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. 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. Plastids accomplish a great variety of metabolic functions and developmental roles in plants and eukaryotic algae. They are distributed in different amounts in each plant tissue but are present in almost all plant cells. These organelles contain their own genomes, but most of their proteins are nuclear encoded and post-translationally imported through a complex machinery located in the plastid envelope. The import process requires a number of cytosolic and stromal soluble factors and several membrane-bound and integral proteins. Most of these proteins have been identified and studied (1Jarvis P. Soll J. Biochim. Biophys. Acta. 2001; 1541: 64-79Crossref PubMed Scopus (99) Google Scholar, 2Keegstra K. Cline K. Plant Cell. 1999; 11: 557-570Crossref PubMed Scopus (321) Google Scholar, 3Chen X. Schnell D.J. Trends Cell Biol. 1999; 9: 222-227Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The significant biochemical and molecular data achieved to date have allowed the creation of working models for the chloroplast protein import mechanism. Precursor proteins containing N-terminal signal sequences are bound to the chloroplast outer membrane probably by interacting initially with lipids and then, by a "docking" step that involves a highly specific protein-protein recognition, and GTP-ATP hydrolysis and/or exchange (4Bruce B.D. Biochim. Biophys. Acta. 2001; 1541: 2-21Crossref PubMed Scopus (185) Google Scholar). The passage of the preprotein through the membranes is initiated by its N terminus. Once the precursor protein has reached the irreversible stage recognized as "early import intermediate," the transit peptide and the N-terminal unfolded portion of the preprotein pass through the outer membrane channel and interact with some of the components of the internal membrane translocation apparatus (5Akita M. Nielsen E. Keegstra K. J. Cell Biol. 1997; 136: 983-994Crossref PubMed Scopus (168) Google Scholar, 6Nielsen E. Akita M. Davila-Aponte J. Keegstra K. EMBO J. 1997; 16: 935-946Crossref PubMed Scopus (233) Google Scholar, 7Waegemann K. Soll J. Plant J. 1991; 1: 149-158Crossref Scopus (167) Google Scholar). The complete translocation of the protein requires high energy availability (>1 mm ATP in in vitro import experiments) to unfold the bound folded precursor and move it across both envelope membranes (8Theg S.M. Bauerle C. Olsen L.J. Selman B.R. Keegstra K. J. Biol. Chem. 1989; 264: 6730-6736Abstract Full Text PDF PubMed Google Scholar, 9Pain D. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3288-3292Crossref PubMed Scopus (107) Google Scholar). Finally, the transit peptide is proteolytically removed (10Robinson C. Ellis R.J. Eur. J. Biochem. 1984; 142: 337-342Crossref PubMed Scopus (152) Google Scholar), and the mature protein is folded to its final conformation. Two possible mechanisms have been proposed for mitochondrial protein import (11Glick B.S. Cell. 1995; 80: 11-14Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 12Pfanner N. Meijer M. Curr. Biol. 1995; 5: 132-135Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 13Neupert W. Hartl F.U. Craig E.A. Pfanner N. Cell. 1990; 63: 447-450Abstract Full Text PDF PubMed Scopus (182) Google Scholar, 14Gaume B. Klaus C. Ungermann C. Guiard B. Neupert W. Brunner M. EMBO J. 1998; 17: 6497-6507Crossref PubMed Scopus (79) Google Scholar). In both models, internal Hsp70s has been postulated to be the main motor of the process. The Brownian ratchet mechanism assumes that the precursor chain diffuses within the translocation pore. Then, Hsp70 molecular chaperones located in the inner side of the organelle bind to sites distributed along the polypeptide chain hampering the reverse movement of the polypeptide, thus promoting an unidirectional movement of the chain into the mitochondrial matrix (13Neupert W. Hartl