©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Domains of the Ferredoxin Transit Sequence Involved in Chloroplast Import (*)

(Received for publication, August 22, 1994; and in revised form, December 12, 1994)

Marinus Pilon (1)(§) Hans Wienk (2) Wendy Sips (3) Martin de Swaaf (2) Irvin Talboom (2) Ron van 't Hof (2)(¶) Gerda de Korte-Kool (2) Rudy Demel (2) Peter Weisbeek (1) (3)(**) Ben de Kruijff (1) (2)

From the  (1)Institute of Biomembranes, the (2)Center for Biomembranes and Lipid Enzymology, Department of Biochemistry of Membranes, and the (3)Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes, oligonucleotides, sequencing kits, the Cap analog G-(5`)-ppp-(5)-G, and Percoll were from Pharmacia, Uppsala, Sweden. T7 polymerase transcription kits were from Epicentre Technologies, Madison, WI. Plasmid pET11-d was purchased from Novagen, United Kingdom. Wheat-germ lysate translation kits were from Promega, Madison, WI. All other chemicals were of the highest grade available.

Plasmid Constructions

The sequences (5`-end to 3`-end) of the oligonucleotides used were: (A), CT-AAA-AAA-CTC-ACC-ATG-GTCTC; (B), C-CAA-GCT-TGG-CTA-GGT-CTC-TAA-AAA-ACT-AGA-ATGGCT-TCT-ACA-A; (C), ATG-GCT-TCT-ACA-CTC-AGC-GCT-TGGTCG-GTG-AGC-GCA-TCG; (D), ACC-CTC-TCG-GTG-TGC-GCA-TCGTTG-TGG-CCA-AAG-CAA-CA; (E), GTC-GCC-TCA-TCG-TGG-CCAACC-AAC-ATG; (F), C-ATG-GGC-CAA-GCC-GGC-TTG-TGG-CCACTG-AAA-GCC-GG; (G), GGA-CTG-AAA-GCC-GGC-TCC-CGT-TCGCGA-TGG-TGC-GCA-ATG-GCC-ACA; (H), GTG-ACT-GCA-ATG-GCTAGC-TAC-AAG-GTT-ACC; (I), AC-AAG-GTT-ACC-TTG-ATT-ACCAGC-GCT-TCA-GGA-ACT. Standard cloning procedures were used (Sambrook et al., 1989). To allow insertion into the pET11-d vector, the existing prefd mutants pFDS-t7 and pFDS-t36 (Smeekens et al., 1989) were supplied with an unique NcoI site using oligonucleotide A as outlined previously for the creation of pSPAF (Pilon et al., 1990). Plasmid pSPAF, now referred to as pSPAF-wt, was used as starting material to create novel mutations. To create a new set of substitution mutants with restriction sites, that are unique in the coding region of prefd and that upon cleavage produce blunt ends all in the same reading frame, oligonucleotide-mediated site-directed mutagenesis was done according to the gapped duplex method (Kramer et al., 1984). Two rounds of mutagenesis were performed. First, new restriction sites were introduced in the transit sequence region using oligonucleotides B-G and I resulting in plasmids pSPAF-200, -105, -104, -103, -102, -101, -100, respectively. Then, pSPAF-wt and pSPAF-105, -104, -103, and -102 were subjected to a second round of mutagenesis employing oligonucleotide H, which replaces the original BalI site as present in pSPAF-wt for an NheI site. These plasmids were named pSPAF-ws, pSPAF-105ws, pSPAF-104ws, etc. In these latter substitution mutants, the BalI site is unique now. The sticky ends generating enzymes BamHI, HindIII, or ClaI which have unique recognition sequences in all plasmids were employed in combination with the various blunt ends creating restriction enzymes to create the deletion mutants. The two most N-terminal deletion mutants were placed under control of the T7 promoter in pBluescript (Sambrook et al., 1989) by insertion in the HindIII/BamHI sites resulting in pBSFD-391 and pBSFD-373. All other mutant clones possess an NcoI site, and these constructs were placed under control of the T7lac promoter in plasmid pET11-d (Studier et al., 1990) after digestion with NcoI and BamHI resulting in plasmid pETFD-wt and the mutant derivatives. The sequences of all the deletion and substitution mutants in the expression plasmids were confirmed by dideoxy sequencing using a primer that hybridizes to the T7 promoter sequence.

Expression in Escherichia coli and Purification of Proteins

pETFD-wt, pETFD-372, pETFD-361, pETFD-342, pETFD323, and pETFD-t7 were transformed into E. coli strain BL21(DE3) (Studier et al., 1990). These strains were used to express, respectively, the full ferredoxin precursor, Delta6-14, Delta15-25, Delta26-34, Delta35-43, and Delta41-47, were expressed. The cultures were grown in Luria broth at 37 °C to an A of approximately 0.3. Then isopropyl-1-thio-beta-D-galactopyranoside was added to a final concentration of 0.4 mM, and the cultures were incubated for another 3 h before harvesting. PreFd and deletion mutants were purified essentially as described (Pilon et al., 1992a). The proteins were stored in surfacil (Pierce) precoated cups at -20 °C.

