Requirement for three membrane-spanning alpha-helices in the post-translational insertion of a thylakoid membrane protein.

The insertion of a protein into a lipid bilayer usually involves a short signal sequence and can occur either during or after translation. A light-harvesting chlorophyll a/b-binding protein (LHCP) is synthesized in the cytoplasm of plant cells as a precursor and is post-translationally imported into chloroplasts where it subsequently inserts into the thylakoid membrane. Only mature LHCP is required for insertion into the thylakoid. To define which sequences of the mature protein are necessary and sufficient for thylakoid integration, fusion and deletion proteins and proteins with internal rearrangements were synthesized and incubated with isolated thylakoids and stroma. No evidence is found for the existence of a short signal sequence within LHCP, and, with the exception of the amino terminus and a short lumenal loop, the entire mature protein with consecutively ordered alpha-helices is required for insertion into thylakoid membranes. The addition of positive charges into stromal but not lumenal segments permits the insertion of mutant LHCPs into isolated thylakoids. Replacement of the LHCP transit peptide with the transit peptide from plastocyanin has no effect on LHCP insertion and does not restore insertion of the lumenal charge addition mutants.     

What is the role of the transit peptide in thylakoid integration of the light-harvesting chlorophyll a/b protein?

Whereas it is widely accepted that the transit peptide of the precursor for the light-harvesting chlorophyll a/b protein (preLHCP) is responsible for targeting this polypeptide to chloroplasts, the signals which govern its intraorganellar targeting appears to be transit peptide-mediated for plastocyanin (Smeekins, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375) and several other nuclear-encoded, thylakoid luminal proteins. To determine whether a similar mechanism operates for LHCP (an integral thylakoid protein), we have used oligonucleotide-directed mutagenesis to delete the proposed transit sequence from a petunia precursor of this polypeptide. Intact preLHCP and the deletion mutant product have been expressed in vitro, and their abilities to integrate into purified thylakoids have been compared. We have found that both polypeptides insert into thylakoids correctly, provided the latter are supplemented with a membrane-free stromal extract and Mg.ATP. Our results clearly demonstrate that whereas the transit peptide is required for transport into chloroplasts, thylakoid integration of preLHCP is determined by mature portions of the polypeptide. In addition, we note that transit peptide removal has little effect on the apparent solubility of the in vitro translation products.     

Transport of proteins into chloroplasts

The import of cytoplasmically synthesized proteins into chloroplasts involves an interaction between at least two components; the precursor protein, and the import apparatus in the chloroplast envelope membrane. This review summarizes the information available about each of these components. Precursor proteins consist of an amino terminal transit peptide attached to a passenger protein. Transit peptides from various precurosrs are diverse with respect to length and amino acid sequence; analysis of their sequences has not revealed insight into their mode of action. A variety of foreign passenger proteins can be imported into chloroplasts when a transit peptide is present at the amino terminus. However, foreign passenger proteins are not imported as efficiently as natural passenger proteins, and some chimeric precursor proteins are not imported into chloroplasts at all. Therefore, the passenger protein, as well as the transit peptide, influences the import process. Import begins by binding of the precursor to the chloroplast surface. It has been suggested that this binding is mediated by a receptor, but evidence to support this hypothesis remains incomplete and a receptor protein has not yet been characterized. Protein translocation requires energy derived from ATP hydrolysis, although there are conflicting reports as to where hydrolysis occurs and it is unclear how this energy is utilized. The mechanism(s) whereby proteins are translocated across either the two envelope membranes or the thylakoid membrane is not known.Abbreviations EPSP 5-enolpyruvyulshikimate-3-phosphate - LHCP Chlorophyll a/b binding protein of the light-harvesting complex - NPT-II Neomycin phosphotransferase II - PC Plastocyanin - Pr Precursor - Rubisco Ribulose-1,5,-bisphosphate carboxylase/oxygenase - SS Small subunit of Rubisco     

Processing of a wheat light-harvesting chlorophyll a/b protein precursor by a soluble enzyme from higher plant chloroplasts