F.U. Craig E.A. Pfanner N. Cell. 1990; 63: 447-450Abstract Full Text PDF PubMed Scopus (182) Google Scholar, 14Gaume B. Klaus C. Ungermann C. Guiard B. Neupert W. Brunner M. EMBO J. 1998; 17: 6497-6507Crossref PubMed Scopus (79) Google Scholar). The second model postulates that Hsp70 associates with the membrane-bound complex (Sec63p or Tim44) and the precursor protein. Hsp70 then undergoes an ATP-dependent conformational change that pulls the precursor through the pore, driving the external unfolding, and impelling the precursor protein to cross the membranes (11Glick B.S. Cell. 1995; 80: 11-14Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 12Pfanner N. Meijer M. Curr. Biol. 1995; 5: 132-135Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 15Matouschek A. Azem A. Ratliff K. Glick B.S. Schmid K. Schatz G. EMBO J. 1997; 16: 6727-6736Crossref PubMed Scopus (124) Google Scholar). The interaction of the transit peptides of mitochondrial and chloroplast precursors with Hsp70, which helps to sustain the above mentioned models, is supported by experimental and theoretical means (14Gaume B. Klaus C. Ungermann C. Guiard B. Neupert W. Brunner M. EMBO J. 1998; 17: 6497-6507Crossref PubMed Scopus (79) Google Scholar, 15Matouschek A. Azem A. Ratliff K. Glick B.S. Schmid K. Schatz G. EMBO J. 1997; 16: 6727-6736Crossref PubMed Scopus (124) Google Scholar, 16Ivey III, R.A. Bruce B.D. Cell Stress Chaperones. 2000; 5: 62-71Crossref PubMed Scopus (36) Google Scholar, 17von Heijne G. J. Mol. Biol. 1984; 173: 243-251Crossref PubMed Scopus (462) Google Scholar, 18Ivey III, R.A. Subramanian C. Bruce B.D. Plant Physiol. 2000; 122: 1289-1300Crossref PubMed Scopus (86) Google Scholar, 19Zhang X.P. Elofsson A. Andreu D. Glaser E. J. Mol. Biol. 1999; 288: 177-190Crossref PubMed Scopus (20) Google Scholar, 20Zhang X.P. Glaser E. Trends Plant Sci. 2002; 7: 14-21Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 21Rial D.V. Arakaki A.K. Ceccarelli E.A. Eur. J. Biochem. 2000; 267: 6239-6248Crossref PubMed Scopus (73) Google Scholar). The Hsp70 molecular chaperones comprise a protein family that is widely distributed among different organisms and subcellular compartments. Several Hsp70 proteins were found to be located in chloroplasts, associated with the envelope, or resident in the stroma. Com70 was detected facing the cytoplasm, Hsp70-IAP was found to be exposed to the intermembrane space between the outer and inner membranes, and at least two more Hsp70 molecular chaperones are located in the stroma, CSS1, for which its cDNA has been isolated and sequenced, and another Hsp70 less characterized (22Marshall J.S. Keegstra K. Plant Physiol. 1992; 100: 1048-1054Crossref PubMed Scopus (58) Google Scholar, 23Marshall J.S. DeRocher A.E. Keegstra K. Vierling E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 374-378Crossref PubMed Scopus (170) Google Scholar, 24Jackson-Constan D. Keegstra K. Plant Physiol. 2001; 125: 1567-1576Crossref PubMed Scopus (118) Google Scholar). The intermembrane and stromal Hsp70s can be considered ideal candidates for providing the driving force during precursor protein import into chloroplasts. However, it is still a matter of discussion which class of chaperones acts as the main molecular motor of the protein import into chloroplasts. It has been proposed that an Hsp93 molecular chaperone (ClpC), a member of the Hsp100 family associated with the inner face of the envelope alone or in collaboration with a plastidic Hsp70, may function as the driving force of the system (4Bruce B.D. Biochim. Biophys. Acta. 2001; 1541: 2-21Crossref PubMed Scopus (185) Google Scholar, 25Jackson-Constan D. Akita M. Keegstra K. Biochim. Biophys. Acta. 2001; 1541: 102-113Crossref PubMed Scopus (84) Google Scholar, 26Keegstra K. Froehlich J.E. Curr. Opin. Plant Biol. 1999; 2: 471-476Crossref PubMed Scopus (62) Google Scholar). In previous work, we have demonstrated that more than 75% of