©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sorting of Nuclear-encoded Chloroplast Membrane Proteins to the Envelope and the Thylakoid Membrane (*)

(Received for publication, March 27, 1995; and in revised form, May 2, 1995)

Susanne Brink (1)(§) Karsten Fischer (1) Ralf-Bernd Klösgen (2) Ulf-Ingo Flügge (1)(¶)

From the  (1)Botanisches Institut der Universität zu Köln, Gyrhofstrasse 15, D-50931 Köln, Germany and the (2)Botanisches Institut der Universität München, Menzingerstrasse 67, D-80638 München, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The spinach triose phosphate/phosphate translocator and the 37-kDa protein are both integral components of the chloroplast inner envelope membrane. They are synthesized in the cytosol with N-terminal extensions, the transit peptides, that are different in structural terms from those of imported stromal or thylakoid proteins. In order to determine if these N-terminal extensions are essential for the correct localization to the envelope membrane, they were linked to the mature parts of thylakoid membrane proteins, the light-harvesting chlorophyll a/b binding protein and the CF(0)II-subunit of the thylakoid ATP synthase, respectively. In addition, the transit peptide of the CF(0)II-subunit that contains signals for the transport across both the envelope and the thylakoid membrane was fused to the mature parts of both envelope membrane proteins. The chimeric proteins were imported into isolated spinach chloroplasts, and the intraorganellar routing of the proteins was analyzed. The results obtained show that the N-terminal extensions of both envelope membrane proteins possess a stroma-targeting function only and that the information for the integration into the envelope membrane is contained in the mature parts of the proteins. At least part of the integration signal is provided by hydrophobic domains in the mature sequences since the removal of such a hydrophobic segment from the 37-kDa protein leads to missorting of the protein to the stroma and the thylakoid membrane.


INTRODUCTION

The majority of the proteins found in higher plant chloroplasts are encoded in the nucleus, synthesized as higher molecular weight precursors in the cytosol, and post-translationally transported to their final destination (for recent reviews, see Flügge(1990), de Boer and Weisbeek(1991) and Soll and Alefsen(1993)). All chloroplast precursor proteins have to be bound to the organelle and, subsequently, either translocated across, or inserted into, the envelope membranes that surround the plastid. For the precursor proteins that are located to the interior of chloroplasts (i.e. stroma, thylakoid membrane, thylakoid lumen), it has been demonstrated that the information for targeting to the chloroplast and translocation across the envelope is present in N-terminal transit sequences (van den Broeck et al., 1985; Schreier et al., 1985). In some instances, they also contain information for the subsequent targeting to or across the thylakoid membrane (Hagemann et al., 1990; Clausmeyer et al., 1993; Ko and Cashmore, 1989).

Much less is known about the intraorganellar sorting of proteins to the inner envelope membrane. In organello import studies on two prominent components of this compartment, i.e. the triose phosphate/phosphate translocator (TPT) (^1)and the 37-kDa protein, had revealed that the post-translational import of these nuclear-encoded proteins involves, similar to the import of stromal or thylakoid proteins, binding of precursors to the outer envelope membrane, ATP-dependent translocation into the organelle, and proteolytic removal of the transit peptide (Flügge et al., 1989; Dreses-Werringloer et al., 1991; Willey et al., 1991).

According to structure predictions, the transit sequences of the TPT and the 37-kDa protein do, however, not possess the typical features of other chloroplast proteins but tend to form a positively charged amphiphilic alpha-helix like mitochondrial presequences (Dreses-Werringloer et al., 1991; Willey et al., 1991). It has been shown recently that these preproteins have the ability to interact in vitro with mitochondrial import receptors and to be imported into mitochondria from fungi (Brink et al., 1994). Therefore, it appears feasible that these transit peptides play a role in determining the targeting to and integration into organellar membranes in general. Alternatively, the transit peptides may function as envelope-membrane-translocation signals, and the actual ``membrane insertion'' signal may be contained in the respective mature proteins as is the case for thylakoid integration of the light-harvesting chlorophyll a/b binding protein (Lamppa, 1988). It is evident that these signals must be different for proteins of the envelope and the thylakoid membranes in order to ensure correct intraorganellar sorting.