A processing activity has been identified in higher plant chloroplasts that cleaves the precursor of the light-harvesting chlorophyll a/b- binding protein (LHCP). A wheat LHCP gene previously characterized (Lamppa, G.K., G. Morelli, and N.-H. Chua, 1985. Mol. Cell Biol. 5:1370- 1378) was used to synthesize RNA and subsequently the labeled precursor polypeptide in vitro. Incubation of the LHCP precursors with a soluble extract from lysed chloroplasts, after removal of the thylakoids and membrane vesicles, resulted in the release of a single 25-kD peptide. In contrast, when the LHCP precursors were used in an import reaction with intact pea or wheat chloroplasts, two forms (25 and 26 kD) of mature LHCP were found. The peptide released by the processing activity in the organelle-free assay comigrated with the lower molecular mass form of mature LHCP produced during import. Properties of the processing activity suggest that it is an endopeptidase. Chloroplasts from both pea and wheat, two divergent higher plants, contain the processing enzyme, suggesting its physiological importance in LHCP assembly into the thylakoids. We discuss the implications of LHCP precursor processing by a soluble enzyme that may be in the stromal compartment.     

The N-terminal portion of the preToc75 transit peptide interacts with membrane lipids and inhibits binding and import of precursor proteins into isolated chloroplasts.

Toc75 is an outer envelope membrane protein of chloroplasts. It is unusual among the outer membrane proteins in that its precursor form has a bipartite transit peptide. The N-terminal portion of the Toc75 transit peptide is sufficient to target the protein to the stromal space of chloroplasts. We prepared a 45 amino-acid peptide containing the stromal targeting domain of the Toc75 transit peptide in Escherichia coli, using the intein-mediated system, and purified it by reverse-phase HPLC. Its identity was confirmed by N-terminal amino-acid sequencing and matrix assisted laser desorption ionization mass spectrometry. In monolayer experiments, the peptide inserted into the chloroplastic membrane lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol and into a nonchloroplastic lipid phosphatidylethanolamine. However, it did not insert into other chloroplastic lipids, such as mono- and digalactosyl diacylglycerol, and phosphatidylcholine. Furthermore, the peptide significantly inhibited binding of radiolabeled precursors of Toc75 and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to intact chloroplasts as effectively as did a bacterially produced precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase. The peptide also inhibited import of radiolabeled precursors into isolated chloroplasts, however, to a lesser extent than did nonlabeled precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase.     

Determinants for cleavage of the chlorophyll a/b binding protein precursor: a requirement for a basic residue that is not universal for chloroplast imported proteins

We demonstrate that the precursor of the major light-harvesting chlorophyll a/b binding protein (LHCP of Photosystem II), encoded by a Type I gene, contains distinct determinants for processing at two sites during in vitro import into the chloroplast. Using precursors from both pea and wheat, it is shown that primary site processing, and release of a approximately 26-kD peptide, depends on an amino-proximal basic residue. Substitution of an arginine at position -4 resulted in an 80% reduction in processing, with the concomitant accumulation of a high molecular weight intermediate. Cleavage occurred normally when arginine was changed to lysine. The hypothesis that a basic residue is a general requirement for transit peptide removal was tested. We find that the precursors for the small subunit of Rubisco and Rubisco activase do not require a basic residue within seven amino acids of the cleavage site for maturation. In the wheat LHCP precursor, determinants for efficient cleavage at a secondary site were identified carboxy to the primary site, beyond what is traditionally called the transit peptide, within the sequence ala-lys-ala-lys (residues 38-41). Introduction of this sequence into the pea precursor, which has the residues thr-thr-lys-lys in the corresponding position, converted it to a substrate with an efficiently recognized secondary site. Our results indicate that two different forms of LHCP can be produced with distinct NH2-termini by selective cleavage of a single precursor polypeptide.     

Properties of a Chloroplast Enzyme that Cleaves the Chlorophyll a/b Binding Protein Precursor : Optimization of an Organelle-Free Reaction