chloroplast transit peptides have at least one putative DnaK (Escherichia coli Hsp70 molecular chaperone) binding site. We have also confirmed that an interaction exists between the transit peptide of ferredoxin-NADP(H) reductase precursor (preFNR) 1The abbreviations used are: preFNRferredoxin-NADP(H) reductase precursorFNRferredoxin-NADP(H) reductase (EC 1.18.1.2)GSTglutathione S-transferase (EC 2.5.1.18)GST-TPGST-transit peptide fusion proteinRubiscoribulose-bisphosphate carboxylase/oxygenase. and Hsp70 molecular chaperones from plant cells in vitro (21Rial D.V. Arakaki A.K. Ceccarelli E.A. Eur. J. Biochem. 2000; 267: 6239-6248Crossref PubMed Scopus (73) Google Scholar). Similar observations were made for the transit peptide of the precursor of the small subunit of Rubisco (18Ivey III, R.A. Subramanian C. Bruce B.D. Plant Physiol. 2000; 122: 1289-1300Crossref PubMed Scopus (86) Google Scholar). ferredoxin-NADP(H) reductase precursor ferredoxin-NADP(H) reductase (EC 1.18.1.2) glutathione S-transferase (EC 2.5.1.18) GST-transit peptide fusion protein ribulose-bisphosphate carboxylase/oxygenase. In this report we have studied the import of wild type and mutant precursors with altered affinity for Hsp70 in their transit peptides into isolated chloroplasts and their interactions with cytosolic and chloroplast Hsp70 molecular chaperones. As a model, the ferredoxin-NADP(H) reductase (FNR) precursor was used. This enzyme catalyzes NADP+ reduction during photosynthesis. The NADPH generated is used for CO2 fixation and other biosynthetic pathways (27Arakaki A.K. Ceccarelli E.A. Carrillo N. FASEB J. 1997; 11: 133-140Crossref PubMed Scopus (139) Google Scholar). preFNR had been previously purified as a tightly folded precursor containing non-covalently bound FAD. The protein was fully active and was efficiently imported to intact pea chloroplasts (28Ottado J. Ceccarelli E.A. Eur. J. Biochem. 1998; 253: 132-138Crossref PubMed Scopus (7) Google Scholar). We used monoclonal and polyclonal antibodies to show that the transit peptide of preFNR interacts preferentially with the chloroplast Hsp70 molecular chaperone CSS1 and not with chaperones from the cytosol. We performed quantitative protein import studies using precursors with decreased Hsp70 binding ability in their transit peptides to challenge the current working models on polypeptide translocation. Here we show that weakening the interaction between the chaperone and the transit peptide does not impair the protein import process. To the contrary, mutations introduced in the transit peptides produced a 2-fold import efficiency increase, whereas they did not affect the precursor binding to the import machinery. Therefore, in chloroplasts the main unfolding force may be applied by molecular chaperones different from stromal Hsp70s, and a simple Hsp70-Brownian ratchet mechanism may not be sufficient to drive chloroplast polypeptide import. These results provide additional insights to the process and are discussed under the light of the current models. Plasmid Constructions, Expression, and Purification of preFNRs— The cDNAs coding for preFNR variants (preFNR-14, preFNR-23, and preFNR-1234) were inserted into a modified pET32 vector (Novagen) and expressed in E. coli as thioredoxin N-terminal fusion proteins. The construction of this modified vector was previously described (29Rial D.V. Lombardo V.A. Ceccarelli E.A. Ottado J. Eur. J. Biochem. 2002; 269: 5431-5439Crossref PubMed Scopus (17) Google Scholar). Then, the PstI-EcoRI cDNA fragments (≅1.3 kbp) coding for pea preFNR mutants from plasmids pGF202-14, pGF202-23, and pGF202-1234 (21Rial D.V. Arakaki A.K. Ceccarelli E.A. Eur. J. Biochem. 