In the work presented, the function of the transit peptides and the mature parts of the TPT and the 37-kDa protein were characterized by gene fusion experiments. We have produced a series of chimeric proteins in which the transit peptides and mature parts of proteins of the envelope and thylakoid membranes were reciprocally exchanged and compared the import and intraorganellar routing of these chimeras with that of the corresponding authentic proteins.


MATERIALS AND METHODS

In Vitro Transcription/Translation

The DNA fragments were cloned into the vector pBSC (Stratagene) and transcribed using T7 or T3 RNA polymerases (Boehringer Mannheim). Translation was performed in a reticulocyte lysate system (Pro-mega, Heidelberg) in the presence of [S]methionine (Amersham, Braunschweig).

Construction of Fusion Proteins

The construction of recombinant cDNAs encoding chimeric proteins was carried out using standard procedures (Sambrook et al., 1989). The coding sequences of the TPT, the 37-kDa protein, and LHCP were amplified from cDNA clones by use of the polymerase chain reaction with Taq polymerase (Perkin Elmer-Cetus) essentially as described (Saiki et al., 1988). To generate the different fusion proteins consisting of reciprocally exchanged transit peptides, the published processing sites were taken as dividing points between the presequences and the mature parts (Willey et al., 1991; Dreses-Werringloer et al., 1991; Cashmore, 1984). The DNA fragments encoding the transit peptide and the mature part of the CF(0)II subunit of the ATP synthase were isolated from the corresponding gene cassettes (^2)that were generated according to the procedure described by Bartling et al.(1990) and Clausmeyer et al.(1993).

TPT-LHCP, TPT-37, 37-LHCP, 37-LHCP, 37-LHCP, 37-LHCP

The fusion proteins comprising the presequences of the TPT (80 amino acid residues) or N-terminal fragments of the 37-kDa protein (21 to 60 amino acid residues, 37, 37, 37, 37), respectively, fused to the mature LHCP or the mature 37-kDa protein (37), respectively, were constructed by polymerase chain reactions as follows. The DNA sequences encoding either the transit peptides or mature proteins were amplified from plasmids containing the TPT-full-length cDNA (1432 base pairs, Flügge et al.(1989)), the cDNA encoding the spinach 37-kDa protein (1223 base pairs, Dreses-Werringloer et al.(1991)), or the LHCP-cDNA (1020 base pairs, kindly provided by Dr. H. Paulsen, Munich, Germany), respectively. As forward and reverse primers in the polymerase chain reactions, oligonucleotides were used that are listed in Table 1. To generate cohesive ends, the amplified DNA fragment for transit peptides or mature proteins were digested with PstI or EcoRI, respectively, ligated reciprocally with each other, and cloned into the PstI/EcoRI-cut pBSC plasmid.



37-LHCP

The fusion protein 37-LHCP, comprising the 66 N-terminal amino acid residues of the 37-kDa protein fused to the mature part of LHCP, was constructed as described above except that a PstI/StuI fragment of the cDNA for the 37-kDa protein was used.

CF(0)II-TPT, CFII-37, 37-CFII, and 37-CFII

The DNA fragment encoding the transit peptide of CF(0)II (75 amino acid residues (Herrmann et al., 1993) was isolated as a EcoRI/NaeI fragment from the corresponding gene cassette,^2 fused in-frame to the DNA fragments encoding the mature parts of the TPT or the 37-kDa protein (37), respectively, and cloned into the SacI/EcoRI-cut pBSC plasmid. The mature CF(0)II was derived as a BamHI/HincII-fragment from the respective gene cassette^2 and fused in-frame to DNA fragments coding for the N-terminal parts of the 37-kDa protein (37, 37, see above) to generate 37-CF(0)II and 37-CF(0)II, respectively.

ATG-37

The DNA encoding the mature 37-kDa protein with an additional start methionine was amplified by a polymerase chain reaction from the pT7T3 plasmid containing the cDNA for the 37-kDa protein (Dreses-Werringloer et al., 1991). The oligonucleotides that were used as primers are listed in Table 1. The DNA fragment produced was treated with Klenow enzyme to get blunt ends and then inserted into the EcoRI site of the pBSC vector.

37

To remove the C-terminal hydrophobic region of the 37-kDa protein, the corresponding full-length cDNA (Dreses-Werringloer et al., 1991) was digested with BanII that cuts twice in the DNA, at nucleotide position 955 (amino acid residue 318) and 6 base pairs downstream of the stop codon. The larger of the two fragments generated was isolated and subsequently self-ligated.