The major light-harvesting chlorophyll a/b binding protein (LHCP) of higher plant chloroplasts is nuclear-encoded, synthesized as a precursor, and processed upon import. We have previously (GK Lamppa, M Abad [1987] J Cell Biol 105: 2641-2648) identified a soluble enzyme that cleaves the LHCP precursor (pLHCP). In this study, we describe the conditions for optimal recovery of the processing activity and provide evidence that the N terminus of pLHCP is indeed cleaved, removing the transit peptide. Two pLHCP deletions were made from a cloned pLHCP gene removing 13 and 21 amino acids, respectively, from the carboxy terminus of the protein. After organelle-free processing, the cleavage products showed a shift in mobility during SDS-PAGE proportional to the size of the precursor truncations, as predicted for N-terminal processing. Unexpectedly, a third truncated precursor lacking 91 residues of the C-terminus was not cleaved although the transit peptide domain was intact, suggesting that this deletion disrupted conformational features of the precursor necessary for processing. The pLHCP processing enzyme is inhibited by 2 millimolar EDTA and the metal chelator 1, 10 phenanthroline at 0.4 millimolar, while being inhibited by EGTA only at high concentrations and insensitive to iodoacetate. Optimal processing occurs at pH 8 to 9, and 26°C. Gel filtration chromatography shows that the pLHCP processing enzyme has an apparent molecular weight of about 240,000. The identical column fractions that process pLHCP also convert the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase to its mature form.     

Current views on chloroplast protein import and hypotheses on the origin of the transport mechanism

Most chloroplastic proteins are synthesized as precursors in the cytosol prior to their transport into chloroplasts. These precursors are generally synthesized in a form that is larger than the mature form found inside chloroplasts. The extra amino acids, called transit peptides, are present at the amino terminus. The transit peptide is necessary and sufficient to recognize the chloroplast and induce movement of the attached protein across the envelope membranes. In this review, we discuss the primary and secondary structure of transit peptides, describe what is known about the import process, and present some hypotheses on the evolutionary origin of the import mechanism.Abbreviations DHFR dihydrofolate reductase - EPSP synthase 5-enolpyrovylshikimate-3-phosphate synthase; hsp heat-shock protein - LHCP II light-harvesting chlorophylla/b binding protein - OEE 16, 23, and 33 the 16-, 23-, and 33-kDa proteins of the oxygen-evolving complex - pr precursor - rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SS rubisco small subunit     

Precursors with altered affinity for Hsp70 in their transit peptides are efficiently imported into chloroplasts

Protein import into chloroplasts is postulated to occur with the involvement of molecular chaperones. We have determined that the transit peptide of ferredoxin-NADP(H) reductase precursor binds preferentially to an Hsp70 from chloroplast stroma. To investigate the role of Hsp70 molecular chaperones in chloroplast protein import, we analyzed the import into pea chloroplasts of preproteins with decreased Hsp70 binding affinity in their transit peptides. Our results indicate that the precursor with the lowest affinity for Hsp70 molecular chaperones in its transit peptide was imported to chloroplasts with similar apparent Km as the wild type precursor and a 2-fold increase in Vmax. Thus, a strong interaction between chloroplast stromal Hsp70 and the transit peptide seems not to be essential for protein import. These results indicate that in chloroplasts the main unfolding force during protein import may be applied by molecular chaperones other than Hsp70s. Although stromal Hsp70s undoubtedly participate in chloroplast biogenesis, the role of these molecular chaperones in chloroplast protein translocation differs from the one proposed in the mechanisms postulated up to date.     

Precursor for the light-harvesting chlorophyll a/b-binding protein synthesized in Escherichia coli blocks import of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase.

When synthesized in Escherichia coli, the light-harvesting chlorophyll a/b-binding protein (LHCP) precursor accumulates in inclusion-like bodies (Abad, M. S., Oblong, J. E., and Lamppa, G. K. (1991) Plant Physiol. 96, 1220-1227). In this study we show that after solubilization in 6 M urea and dialysis into 20 mM Tris-HCl (pH 8.0) the recombinant LHCP precursor (preLHCP) was not found as a monomer (31 kDa), but instead produced a heterogeneous population of oligomeric complexes, ranging from 60-300 kDa as determined by gel filtration chromatography. Circular dichroism analysis indicated that the oligomers had folded structure, and that it was composed of both alpha-helix and beta-sheet. Approximately half of recombinant preLHCP found in these complexes was cleavable at the transit peptide-mature protein junction by a soluble chloroplast-processing enzyme in an organelle-free reaction. At 1.5 microM the recombinant precursor inhibited the import of radiolabeled preLHCP and the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase generated by reticulocyte lysate translations. When chloroplasts were preincubated with the precursor, followed by their reisolation, import was still blocked, providing evidence that competition between recombinant preLHCP and these substrates occurred at the chloroplast per se. Recombinant preLHCP was visualized on the envelope by immunofluorescence microscopy, and its presence there was mediated by a thermolysin-sensitive factor.     