2000; 267: 6239-6248Crossref PubMed Scopus (73) Google Scholar) were subcloned into the modified pET32b vector digested with PstI and EcoRI. To ensure that no sequence artifacts were introduced during protein expression E. coli BL21(DE3)pLysS cells were transformed each time with the different sequenced plasmids and then grown in Luria-Bertani medium containing antibiotics at 37 °C up to the suspension reached an absorbance of 0.7 at 600 nm. Expression and purification of 35S-labeled preFNRs were performed as described previously (29Rial D.V. Lombardo V.A. Ceccarelli E.A. Ottado J. Eur. J. Biochem. 2002; 269: 5431-5439Crossref PubMed Scopus (17) Google Scholar). Binding of Hsp70 Chaperones to GST-TP Fusion Proteins—In vitro binding of plant Hsp70 chaperones to GST-TP or GST-TP-1234 was performed essentially as described (21Rial D.V. Arakaki A.K. Ceccarelli E.A. Eur. J. Biochem. 2000; 267: 6239-6248Crossref PubMed Scopus (73) Google Scholar), using cytosol or chloroplast extracts. Briefly, GST-TP fusion proteins used for these studies were expressed and purified from the E. coli strain BB1553 (30Kanemori M. Mori H. Yura T. J. Bacteriol. 1994; 176: 5648-5653Crossref PubMed Google Scholar), which does not contain the E. coli Hsp70 molecular chaperone DnaK. GST-TP fusion proteins (0.3 nmol) loaded in 30-μl fractions of glutathione-agarose were incubated with extracts (2.2 mg/ml final concentration of total proteins) in a final volume of 180 μl in buffer A (50 mm Tris/HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, and 5 mm dithiothreitol). After 5 min of incubation in an ice-cold bath, each sample was recovered by centrifugation (2 min, 3000 × g), washed twice with 120 μl of buffer A, and eluted in two 40-μl steps of the same buffer containing 10 mm glutathione (Fig. 2, A and B). In the binding assays shown in Fig. 2C, the loaded resin was washed three times and eluted in two 50-μl steps of Buffer A containing 10 mm glutathione. In each experiment aliquots of 20 μl of the eluted samples were subjected to SDS-PAGE and Western blotting. To estimate the binding affinity of E. coli Hsp70 to the different transit peptides, wild type or mutant GST-TP·Hsp70 complexes were bound to glutathione-agarose beads from cell lysates as described previously (21Rial D.V. Arakaki A.K. Ceccarelli E.A. Eur. J. Biochem. 2000; 267: 6239-6248Crossref PubMed Scopus (73) Google Scholar). Then, the loaded matrix containing the in vivo formed GST-TP·Hsp70 complexes was divided equally into several Eppendorf tubes (25-μl matrix/tube containing about 0.2 nmol of fusion protein in a total volume of 175 μl), and different volumes of Buffer A were added. After equilibrium was established, the beads were sedimented, and the amounts of GST-TP, the chaperone complexed with GST-TP (associated with the beads) and free in solution, were quantified using specific antibodies. The Kd values were determined by fitting the data obtained using Sigmaplot (Jandel Scientific). Antibodies against E. coli and human Hsp70 were purchased from Stressgen, Canada (Products SPA-880 and SPA-820). Antisera against CSS1 (a chloroplast stroma Hsp70 homolog) were kindly provided by Drs. J. E. Froehlich and K. Keegstra. Binding and Import into Chloroplasts—Intact chloroplasts were isolated from 10- to 14-day-old pea seedlings (Pisum sativum cv. little marvel) as described (31Schreier P.H. Reiss B. Kuntz M. Gelvin S.B. Schilperoort R.A. Verma D.P.S. Plant Molecular Biology Manual. Kluwer Academic Press, Dordrecht1988: 1-22Google Scholar) and suspended in import buffer (50 mm Hepes/KOH, pH 7.3, 330 mm Sorbitol, 0.1% (w/v) bovine serum albumin, 2 mm EDTA, 1 mm MgCl2, and 1 mm MnCl2) at a chlorophyll concentration of 0.6 mg/ml. Import reactions were carried out essentially as described (32Serra E.C. Krapp A.R. Ottado J. Feldman M.F. Ceccarelli E.A. Carrillo N. J. Biol. Chem. 1995; 270: 19930-19935Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Radioactive precursor proteins were precipitated with ammonium sulfate at 66% final concentration and centrifuged for 10 min at 4 °C and 10,000 × g, and the pellet was dissolved in import buffer. A typical import reaction