Standard Protein Import Assay

The in vitro synthesized S-labeled proteins were used for protein uptake studies into isolated spinach chloroplasts (in organello experiments). The radiolabeled protein was added in a volume of reticulocyte lysate corresponding to 2% of the final volume of the import assay reaction. The import assay contained 250 mM sorbitol, 10 mM methionine, 25 mM potassium gluconate, 0.2% (w/v) bovine serum albumin, 2 mM MgSO(4), 50 mM HEPES-KOH (pH 8.0), 2 mM ATP, and intact, purified spinach chloroplasts equivalent to 200 µg of chlorophyll in a final volume of 300 µl. The import reaction was allowed to proceed for 20 min at 25 °C. Successful import was verified by subsequent treatment of the chloroplasts with protease as described in the legend to Fig. 1. For subfractionation of the chloroplasts into the different compartments, the chloroplasts were washed twice in medium B (0.33 M sorbitol, 50 mM HEPES-KOH (pH 8.0), 5 mM EGTA, 2 mM EDTA) and were then osmotically shocked by the addition of 500 µl of medium C (10 mM Tricine (pH 7.6), 0.6 mM MgCl(2), 1 mM phenylmethylsulfonyl fluoride). The chloroplasts were centrifuged for 30 s at 6700 g. The pellet representing the thylakoids was washed twice with medium B, resuspended in 100 µl of medium B, and 14 µl were analyzed by SDS-PAGE. The supernatant containing stroma and envelope membranes was centrifuged for 30 min at 100,000 g. The pellet representing enriched envelope membranes was washed once with medium B. The entire envelope membrane fraction (corresponding to 200 µg of chlorophyll), 14 µl of the thylakoid fraction (corresponding to 28 µg of chlorophyll), and 70 µl of the stromal fraction (corresponding to 28 µg of chlorophyll), respectively, were subsequently analyzed by SDS-PAGE and fluorography. SDS-PAGE, fluorography, and laser densitometry were done as published (Laemmli, 1970; Bonner and Laskey, 1974; Pfanner et al., 1987). To assess the membrane association of imported proteins, envelope membrane pellets were resuspended either in 200 µl of 100 mM Na(2)CO(3) (pH 11.5) or in 100 µl of 100 mM NaOH and incubated for 30 min at 0 °C. All samples were then centrifuged at 166,000 g for 60 min. The supernatant of the NaOH sample was subsequently neutralized by the addition of 100 µl of 100 mM HCl. The pellets and the proteins that had been precipitated from the supernatants by trichloroacetic acid (final concentration, 10%, v/v) were analyzed by SDS-PAGE and fluorography.


Figure 1: The transit peptide of the TPT functions as a stroma-targeting signal. In vitro-synthesized S-labeled precursor proteins (TPT, TPT-LHCP) were imported into intact chloroplasts as described under ``Materials and Methods.'' Before or after thermolysin treatment (100 µg/ml in the presence of 2 mM CaCl(2) for 30 min at 0 °C; protease -, +), EGTA was added (final concentration, 5 mM) and the chloroplasts were reisolated. Thylakoids (T) and envelope membranes (E) were isolated from the import assays, and aliquots were analyzed by SDS-PAGE (see ``Materials and Methods'') and fluorography. In lanes (t), 0.2 µl of the translation mixture were loaded. The positions of the precursor proteins (p) and of the mature proteins (mTPT, mLHCP) are indicated by arrows.



Processing Studies

The stromal processing protease was partially purified from pea chloroplasts according to Robinson and Ellis(1984). In vitro processing of radiolabeled proteins was carried out with 30 µl of the stromal extract containing the partially purified stromal processing protease and 10 µl of the translation mixture in a final volume of 50 µl of processing buffer (10 mM HEPES (pH 8.5), 0.4% chloramphenicol, 2 mM methionine). The processing reaction was carried out for 2 h at 28 °C, terminated by the addition of SDS-sample buffer (14 mM Tris/HCl (pH 6.8), 20% glycerol, 5% SDS, 2% 2-mercaptoethanol, 0.002% bromphenol blue), and analyzed by SDS-PAGE and fluorography.