Synthetic analogues of a transit peptide inhibit binding or translocation of chloroplastic precursor proteins

Although amino-terminal transit peptides of chloroplastic precursor proteins are known to be necessary and sufficient for import into chloroplasts, the mechanism by which they mediate this process is not understood. Another important question is whether different precursors share a common transport apparatus. We used 20-residue synthetic peptides corresponding to regions of the transit peptide of the precursor to the small subunit of ribulose bisphosphate carboxylase (prSS) as competitive inhibitors for the binding and translocation of precursor proteins into chloroplasts. Synthetic peptides with sequences corresponding to either end of the transit peptide had little to no effect on binding of prSS to chloroplasts, but significantly inhibited its translocation. Synthetic peptides corresponding to the central region of the transit peptide inhibited binding of prSS to chloroplasts. Each of the peptides inhibited binding or translocation of precursors to light-harvesting chlorophyll a/b protein, ferredoxin, and plastocyanin in the same manner and to a similar extent as prSS transport was inhibited. The results presented in this paper suggest that the central regions of the transit peptide of prSS mediate binding to the chloroplastic surface, whereas the ends of this transit peptide are more important for translocation across the envelope. Furthermore, all of the precursors tested appear to share components in the transport apparatus even though they are sorted to different chloroplastic compartments.     

Light-Harvesting Chlorophyll a/b Protein : Membrane Insertion, Proteolytic Processing, Assembly into LHC II, and Localization to Appressed Membranes Occurs in Chloroplast Lysates

The apoprotein of the light-harvesting chlorophyll a/b protein (LHCP) is a major integral thylakoid membrane protein that is normally complexed with chlorophyll and xanthophylls and serves as the antenna complex of photosystem II. LHCP is encoded in the nucleus and synthesized in the cytosol as a higher molecular weight precursor that is subsequently imported into chloroplasts and assembled into thylakoids. In a previous study it was established that the LHCP precursor can integrate into isolated thylakoid membranes. The present study demonstrates that under conditions designed to preserve thylakoid structure, the inserted LHCP precursor is processed to mature size, assembled into the LHC II chlorophyll-protein complex, and localized to the appressed thylakoid membranes. Under these conditions, light can partially replace exogenous ATP in the membrane integration process.     

Soluble Chloroplast Enzyme Cleaves preLHCP Made in Escherichia coli to a Mature Form Lacking a Basic N-Terminal Domain

We have investigated the specificity of a chloroplast soluble processing enzyme that cleaves the precursor of the major light-harvesting chlorophyll a/b binding protein (LHCP). The precursor of LHCP (preLHCP) was synthesized in Escherichia coli and recovered from inclusion-like bodies. It was found to be a substrate for proteolytic cleavage by the soluble enzyme in an organelle-free reaction, yielding a 25 kilodalton peptide. This peptide co-migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the smaller of the forms (25 and 26 kilodalton) produced when either the E. coli-synthesized precursor, or preLHCP made in a reticulocyte lysate, was imported into chloroplasts. N-Terminal sequence analysis of the E. coli-generated precursor showed that it lacked an N-terminal methionine. N-Terminal sequencing of the 25 kilodalton peptide produced in the organelle-free reaction indicated that processing occurred between residues 40 and 41, removing a basic domain (RKTAAK) thought to be at the N-terminus of all LHCP molecules of type I associated with photosystem II. To determine if the soluble enzyme involved also cleaves other precursor polypeptides, or is specific to preLHCP, it was partially purified, and the precursors for Rubisco small subunit, plastocyanin, Rubisco activase, heat shock protein 21, and acyl carrier protein were tested as substrates. All of these precursors were cleaved by the same chromatographic peak of activity that processes preLHCP in the organelle-free reaction.     