contained the amount of chloroplasts suspension equivalent to 30 μg of chlorophyll in a final reaction volume of 150 μl. Precursors were incubated with the chloroplast suspension and5mm MgATP in import buffer at 25 °C in the light. At the end of the import reaction 2.5 μg of thermolysin was added to each sample. After a 30-min treatment on ice, EDTA was added to a final concentration of 10 mm, and chloroplasts were recovered by centrifugation through silicone oil layers (AR200). For studying the import of unfolded precursors, the preproteins were incubated with 8 m urea for 10 min after the precipitation with ammonium sulfate. Prior to their incubation with intact chloroplasts, the precursors were diluted 20 times in import buffer. Binding reactions were carried out in a final volume of 150 μl during 15 min on ice at different precursor concentrations in the presence of 50 μm MgATP. Immediately after reaction, intact chloroplasts were recovered by centrifugation through silicone oil layers. Samples were analyzed by 10% SDS-PAGE, followed by blotting and autoradiography. Urea Gradient Gel Electrophoresis—Urea gradient gels were prepared as described previously (33Calcaterra N.B. Pico G.A. Orellano E.G. Ottado J. Carrillo N. Ceccarelli E.A. Biochemistry. 1995; 34: 12842-12848Crossref PubMed Scopus (30) Google Scholar). Briefly, polyacrylamide gels containing a horizontal linear gradient of 0–8 m urea and a compensatory inverse gradient of 7.0–5.5% (w/v) acrylamide were used. The gradient uniformity among gels was maintained by the use of a casting box that allows the simultaneous preparation of several gels. Samples were incubated in 50 mm Tris/HCl, pH 8.0, 2 mm EDTA, 10 mm dithiothreitol for 20 min at 30 °C to reduce any oxidized state of the proteins, then diluted to 0.2 mg/ml in 50 mm Tris/HCl, pH 8.0, 10% (v/v) glycerol, 20 μg/ml bromphenol blue, and finally applied onto the gels and electrophoresed for 600 min at 10 V at 4 °C. After the electrophoresis was completed, gels were subjected to electroblotting and immunoreaction with rabbit anti-FNR antibodies. Rate constants for unfolding at the transition midpoints were estimated from the continuity of the gel bands, which depends upon the rate of unfolding. The observed distributions of protein at the midpoint were compared with those predicted for different arbitrary values of the unfolding rate constants (34Goldenberg D.P. Creighton T.E. Anal. Biochem. 1984; 138: 1-18Crossref PubMed Scopus (144) Google Scholar). Analytical Procedures—Quantification of labeled protein bands was performed using Gel-Pro Analyzer Software 3.1 (Media Cybernetics). Different amounts of purified labeled preFNR proteins were included in each experiment as standard. Import rates and kinetics parameters were determined by fitting the data obtained to a first order equation using Sigmaplot. FNR-dependent diaphorase activity was determined by published methods (35Zanetti G. Biochim. Biophys. Acta. 1976; 445: 14-24Crossref PubMed Scopus (51) Google Scholar) using ferricyanide reduction at 420 nm (ϵ420 = 1 mm–1·cm–1). Purification of FNR Precursors Containing Mutations in Their Transit Peptides—Four amino acid substitutions were successfully introduced previously into the preFNR transit peptide sequence by PCR techniques (Fig. 1A, see also Ref. 21Rial D.V. Arakaki A.K. Ceccarelli E.A. Eur. J. Biochem. 2000; 267: 6239-6248Crossref PubMed Scopus (73) Google Scholar). Mutations were designed to produce a major increase of ΔΔGK for each region, thus decreasing the putative E. coli Hsp70 (DnaK) binding with a minimal sequence variation. It was experimentally determined that transit peptides carrying these mutations exhibit an increased release of bound DnaK. Moreover, the effect introduced by each mutation was accumulative (21Rial D.V. Arakaki A.K. Ceccarelli E.A. Eur. J. Biochem. 