RESULTS

The Transit Peptides of Chloroplast Envelope Membrane Proteins Possess Stroma-targeting Function

To assess the function of the transit peptides of envelope membrane proteins, we first generated a chimeric protein consisting of the transit peptide of the TPT (80 amino acid residues) fused to the mature part of the light-harvesting chlorophyll a/b binding protein (LHCP, 26 kDa; Clark and Lamppa(1991)), an integral component of the thylakoid membrane. LHCP is directed to the chloroplasts by a stroma-targeting transit peptide that is not required for the subsequent integration of the protein into the thylakoid membrane. This integration is mediated by signals present in the mature part of the protein (Lamppa, 1988; Viitanen et al., 1988; Hand et al., 1989; Auchincloss et al., 1992). The chimeric protein, TPT-LHCP, was synthesized by in vitro transcription/translation and imported into isolated chloroplasts. After the import reaction, the chloroplasts were fractionated into envelope membranes, stroma, and thylakoid membranes. Each of the fractions was subsequently analyzed for the presence of the imported protein. Since this fractionation inevitably results in a loss of envelope membranes to the thylakoid fraction (Murakami and Strotmann, 1978; Andrews and Keegstra, 1983), the authentic TPT was used as a control. TPT is exclusively located in the chloroplast envelope membrane, and, consequently, the amount of TPT found in the thylakoid fraction must be caused by contamination of the thylakoids with envelope membranes (roughly 70% of the envelope membranes are recovered with the thylakoids; Fig. 1). The chimeric protein TPT-LHCP of approx35 kDa was imported into chloroplasts and processed to a product of approx26 kDa, a size similar to that of the authentic LHCP mature protein (Fig. 1). Within the chloroplasts, the protein accumulated exclusively (quantitatively) in the thylakoid membrane, but not in the envelope membrane, indicating that, in combination with mature LHCP, the TPT-transit peptide acts as a chloroplast import signal and not as an envelope targeting signal.

To further check the targeting function of transit sequences of envelope membrane proteins, N-terminal parts of the 37-kDa inner envelope membrane protein were fused to the mature parts of LHCP and TPT, respectively. Previous studies suggested that the cleavage site within the 37-kDa protein is located between amino acid residues 21 and 22 of the precursor (Dreses-Werringloer et al., 1991). The N-terminal 21 amino acid residues were therefore used for the construction of the fusion proteins, 37-LHCP and 37-TPT, respectively. Import of the chimeric proteins into isolated chloroplasts and subsequent treatment of the chloroplasts with protease revealed that the proteins were only bound to, but not imported into, chloroplasts. In contrast to the authentic 37-kDa protein, they were neither inserted into the envelope membrane (Fig. 2A) nor imported into the other suborganellar fractions (not shown). The first 21 amino acid residues obviously do not contain sufficient information for the import of proteins into chloroplasts which suggests that the actual transit peptide of the 37-kDa protein may comprise more than the first 21 amino acid residues that were used in these chimeras. This assumption was confirmed by the analysis of a truncated 37-kDa protein lacking the N-terminal 21 amino acid residues (but containing an additional start methionine), ATG-37. Fig. 2B shows that (i) this protein was indeed larger than the mature 37-kDa protein (lanes 1 and 4) and (ii) it was processed in vitro by a partially purified stromal processing protease to a product that was identical in size not only to the in vitro processing product obtained with the authentic 37-kDa precursor protein (lanes 2 and 4) but also to the native 37-kDa protein isolated from envelope membranes (not shown). The functional transit peptide of the 37-kDa protein thus comprises more than 21 amino acid residues as previously thought (Dreses-Werringloer et al., 1991). Three N-terminal parts of increasing length (48, 54, and 60 amino acid residues) of the 37-kDa precursor protein were therefore used in subsequent experiments as ``transit peptides'' and fused to the LHCP mature protein.


