a specific secondary or tertiary structure. The N-terminal domain contains the information required for efficient interaction with lipids, especially the neutral mono-galactolipid MGDG. An attractive hypothesis thus could be that this domain, which is one of the more hydrophobic regions of the transit sequence, serves to anchor the precursor in the lipid phase of the outer membrane during initial stages of the import process. Since the chloroplast outer membrane is the only membrane exposed to the cytosol that contains galactolipids (Douce and Joyard, 1990) the preference of the N-terminal part of the transit sequence for interaction with these molecules is expected to contribute to targeting. It has been proposed that due to its amino acid composition the transit sequence can participate in a hydrogen bonding network in the interface between the acyl chains and the polar residues of the glycerol backbone and lipid head groups (Van't Hof et al., 1993).
The second domain of the ferredoxin transit
sequence corresponds to the region containing residues 15-25.
Deletions in this region strongly affect in vitro chloroplast
import although recognition still occurs. The significance of this
binding is indicated by the observation that an excess of deletion
mutant 15-25 still efficiently competes for binding sites on
the chloroplast and thus inhibits import of the radiolabeled wild-type
precursor. The lack of full import is also not due to a loss of
interaction of this region with lipids. Two possibilities can be
thought of to explain the loss of import observed for this mutant.
Either the region between residues 15 and 25 contains a recognition
motif which is recognized by proteins at later stages of translocation.
Or this region serves to provide enough flexibility to the transit
sequence needed for other regions to function at later stages of
translocation. The lack of primary sequence conservation, except for
the presence of several helix-breaking proline residues (see Fig. 1) together with the low probability of secondary structure
(Pilon et al., 1992b) for this part of the sequence, could
point to a role as a flexible connector allowing optimal spatial
arrangement of other parts of the transit sequence during later stages
of translocation. It is of interest to note that proline is present in
the corresponding area of most transit sequences.
The third region
roughly consists of residues 26-38. The deletions in this area
indicate that the region contributes to the overall efficiency of
translocation whereas processing is not affected. It should be noted,
however, that relative to the large size of the deletions
26-32 and
26-34 have a limited effect on the
import process suggesting that this region is relatively unimportant
for transit sequence functioning in vitro. The loss in import
efficiency of
26-34 can be contributed to the lower
efficiency of binding. This deleted part of the transit sequence
corresponds to a hydrophobic region (see Fig. 12) which is,
however, not required for interaction with the lipids (Fig. 11).
From this we can conclude that lipid insertion not simply depends on
the presence of hydrophobic domains but that the position within the
sequence is important as well.
Residues 26-38 are a part of
the middle region as defined by von Heijne et al.(1989). It is
also very enriched in turn promoting residues like G and N and contains
the semi-conserved sequence motif FGLK (Fig. 1). In the preFd
transit sequence, this motif is part of a predicted -helix
stretching from residue 29 to 38 (Pilon et al., 1992b). The
FGLK motif is slightly altered to WPLK, and an extra G is inserted in
substitution mutant 33G/F34W/G35P. These alterations are predicted to
interrupt the secondary structure and to increase structural
flexibility (Chou and Fasman, 1978). Nevertheless, they improve the
import efficiency. This suggests that structural flexibility rather
than a stable secondary structure is required for optimal functioning
of this part of the sequence. Note that in
26-32 and
26-34 the sequence motif F/W-G/P-L-K is restored. In
35-38 this motif is also partly restored, and considering
the effect of the substitutions in amino acids 42, 44, and 45 this
mutant still imports quite efficiently. The F/W-G/P-L-K motif is
completely removed in
26-45, a large deletion which
completely blocks import. However, because of the large size of this
deletion this is not easily contributed to any particular part of the
sequence. This is not the case for
33-34, in which the
F/W-G/P-L-K motif is disrupted and which, despite the fact that it
contains more of the original amino acid sequence than mutant
26-34, is imported with a lower efficiency suggesting that
the deleted amino acids functioned in the context of their neighboring
sequence. The F/W-G/P-L-K motif thus might contribute to targeting. In
view of this it is also of interest to note that the peptides that were
shown to inhibit chloroplast import and binding of a set of in
vitro translated precursor proteins all shared a motif similar to
F/W-G/P-K/R (Schnell et al., 1991; Perry et al.,
1991). But for the full in vitro import of preFd into pea
chloroplasts the latter motif has a less essential function than, for
instance, a less conserved part of the N terminus. As our in vitro experiments do not clearly indicate a function for this region, we
are not yet sure about its importance in protein sorting in the plant
cell. Perhaps it is only serving as a flexible spacer sequence needed
to connect other recognition motifs. Another option that cannot be
tested in vitro is that certain parts of the transit sequence
might be involved in the prevention of mis-targeting to other
organelles, as was proposed by Huang et al.(1990).
The
fourth domain of the transit sequence consists of the C-terminal
region. The C-terminal deletions in the transit sequence
(35-42,
39-42,
41-47, and
43-45) all cause two effects. They significantly lower the
import efficiency due to a decreased initial interaction. Secondly,
they affect processing. This area thus has a dual role in both
recognition and in processing. It has been shown previously that amino
acids 46-47 are not essential for any aspect of transit sequence
functioning (Smeekens et al., 1989). Nevertheless, the
deletion of these 2 amino acids plus the first 10 amino acids of the
mature sequence results in a reduction of the import efficiency and
prevents processing. A possible explanation for this result can be that
alteration of the mature sequence produces a less stable form of the
imported protein or that this region is important in providing exposure
of the transit sequence.