Processing of the Precursors for the Light-Harvesting Chlorophyll-Binding Proteins of Photosystem II and Photosystem I during Import and in an Organelle-Free Assay

We have investigated whether the precursors for the light-harvesting chlorophyll a/b binding proteins (LHCP) of photosystems II and I (PSII and PSI) are cleavable substrates in an organelle-free reaction, and have compared the products with those obtained during in vitro import into chloroplasts. Representatives from the tomato (Lycopersicon esculentum) LHCP family were analyzed. The precursor for LHCP type I of PSII (pLHCPII-1), encoded by the tomato gene Cab3C, was cleaved at only one site in the organelle-free assay, but two sites were recognized during import, analogous to our earlier results with a wheat precursor for LHCPII-1. The relative abundance of the two peptides produced was investigated during import of pLHCPII-1 into chloroplasts isolated from plants greened for 2 or 24 hours. In contrast to pLHCPII-1, the precursors for LHCP type II and III of PSI were cleaved in both assays, giving rise to a single peptide. The precursor for LHCP type I of PSI, encoded by gene Cab6A, yielded two peptides of 23.5 and 21.5 kilodaltons during import, whereas in the organelle-free assay only the 23.5 kilodalton peptide was found. N-terminal sequence analysis of this radiolabeled peptide has tentatively identified the site cleaved in the organelle-free assay between met40 and ser41 of the precursor.     

Chloroplast Import of Light-Harvesting Chlorophyll a/b-Proteins with Different Amino Termini and Transit Peptides

We have previously isolated and sequenced two genes encoding light-harvesting chlorophyll a/b-proteins (LHCP) from Lemna gibba. One of these, AB30, encodes a protein that is highly homologous to LHCP sequences reported from other species, but the second, AB19, encodes a protein that has a transit peptide and first 12 amino-terminal residues of the mature protein that are substantially different. Despite these differences, we can demonstrate that AB19 encoded protein synthesized in vitro can be imported into isolated chloroplasts, and we provide evidence that at least some of the imported molecules are assembled into the light-harvesting complex of photosystem II. Thus, our results are consistent with the possibility that there are two functional forms of LHCP.     

Loss of efficient import and thylakoid insertion due to N- and C-terminal deletions in the light-harvesting chlorophyll a/b binding protein.

C-terminally truncated precursors of wheat light-harvesting chlorophyll a/b binding protein (LHCP) were synthesized to investigate the origin of the two forms (about 25 kD and 26 kD) of the mature protein observed upon in vitro import into the chloroplast. Precursors p delta 13 and p delta 27, lacking 13 and 27 amino acids, respectively, were successfully imported, and both gave rise to two smaller forms proportional to the size of their deletions. These results demonstrate that there are two N-terminal sites that are cleaved during import of the LHCP precursor, undoubtedly contributing to the heterogeneity of LHCP found in vivo. Significantly, p delta 27 yielded only 50% of mature LHCP when compared with wild type. Although the products of p delta 27 import were localized to the thylakoids, in contrast to p delta 13 they were not correctly inserted into the membranes, indicating that residues essential for this step are missing. p delta 27 is distinguished from p delta 13 by lacking the carboxy end of a domain highly conserved between LHCP of photosystems II and I. Other specific precursor mutants with larger C-terminal deletions were not efficiently transported into the organelle in time course experiments, nor did they insert directly into the thylakoids using chloroplast lysates, in an assay independent of translocation across the envelope. In addition, the mutant p delta 18n, lacking the first 18 amino acids of mature LHCP, was only found bound to the chloroplast envelope. However, both p delta 18n and the mature protein, i.e., LHCP, synthesized in vitro without its 34-amino acid transit peptide inserted into the thylakoids in chloroplast lysates. The overall conformation of the mutant precursor polypeptides was probed using the chloroplast soluble processing enzyme in an organelle-free reaction optimized for the LHCP precursor and the more general protease trypsin. A tightly folded, protease-resistant conformation was not apparent to explain the loss of efficient import.     