Figure 2: A, the N-terminal 21 amino acid residues of the 37-kDa protein are not sufficient for translocation into chloroplasts. In vitro-synthesized S-labeled precursor proteins (37-kDa protein (37), 31-TPT, 31-LHCP) were imported into intact chloroplasts as described under ``Materials and Methods.'' Before or after thermolysin treatment (protease -, +), the envelope membranes were isolated from the import assays and analyzed by SDS-PAGE and fluorography. The positions of the precursor proteins (p) and of the mature proteins (m) are indicated by arrows. B, in vitro processing of the N-terminally truncated 37-kDa protein (ATG-37) and of the 37-kDa precursor protein by the stromal processing protease. Processing of the in vitro-synthesized S-labeled proteins by a partially purified stromal processing protease was carried out as described under ``Materials and Methods.'' At the times indicated, the reactions were terminated by the addition of SDS-sample buffer and analyzed by SDS-PAGE and fluorography. The position of the mature 37-kDa protein is indicated by an arrow. C, N-terminal parts of the 37-kDa protein contain stroma-targeting information. Import of the 37-kDa precursor protein (37) and of the chimeric proteins (37-LHCP, 37-LHCP, 37-LHCP) into chloroplasts was carried out as described under ``Materials and Methods.'' After treatment of the chloroplasts with thermolysin (100 µg/ml for 30 min), envelope membranes (E) and thylakoids (T) were isolated from the import assays and analyzed by SDS-PAGE and fluorography. The positions of the mature 37-kDa protein (m37) and of the mature LHCP protein (LHCP) are indicated by arrows.



Incubation of these chimeric precursor proteins (37-LHCP, 37-LHCP, 37-LHCP) with isolated chloroplasts demonstrated that each of the N-terminal extensions was able to translocate the passenger protein, LHCP, into the organelle. As shown in Fig. 2C, in all three instances the proteins accumulated as processing products in the thylakoids, but not in the envelope membrane. These results indicate that the transit peptide of the 37-kDa protein, like that of TPT, is a stroma-targeting import signal which is not sufficient to target a protein into the envelope membrane. The processing products obtained showed slightly increasing sizes for the hybrid proteins 37-LHCP, 37-LHCP, and 37-LHCP, respectively (Fig. 2C), indicating that in all three instances processing occurred most likely at the same cleavage site within the common N-terminal 48 residues that were derived from the 37-kDa precursor protein. Initial evaluation of the putative processing site of the 37-kDa precursor protein had been performed by radiosequencing of the in vitro-synthesized and processed protein since the N-terminal amino acid residue was found to be blocked (Dreses-Werringloer et al., 1991). Maximum values for the radioactivity released per cycle were obtained for cycles 2 and 15. Reinspection of the amino acid sequence of the protein revealed that these data were also compatible with a cleavage site between amino acid residues 46 and 47 (-Asn-Ser-ArgAsn-Leu-Arg) with leucine residues at amino acid positions 48 and 61. In conjunction with the experiments shown below (see Fig. 3), we suggest that the processing site of the 37-kDa protein is located between amino acid positions 46 and 47. Unfortunately, determination of the molecular mass of the mature protein by MALDI-TOF-MS, a procedure that would lead to an independent estimation of the cleavage site, was unsuccessful so far.


Figure 3: A, 37-CF(0)II accumulates in the thylakoid membrane after import into isolated chloroplasts. Import of the authentic CF(0)II protein (CF(0)II) and the chimeric 37-CF(0)II protein into chloroplasts was carried out as described under ``Materials and Methods.'' The chloroplasts were subsequently treated with thermolysin and fractionated into the thylakoids (T), envelope membranes (E), and the stroma (S). The different fractions were analyzed by SDS-PAGE and fluorography. In lanes (t), 0.2 µl of the translation mixture were loaded. The position of the precursor proteins (p) and of the processed proteins (mCF(0)II, mCF(0)II*) are indicated by arrows. B, 37-CF(0)II is processed by the stromal processing protease. In vitro processing experiments were carried out with in vitro-synthesized S-labeled precursor proteins (authentic CF(0)II precursor and 37-CF(0)II protein) as described under ``Materials and Methods.'' The reactions were terminated at the times indicated (in minutes) by the addition of SDS-sample buffer and analyzed by SDS-PAGE and fluorography. C, 37-CF(0)II is correctly integrated into the thylakoid membrane. In vitro-synthesized S-labeled precursor proteins (CF(0)II, 37-CF(0)II) were imported into intact isolated chloroplasts as described under ``Materials and Methods.'' The chloroplasts were osmotically lysed, and the thylakoid membranes obtained were incubated without or in the presence of thermolysin (100 µg/ml for 30 min; protease -, +). All samples were subsequently analyzed by SDS-PAGE (10-20% polyacrylamide) and fluorography. The positions of the precursor proteins (p) and mature proteins (m), as well as the specific protease-protected degradation products (m*), are indicated by arrows.