In Vitro Transcription-Translation

Plasmids used as a template for transcription were linearized with BamHI. Synthetic capped mRNAs were transcribed from the T7 promoter using T7 polymerase according to the manufacturer's instructions. In vitro translation in lysates in the presence of ^3H-labeled leucine (specific activity 140-154 Ci/mmol, Amersham, U.K.) were performed according to suggested protocols (Promega). Preparation of post-ribosomal supernatants were performed as described by Pilon et al. (1992a). Typically, 25% of the added label was incorporated into trichloroacetic acid-precipitable counts in the post-ribosomal supernatants.

Import, Binding, and in Vitro Processing Experiments

Chloroplasts were isolated from 9 to 11-day-old pea seedlings, cv. Feltham First, as described (Pilon et al., 1992a). Import assays were performed in import buffer, 50 mM HEPES/KOH, pH 8.0, 330 mM Sorbitol, that was supplied with 2 mM MgCl(2), 0.5 mM dithiothreitol, 200 µg/ml antipain, and 2 mM Mg-ATP. Samples were prepared on ice, and 7.5 µl of translation mixture containing approximately 200,000 trichloroacetic acid precipitable counts was added to a 150-µl reaction. The chloroplasts (30 µg chlorophyll/150 µl incubation volume) were added last. Samples were incubated at 25 °C in the light to allow import. For binding assays identical protocols were used (2 mM Mg-ATP) except that incubations were performed for 30 min at 4 °C in the dark. For competition experiments, purified precursor proteins were added from 8 M urea, 1 mM dithiothreitol. In these experiments the final urea concentration was adjusted to 150 mM. A concentration of urea below 200 mM was shown previously not to effect the efficiency of protein import in vitro (Pilon et al., 1990). The incubations for competition experiments contained 5 µl of translation mixture containing ^3H-labeled preFd (approximately 200,000 counts/min trichloroacetic acid precipitable counts). The incubation time was 20 min. We did not observe any unspecific damage to the chloroplasts by the addition of the unlabeled precursors as the same amount of chloroplasts that was recovered in all the incubations was the same, and no differences were seen in the protein compositions as judged from SDS-PAGE and staining of the gels with Coomassie Brilliant Blue (not shown). Import, competition and binding incubations were terminated by transferring the incubations to ice and the addition of five volumes of ice-cold import buffer. From this point all manipulations were done at 4 °C under dim green light. The chloroplasts were washed, protease treated, reisolated through 40% Percoll cushions, and processed for analysis by SDS-PAGE as described (Pilon et al., 1992a). Radioactivity present in protein bands was quantified as described, after excision of the bands from the gel (Pilon et al., 1990). The number of precursors or mature proteins was calculated from these data and from the number of leucine residues in the sequences and the specific activity of the added [^3H]leucine present during translation. We thus assumed for this calculation that no unlabeled leucine was incorporated. In a standard import reaction up to 30% of the added wild-type ferredoxin precursor was found to be processed, protease protected, and recovered with the chloroplasts. Because in these experiments only approximately 65% of the chloroplasts is recovered, which is due to aspecific losses, this means that up to 50% of the added wild-type precursors takes part in the import under these conditions. We choose not to correct for chloroplast losses as this would only increase the error in the experiments because of the experimental difficulty to reproducibly take homogeneous samples from the last chloroplast wash. In addition, for most experiments the efficiency is compared to the wild-type protein. To analyze the effects of the different incubation conditions on the chloroplasts, the recovery was monitored by comparing protein patterns on Coomassie Brilliant Blue-stained gels before autoradiography and by including parallel experiments from which samples were drawn for protein assays.

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 times 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.

Lipids and Monolayer Experiments

Chloroplast outer envelope membrane lipids were isolated and characterized as described (van't Hof et al., 1993). 1,2-Dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) was synthesized according to established methods (Van Deenen and de Haas, 1964). 1,2-Dioleoyl-3-O-(beta-D-galactopyranosyl)-rac-glycerol) was synthesized as described by Chupin et al.(1994). Monolayer experiments were performed as described by van't Hof et al.(1993). The subphase used was composed of 10 mM PIPES, 50 mM NaCl, 1 mM dithiothreitol, pH 7.4. All experiments were done at room temperature. Proteins were added to the subphase from concentrated stock solutions in 8 M urea to the subphase. A subphase concentration of 0.2 µM was used for all the proteins, which was found to be saturating with respect to surface pressure increase upon injection underneath a DOPG monolayer with an initial surface pressure of 20 mM/m.