2000; 267: 6239-6248Crossref PubMed Scopus (73) Google Scholar). It has been stated that predicted binding specificity for E. coli Hsp70 could be valid for a broad range of Hsp70s (20Zhang X.P. Glaser E. Trends Plant Sci. 2002; 7: 14-21Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 21Rial D.V. Arakaki A.K. Ceccarelli E.A. Eur. J. Biochem. 2000; 267: 6239-6248Crossref PubMed Scopus (73) Google Scholar). Moreover, the above mentioned mutations were proved to affect the interaction of the preFNR transit peptide with plant Hsp70 chaperones (21Rial D.V. Arakaki A.K. Ceccarelli E.A. Eur. J. Biochem. 2000; 267: 6239-6248Crossref PubMed Scopus (73) Google Scholar). Three different preFNRs carrying the mutations described above and the wild type precursor were cloned as thioredoxin N-terminal fusion proteins, expressed in E. coli, and purified by affinity chromatography using a nickel-nitrilotriacetic acid-agarose resin. Precursor proteins were released of Hsp70 molecular chaperones as described previously (36Rial D.V. Ceccarelli E.A. Protein Expr. Purif. 2002; 25: 503-507Crossref PubMed Scopus (58) Google Scholar). Then, precursors were separated from thioredoxin by digestion with the endoprotease thrombin. Purified samples are shown in Fig. 1B. Precursor preparations used were >90% pure as judged by SDS-PAGE electrophoresis and scanning densitometry. Faint minor bands were reactive against anti FNR antibodies. They could be truncated proteins or the result of some proteolytic digestion of the fusion protein. No Hsp70 molecular chaperones were detected by the monoclonal and polyclonal anti-Hsp70 antibodies used in this work copurifying with the isolated precursor proteins. Interaction of Stromal Hsp70s with Precursor Proteins— Models of post-translational protein translocation assume that interaction of the incoming polypeptide with the chaperones responsible for its import occurs only at the luminal side of the endoplasmic reticulum, or the inner side of the organelles (37Elston T.C. Biophys. J. 2000; 79: 2235-2251Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 38Liebermeister W. Rapoport T.A. Heinrich R. J. Mol. Biol. 2001; 305: 643-656Crossref PubMed Scopus (47) Google Scholar, 39Chauwin J.F. Oster G. Glick B.S. Biophys. J. 1998; 74: 1732-1743Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Considering that homologs of the Hsp70 chaperone family are present at both sides of the chloroplast envelope, differences in affinities or specificities should be required at both sides of the membranes to uphold these models of protein import. Thus, we have analyzed the binding preference of the preFNR transit peptide for molecular chaperones from cell cytosol or chloroplast stroma. In the case of cellular extracts, chaperones from cytosol and from organelles were present, because a considerable breakage of plastids probably occurs during the mechanical disruption of the plant tissues. To the contrary, intact chloroplasts were isolated through a Percoll gradient, a method that had been shown to avoid contamination of chloroplasts with cytosolic factors involved in protein import (40Waegemann K. Paulsen H. Soll J. FEBS Lett. 1990; 261: 89-92Crossref Scopus (95) Google Scholar). Thus, the stroma obtained from them did not contain cytosolic or envelope factors and will mainly have stromal Hsp70 molecular chaperones. We incubated GST-TP fusion proteins with whole cellular extracts from pea leaves or chloroplast stroma at identical final protein concentration. Then, complexes between GST-TPs and the interacting proteins from the extracts were purified, washed, resolved in SDS-PAGE, and analyzed by Coomassie Brilliant Blue staining, or Western blotting using two different antibodies. One of them (anti-Hsp70) is a monoclonal antibody raised against human Hsp70 that also reacts against plant Hsp70s. The other is a polyclonal antibody prepared against purified recombinant CSS1, a chloroplast Hsp70 also known as Hsp78. As shown in Fig. 2A (lanes 1 and 