The CF(0)II Transit Peptide Can Functionally Be Replaced by the N-terminal Part of the 37-kDa Protein

To further characterize the targeting properties of the transit peptide from the 37-kDa protein, N-terminal fragments of 54 and 60 amino acid residues of the 37-kDa precursor were fused to the CF(0)II-subunit of the thylakoid ATP synthase. CF(0)II is an integral, bitopic thylakoid protein with a single membrane-spanning segment close to its lumen-located N terminus (Herrmann et al., 1993). It is synthesized in the cytosol as a precursor with a bipartite transit peptide carrying signals for the transport across the envelope membranes and the thylakoid membrane in tandem (Michl et al., 1994). Incubation of the resulting hybrid proteins (37-CF(0)II and 37-CF(0)II, respectively) with isolated intact chloroplasts showed that 37-CF(0)II was not imported into the organelle, whereas 37-CF(0)II accumulated after import almost exclusively in the thylakoid membrane (Fig. 3A), i.e. in the same location as the native CF(0)II protein. The size of the 37-CF(0)II chimera after import (mCF(0)II*, approx20 kDa) was approx1.5 kDa larger than the authentic mature CF(0)II of approx18.5 kDa (mCF(0)II, Fig. 3A). This corresponds well with the assumption that, after removal of the putative transit peptide of 46 residues by stromal processing protease (see above), 14 amino acid residues of the 37-kDa protein remained at the N terminus of CF(0)II. In vitro processing experiments using partially purified stromal processing protease confirmed this result: 37-CF(0)II protein was processed to a product identical in size to that obtained by import into isolated chloroplasts, whereas the authentic CF(0)II precursor which cannot be cleaved by stromal processing protease (Michl et al., 1994) remained unprocessed in this assay (Fig. 3B).

To test if the chimeric protein was properly integrated into the membrane, thylakoids were treated with protease after the import reaction. In the case of the authentic CF(0)II protein, this treatment led to a degradation of the stroma-exposed part and resulted in the appearance of a specific and indicative product of approx3 kDa that represents the membrane span and the lumen-located N terminus of the protein (m*, Fig. 3C).^2 After import of the chimeric 37-CF(0)II protein, protease treatment of the thylakoids yielded a degradation product of approx4.5 kDa (m*, Fig. 3C). The size of this degradation product correlates well with the approx3-kDa product observed for the native CF(0)II protein plus the additional 14 amino acid residues from the 37-kDa protein remaining at the N terminus of CF(0)II after processing suggesting that the chimera was correctly integrated into the thylakoid membrane.

The correct integration of 37-CF(0)II into the thylakoid membrane is remarkable in two respects. First, it confirms that the N-terminal region of the 37-kDa precursor protein is capable of importing hydrophobic passenger proteins into the chloroplast, and, second, it shows that it can functionally replace the bipartite transit peptide of the CF(0)II protein. Since this N-terminal segment does not have the typical structure of a bipartite transit peptide (von Heijne et al., 1989), it appears unlikely that it has thylakoid-targeting properties. Instead, we assume that the residual 14 residues lead, for as yet unknown reasons, to the suspension of the otherwise strict requirement for a transient hydrophobic domain during membrane integration of CF(0)II.^2 This phenomenon is currently under investigation.

The Mature Parts of Inner Envelope Proteins Determine Their Localization to the Chloroplast Envelope Membrane

The previous experiments show that the transit peptides of two envelope membrane proteins are capable of targeting passenger proteins into the chloroplast. Specific targeting of these proteins to the envelope membrane must therefore be assumed to be an inherent function of the mature proteins. To elucidate the targeting function of the mature envelope membrane proteins, mature TPT and a truncated 37-kDa precursor lacking the N-terminal 21 amino acid residues were fused to the thylakoid-targeting transit peptide of CF(0)II resulting in the chimeric proteins CF(0)II-TPT and CF(0)II-37, respectively.