General Methods

Sequence alignments were performed with the aid of the PC/GENE software (IntelliGenetics, Inc, Mountain view, CA) using the clustal algorithm with the recommended default parameters. Sequences with known cleavage sites were withdrawn from the EMBL Data library. Published methods were used for SDS-PAGE (Laemmli, 1970) and protein assays (Bradford, 1976).


RESULTS

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 (Delta12-47) and t7 (Delta41-47), 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 [^3H]leucine to generate radioactive precursor proteins. For all the clones the translation of mRNA was efficient and with comparable efficiency, except for two mutants Delta21-25 and Delta45-57 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 Delta43-45 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 Delta15-25 andDelta 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, Delta43-45; open triangles, Delta39-42; closed squares, Delta35-38; closed circles, Delta26-34; closed triangles, Delta11-14. 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 Delta12-47 and Delta26-45 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 Delta15-25 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 Delta15-21. This clone was constructed by duplicating amino acids 11-14(ASLW) present in mutant Delta15-25. 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, Delta26-32 and Delta26-34, are imported with relatively good efficiency. Compared to these two larger deletions the small deletion mutant Delta33-34 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 Delta26-34). 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 Delta35-38, Delta35-42, and Delta39-42 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 Delta35-38 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, Delta43-45 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 Delta35-42, Delta39-42, and Delta41-47 are imported with a strongly reduced efficiency. They become localized in the stroma but are not processed (see Fig. 4). The observed import of Delta41-47 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 Delta43-57 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). Delta46-57, 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 Delta43-45 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 (Delta43-45) 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 Delta15-21 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-beta-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 Delta26-34 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 Delta35-43 should be marked Delta35-42.



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 Delta6-14 and Delta41-47 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 Delta15-25, Delta26-34, and Delta35-45 were nearly as efficient competitors as the full precursor (see Fig. 10B) suggesting that these proteins are capable of entering the import machinery. Interestingly, Delta15-25 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 ^3H-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, Delta6-14; open squares, Delta15-25; closed squares, Delta26-34; open triangles, Delta35-42; closed triangles, Delta41-47.



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 (Delta) 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 Delta 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/Delta 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 Delta6-14, Delta35-42, and Delta 41-47. The loss of insertion is not simply due to the shortening of the transit sequence by the deletion because mutant Delta26-34 inserts with an efficiency comparable to the wild-type protein. An even stronger interaction is seen with Delta15-25 which is the largest deletion studied here. Delta15-25 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 Delta6-14 and Delta41-47 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/Delta 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 Delta6-14 is especially blocked in the ability to penetrate into the MGDG monolayer. The most C-terminal deletion mutant Delta41-47 is mainly disturbed in its capacity to interact with the negatively charged DOPG. Mutant Delta35-42 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 Delta41-47 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 beta-strand was found as the most likely structure for residue 3-14, coinciding with hydrophobic region A. Two alpha-helices are predicted, residues 20-25 and 29-38, coinciding with hydrophobic regions C and D. A beta-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 beta-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 Delta6-14, which lacks the capacity to recognize the chloroplast, the possible beta-strand and hydrophobic region A are removed. In Delta15-25, 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 beta-strand. In Delta26-34, 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 Delta35-42, 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 Delta41-47. 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.


DISCUSSION

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 Delta15-25 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 Delta26-32 and Delta26-34 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 Delta26-34 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 alpha-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 Delta26-32 and Delta26-34 the sequence motif F/W-G/P-L-K is restored. In Delta35-38 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 Delta26-45, 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 Delta33-34, 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 Delta26-34, 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 (Delta35-42, Delta39-42, Delta41-47, and Delta43-45) 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 Delta35-42 is contrasted by the lack of competition observed for the partly overlapping deletion mutant Delta41-47. 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 Delta41-47.

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 Delta40-47 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 Delta35-38 up to Delta46-57 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 Delta35-42 and Delta39-42 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 Delta43-45 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. (^2)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.




FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, Div. of Biochemistry and Molecular Biology, 630 Barker Hall, University of California, Berkeley, CA 94720. Tel: 510-643-5035.

Supported by the Netherlands Foundation for Biological Research (Bion), with financial aid from the Netherlands Organization for Scientific Research (NWO).

**
Supported by a grant from the Human Frontier Science Program Organization.

(^1)
The abbreviations used are: pSSU, precursor to the small subunit of ribulose bis-phosphate carboxylase; DOPG, 1,2-di-oleoyl-sn-glycero-3-phosphoglycerol; MGDG, Monogalactosyl-diacylglycerol; mN, milliNewton; PAGE, polyacrylamide gel electrophoresis; pLHCP, precursor to light harvesting complex protein; preFd, precursor of ferredoxin; PIPES, 1,4-piperazinediethanesulfonic acid.

(^2)
T. America and P. Weisbeek, unpublished observations.


ACKNOWLEDGEMENTS

We gratefully acknowledge the aid of Dick Smit in preparing the figures for the manuscript.


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