The part of the C terminus of the transit
sequence that contributes most to translocation includes residues
39-45. The rather efficient competitive inhibition of the import
of the wild-type protein observed for 35-42 is contrasted by
the lack of competition observed for the partly overlapping deletion
mutant
41-47. The most simple interpretation is that the
non-overlapping residues (43, 44, 45, 46, 47) are
required for competition. Remarkably, both deletion mutants are
imported with a similar (low) efficiency. Interestingly, when residues
43-45 were deleted this resulted in a severe reduction of binding
but not of full import. Additionally, the analysis of the time course
of import revealed that the kinetics of import observed with this
precursor are different from the wild-type preFd. It is possible that
this C-terminal region thus contains a sequence required to lock the
precursor in an initial step of the import process, which would
normally be followed by a release leading to full translocation. Such
an initial recognition step is possibly also bypassed by
41-47.
The region from residue 35-47 is required
for lipid insertion. A lipid specificity is especially observed for the
most C-terminal region. The deletion mutant 40-47 is
severely restricted in its possibility to interact with anionic lipids
possibly due to the removal of 2 positively charged residues. In most
transit sequences a positively charged C terminus can be found (von
Heijne et al., 1989). Possibly this charged region can serve
to help anchor the precursor temporarily in the target membrane.
A
surprisingly large part of the precursor is required for processing.
Deletions 35-38 up to
46-57 all affect
processing. In most of these deletions positively charged residues were
removed. Gavel and von Heijne(1990) have proposed the existence of a
loosely conserved processing site. In a substitution mutant
(G42S/V44W/T45C) which was correctly processed, the loosely conserved
cleavage site motif was disrupted. The import efficiency of this mutant
was reduced relative to wild-type preFd. Archer and Keegstra(1993) have
reported that amino acid substitutions in the C terminus of the pSSU
transit sequence which are predicted to lower the turn propensity
caused a decrease in import efficiency. However, despite the removal of
a glycine residue, for substitution mutant G42S/V44W/T45C no
significant change in secondary structure prediction according to Chou
and Fasman(1978) is calculated. The processing of deletion mutants
35-42 and
39-42 is blocked although they possess
the actual processing site which was recognized in the substitution
mutant G42S/V44W/T45C. It thus seems that the decision to process is
blocked. Mutant
43-45 is peculiar as it is partly processed,
but a site is used which produces a smaller mature protein. Our
deletion analysis thus suggests that processing is specified by a
cleavage site motif which must lie a few amino acids more to the C
terminus from a positively charged region. The preFd transit sequence
deletion mutants that are not processed all have in common that
arginine 41 is removed. An arginine located at position -4, C
terminally from the processing site, was also shown to be of importance
for the processing of pLHCP (Clark and Lamppa, 1991) and of pSSU
(Archer and Keegstra, 1993).
Prefd was shown previously not to
require cytosolic factors for import. The recognition process thus
involves direct interactions of the precursor and the chloroplast
envelope. The identification of domains in the topogenic sequence each
involved in specific aspects of the translocation reaction might imply
the existence of different components of the import machinery that
serve as interacting partners for each of these regions. An intriguing
task will be to identify these components and find out how they
interact with the precursor and each other in order to function as a
selective translocation machinery across the two membranes of the
chloroplast envelope for precursors. Using a photo-cross-link approach,
it was shown that only the precursor to the small subunit of ribulose
bis-phosphate carboxylase but not the mature protein could be
cross-linked to two distinct envelope proteins. Association with a
75-kDa protein was observed at later stages of translocation and only
when ATP was present whereas association to a 86-kDa protein was also
observed without ATP. These observations suggest functions in different
aspects of translocation (Perry and Keegstra, 1994), and possibly this
involves the interaction of the import machinery with different parts
of the transit sequence. Interestingly, preFd bound to pea chloroplasts
in the presence of ATP could also be cross-linked to two protein
species in the 70-90 kDa molecular mass range. ()However, since in both cases the cross-linking moieties
were attached to cysteine residues in the mature sequence any
involvement of the transit sequence in recognizing the identified
components is not certain. The capability to recognize the chloroplast
is correlated with the capacity to insert into the total lipid extract
of the chloroplast outer envelope membrane at lateral pressures that
are believed to be physiologically relevant. Our results thus suggest
an important role both for transit sequence-lipid interactions and
transit sequence-envelope protein interactions in the chloroplast
protein uptake process.
How can we now envisage that the transit sequence fulfills its role in recognition? Based on the present knowledge we propose a model for the functioning of the transit sequence (see Fig. 13). The low protein content of the chloroplast outer membrane (Douce and Joyard, 1990) should provide space for precursors to insert. Both the C- and N-terminal parts of the transit sequence harbor lipid interacting domains. These anchor regions can serve to help target the precursor to the outer membrane of the chloroplast. It has been shown previously that the insertion into a membrane containing negatively charged lipids results in an induction of secondary structure in the transit peptide (Horniak et al., 1993). The insertion thus might give rise to changes in secondary structure which might expose new recognition motifs that fit to protein components of the import machinery. Flexible regions may be required to allow optimal fitting of recognition motifs. In the absence of such a region, competition for sites might still occur, but transfer to a later stage in the translocation process might be blocked.
Figure 13: Model for the functioning of the transit sequence in the import process. Regions I-III represent hypothetical domains that contribute to recognition. Upon interaction with the lipids in the outer membrane, structural changes occur that allow the transit sequence to fit to a hypothetical protein component of the translocation machinery, P.