Incubation of isolated, intact spinach chloroplasts with CF(0)II-TPT showed that the protein accumulated exclusively as the unprocessed precursor in the organelles (Fig. 4A). This indicates that CF(0)II-TPT has probably not reached the thylakoids, because the transit peptide of CF(0)II can only be removed by thylakoid processing protease (Michl et al., 1994). This was confirmed by fractionation of the chloroplasts after the import reaction which showed that the hybrid protein was found in exactly the same fractions as the authentic TPT which was analyzed in parallel. Both proteins were apparently quantitatively integrated into the envelope membrane, in contrast to CF(0)II which was found exclusively in the thylakoids after import (Fig. 4A). Thus, in spite of the thylakoid-targeting transit peptide at its N terminus, CF(0)II-TPT was efficiently integrated into the envelope membrane, proving that mature TPT carries all the information necessary for its envelope membrane-specific insertion.


Figure 4: A, the mature TPT contains information for membrane integration. In vitro-synthesized S-labeled precursor proteins (CF(0)II, CF(0)II-TPT, TPT) were imported into intact chloroplasts as described under ``Materials and Methods.'' After the import reaction, the chloroplasts were treated with thermolysin (100 µg/ml) for 30 min at 0 °C. Stroma (S), thylakoids (T), and envelope membranes (E) were isolated from the import assays and analyzed by SDS-PAGE and fluorography. The positions of the precursor protein (pCF(0)II-TPT) and of the mature proteins (mCF(0)II, mTPT) are indicated by arrows. B, The mature 37-kDa protein contains information for membrane integration. Import of the authentic 37-kDa protein (37) and of the chimeric CF(0)II-37 protein into chloroplasts was carried out as described under ``Materials and Methods.'' After treatment of the chloroplasts with thermolysin (100 µg/ml for 30 min), the chloroplasts were fractionated into thylakoids (T), envelope membranes (E), and the stroma (S), and the different fractions were analyzed by SDS-PAGE and fluorography. In lanes (t), 0.2 µl of the translation mixture were loaded. The position of the mature 37-kDa protein (m37) is indicated by an arrow. C, CF(0)II-37 is integrated into the envelope membrane. In vitro-synthesized S-labeled precursor proteins (37-kDa protein (37), CF(0)II-37) were imported in intact chloroplasts as described under ``Materials and Methods.'' The chloroplasts were subsequently treated with thermolysin (100 µg/ml for 30 min). Envelope membranes were isolated from the import assays and were treated either with 100 mM Na(2)CO(3) (pH 11.5) or 100 mM NaOH as described under ``Materials and Methods.'' The membrane pellets (p) and the supernatants (s) were subsequently analyzed by SDS-PAGE and fluorography. D, CF(0)II-37 is processed by the stromal processing protease. In vitro-synthesized S-labeled precursor proteins (CF(0)II, CF(0)II-37, TPT) were imported into intact chloroplasts as described under ``Materials and Methods'' in the absence(-) or presence (+) of 2 mM EDTA. After the import reaction, chloroplasts were treated with thermolysin (100 µg/ml) for 30 min at 0 °C. Thylakoids (T) or envelope membranes (E), respectively, were subsequently isolated from the import assays and analyzed by SDS-PAGE and fluorography. The positions of the precursor proteins (p) and mature proteins (m) are indicated by arrows.



Analogously, we studied the translocation of CF(0)II-37 into chloroplasts. As shown in Fig. 4B, CF(0)II-37 was processed to the 37-kDa mature protein and, like the authentic 37-kDa precursor protein, targeted exclusively to the envelope membrane. Thus, similar to CF(0)II-TPT, the imported protein reached its correct destination even if the import was mediated by the thylakoid-targeting transit peptide of CF(0)II. The protein was apparently correctly integrated into the envelope membrane, because it showed the same characteristics upon treatment of the membranes with alkaline reagents like the 37-kDa protein that was obtained from import of the authentic precursor. Both proteins were almost unaffected by 0.1 M carbonate treatment (pH 11.5) and were partially (approx70%) extracted by 0.1 M NaOH (Fig. 4C). Such treatments are known to extract soluble and peripheral proteins only, while integral membrane proteins remain associated with the membrane sheets (Fujiki et al., 1982). In contrast to the chimera CF(0)II-TPT, CF(0)II-37 was correctly processed to the size of the mature polypeptide. According to the results shown in Fig. 3B, this processing was probably performed by stromal processing protease. This was confirmed by import experiments in the presence of EDTA. Under these conditions, the maturation of the authentic CF(0)II precursor protein (that is cleaved by thylakoid processing protease only) was not affected due to the cation independence of the protease (Musgrove et al., 1989) (Fig. 4D). On the other hand, the processing of both the TPT precursor protein and CF(0)II-37 was inhibited by EDTA to a similar extent, confirming that maturation of CF(0)II-37 by stromal processing protease occurs at a processing site derived from the 37-kDa protein. Additional support for this conclusion comes from the observation that the size of this processed product was identical to that obtained with the authentic 37-kDa precursor protein (see Fig. 4B).