4), the human monoclonal antibody reacted with the Hsp70s present in whole cellular extracts but did not recognize Hsp70 molecular chaperones present in purified chloroplast stroma. To the contrary, anti-CSS1 detected a chloroplast chaperone present in stroma and whole cellular extracts (Fig. 2B, lanes 1 and 4). When the isolated complexes were examined, we observed that the protein of ∼70 kDa, which always co-purified with GST-TP, was reactive only with anti-CSS1 antibodies (Fig. 2B, lanes 2 and 3). It had been previously reported that the molecular mass of mature CSS1 is about 69 kDa (22Marshall J.S. Keegstra K. Plant Physiol. 1992; 100: 1048-1054Crossref PubMed Scopus (58) Google Scholar). No reaction of the purified samples was observed using antibodies against cytoplasmic Hsp70 (Fig. 2A, lanes 2 and 3). The reactive band in Fig. 2B (lanes 2 and 3) was of identical molecular weight to the one observed when chloroplast stroma was analyzed with the same antibodies (Fig. 2B, lane 1). Identical results were obtained when the stroma used in the incubations was purified from chloroplasts that were previously treated with thermolysin, a protease that does not penetrate the chloroplast outer membrane, thus excluding the possibility that the chaperone bound to the fusion protein was a cytoplasmic contaminant (not shown). When the complexes were analyzed by SDS-PAGE using Coomassie Brilliant Blue staining (Fig. 2C, lanes 1 and 2), a band of ∼70 kDa with identical molecular mass to the one detected by Western blotting was observed. These results show that, even in the presence of cytosolic Hsp70 molecular chaperones, the transit peptide of preFNR specifically binds to organellar Hsp70. When we incubated GST-TP-1234, in which all its Hsp70 binding sites were modified by site directed mutagenesis (see Fig. 1A and Ref. 21Rial D.V. Arakaki A.K. Ceccarelli E.A. Eur. J. Biochem. 2000; 267: 6239-6248Crossref PubMed Scopus (73) Google Scholar), with chloroplast stroma or whole cellular extracts, we detected a significant decrease in the amount of Hsp70 chaperone bound compared with the wild type form (Fig. 2, C (lanes 3 and 4) and D (lane 2)). Using a solid-phase binding assay, the affinity of the E. coli Hsp70 to the wild type and mutated transit peptides were estimated (Table I). A high affinity binding (Kd 125 nm) occurs with the wild type transit peptide, meanwhile introduction of the four mutations decreased about seven times the Kd (880 nm for the GST-TP-1234·Hsp70 complex).Table IParameters characterizing the import kinetics and unfolding of preFNRsPrecursorKmaKinetic parameters were determined by fitting the experimental data to a first order equation as described under "Experimental Procedures."VmaxaKinetic parameters were determined by fitting the experimental data to a first order equation as described under "Experimental Procedures."CmbMidpoint of the urea unfolding curves.mcSlope of ΔG versus urea concentration at Cm.FNR activitydFNR-dependent diaphorase activity was determined as stated under "Experimental Procedures."KdeKd values for the complex of GST-TPs and DnaK (E. coli Hsp70) determined as described under "Experimental Procedures."nmmolecules/chl·minmkcal/mol·mmmol/mg·min.nmpreFNR406 ± 19410,195 ± 1,8884.00 ± 0.521.11 ± 0.28149125preFNR-14477 ± 13812,291 ± 1,5574.00 ± 0.270.90 ± 0.18141232preFNR-23399 ± 5911,650 ± 6934.00 ± 0.231.25 ± 0.09150540preFNR-1234380 ± 8019,989 ± 1,4764.10 ± 0.311.42 ± 0.11145880FNR4.00 ± 0.581.38 ± 0.21156a Kinetic parameters were determined by fitting the experimental data to a first order equation as described under "Experimental Procedures."b Midpoint of the urea unfolding curves.c Slope of ΔG versus urea concentration at Cm.d FNR-dependent diaphorase activity was determined as stated under "Experimental Procedures."e Kd values for the complex of GST-TPs and DnaK (E. coli Hsp70) determined as described under "Experimental Procedures."