Mutations at the transit peptide-mature protein junction separate two cleavage events during chloroplast import of the chlorophyll a/b-binding protein

We have shown previously that during in vitro import into chloroplasts, the precursor of the major light-harvesting chlorphyll a/b-binding protein (LHCP) generated from a wheat gene gives rise to two mature forms (25 and approximately 26 kDa) which are inserted into the thylakoids. However, during incubation of the LHCP precursor with a chloroplast-soluble extract in an organelle-free processing reaction, the NH2 terminus is cleaved, yielding only a 25-kDa peptide. In the present study, mutations at the transit peptide-mature protein junction were introduced in the LHCP precursor to investigate the relationship between the two peptides and the determinants of proteolytic processing. Mutant p delta 3 lacks 3 amino acids including Met34 at the primary cleavage site thought to give rise to the 26-kDa peptide. It is still processed during import and in the organelle-free reaction yielding in both assays only a 25-kDa peptide. Mutant p + 4 has 4 amino acids inserted immediately after Met34 and a proline that disrupts the alpha-helix predicted by the Garnier-Osguthorpe-Robson method (Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120, 97-120) to extend through this region. Although p + 4 is imported, it is inefficiently processed; both a 25- and 26-kDa peptide are found, but at least 60% of the imported precursor remains uncleaved. Less than 5% is processed in the organelle-free assay. Replacement of the predicted alpha-helix in the mutant p + 4 alpha restores processing upon import into the chloroplast, but this mutant, which also has a 4-amino acid insert, yields only a 26-kDa peptide. p + 4 alpha is not processed in the organelle-free reaction. These results provide evidence that the two forms of LHCP obtained during import are the result of independent processing at two cleavage sites: the first site at Met34, and a second approximately 10 amino acids downstream within what has been designated the NH2 terminus of the mature protein. Whereas p delta 3 has the first site removed but retains a functional second site, in p + 4 alpha only the first site, or one very near it, is accessible to the processing enzyme during import. The conditions of the organelle-free reaction are specific for processing at only the secondary site. We discuss the implications of these findings in terms of the heterogeneity of LHCP in vivo.     

An imported thylakoid protein accumulates in the stroma when insertion into thylakoids is inhibited

The light-harvesting chlorophyll a/b protein (LHCP) is synthesized in the cytosol as a precursor (pLHCP) that is imported into chloroplasts and assembled into thylakoid membranes. Under appropriate conditions, either pLHCP or LHCP will integrate into isolated thylakoids. We have identified two situations that inhibit integration in this assay. Ionophores and uncouplers inhibited integration up to 70%. Carboxyl-terminal truncations of pLHCP also interfered with integration. A 22-residue truncation reduced integration to about 25% of control, whereas a 93 residue truncation completely abolished it. When pLHCP was imported into chloroplasts in the presence of uncouplers or when truncated forms of pLHCP were used, significant amounts of the imported proteins failed to insert into thylakoids and instead accumulated in the aqueous stroma. Accumulation of stromal LHCP occurred at uncoupler concentrations required to dissipate the trans-thylakoid proton electrochemical gradient and was enhanced at reduced levels of ATP. The latter effect may be a secondary consequence of a reduction in ATP-dependent degradation within the stroma. These results indicate that the stroma is an intermediate location in the LHCP assembly pathway and provide the first evidence for a soluble intermediate during biogenesis of a chloroplast membrane protein.     

Chloroplast Hsp70s are not involved in the import of ferredoxin-NADP+ reductase precursor

Heat-shock protein 70 (Hsp70) chaperones function as molecular motors pulling precursor proteins across membranes. Although several Hsp70s have been identified in chloroplasts, their participation in protein translocation is still uncertain. A phylogenetic analysis of the peptide-binding domain from plant Hsp70s shows that they can be classified into defined groups related to their subcellular localizations, allowing differences in substrate specificities to be inferred. Using an algorithm developed by Blond-Elguindi et al. we detected three regions in the transit peptide of the pea ferredoxin-NADP⁺ reductase precursor (preFNR) that are related to binding with immunoglobulin heavy-chain binding protein (BiP), one of the members of the Hsp70 family resident in the endoplasmic reticulum. We constructed a mutant transit peptide in which prolines 18, 20 and 28 were substituted by serines. Thus, the theoretical probability of BiP-type binding of the peptide was abolished without modifying the sites for Hsp70 with DnaK-type binding. The stromal Hsp70 homolog CSS1 displayed lower affinity for this mutant transit peptide than for the wild-type presequence. Nevertheless, preFNR containing the mutant transit peptide was imported into isolated chloroplasts from pea with initial rates similar to that observed for the wild-type precursor, and only an 18% decrease in the total number of imported molecules was observed after 20 min of reaction. Our results support an import model for the preFNR in which neither DnaK- nor BiP-like Hsp70 molecular chaperones play a central role as motor of the translocation machinery in chloroplasts.