The C-terminal Hydrophobic Region of the 37-kDa Protein Is Involved in the Integration into the Envelope Membrane

The 37-kDa protein contains only a single hydrophobic segment that is long enough to span the membrane (Dreses-Werringloer et al., 1991). This segment is located near the C terminus and is assumed to anchor the protein within the inner envelope membrane. To test this hypothesis, a truncated protein (37) was generated lacking this hydrophobic segment. Import experiments showed that the protein accumulated in the chloroplasts as a processing product of approx36 kDa the size of which corresponds well with that expected after removal of the transit peptide. Within the chloroplasts, the truncated ``mature'' protein was found associated with both the thylakoid and the envelope membranes and, in part, even free in the stroma fraction (Fig. 5A), in contrast to the quantitative integration into the envelope membrane that is generally observed after import of the authentic 37-kDa precursor (Fig. 4B). Thus, it appears that the lack of the putative membrane anchor results in a loss of the specificity in the inner plastidar sorting of the 37-kDa protein. After its import into the chloroplast stroma, the truncated protein was apparently equally distributed to both kinds of chloroplast membranes. Since the lack of the membrane anchor must be assumed also to affect the quality of interaction with membranes, the question arises as to how deeply the truncated protein was still embedded into the envelope membrane. This was analyzed by incubation of the envelope membranes at alkaline pH after import reactions. Whereas the authentic 37-kDa protein was quantitatively recovered with the membrane pellet after treatment with 0.1 M Na(2)CO(3), the truncated protein was partially extracted (approx30%) by Na(2)CO(3) and became even totally soluble after treatment with 0.1 M NaOH (Fig. 5B). Thus, the C-terminal part of the protein plays a crucial role in both the correct intraorganellar sorting to and the anchoring of the protein within the envelope membrane of chloroplasts.


Figure 5: A, the C-terminal hydrophobic region of the 37-kDa protein is required for targeting to the inner envelope membrane. Import of the 37-kDa precursor protein (37) and of the C-terminally truncated 37-kDa protein (37) into chloroplasts was carried out as described under ``Materials and Methods.'' The chloroplasts were subsequently treated with thermolysin (100 µg/ml for 30 min) and fractionated into the stroma (S), the envelope membranes (E), and the thylakoids (T). The different fractions were analyzed by SDS-PAGE and fluorography. B, the C-terminal hydrophobic region of the 37-kDa protein anchors the protein in the envelope membrane. Envelope membranes containing the imported 37-kDa protein (37) or the C-terminally truncated 37-kDa protein (37) (see A) were treated either with 100 mM Na(2)CO(3) (pH 11.5) or 100 mM NaOH as described under ``Materials and Methods.'' The membrane pellets (p) and the supernatants (s) were subsequently analyzed by SDS-PAGE and fluorography.




DISCUSSION

In this paper, we address the question of how two nuclear-encoded inner envelope membrane proteins, the TPT and the 37-kDa protein, are targeted to the chloroplasts and which parts of the precursors, transit peptides or mature polypeptides, carry the signals that are responsible for the correct targeting to and into the inner envelope membrane. The results presented show unambiguously that the transit sequences of both envelope membrane proteins are able to import LHCP as a passenger protein into the chloroplast stroma from where it subsequently integrates into the thylakoid membrane. These stroma-targeting properties of the two transit peptides are particularly remarkable, because they do not possess the typical features of transit sequences of higher plant proteins which are destined to the stroma or the thylakoid membrane (von Heijne et al., 1989), but instead show the potential to form an amphiphilic alpha-helix like is frequently found in mitochondrial presequences. We have therefore proposed earlier that this structural element may serve as a ``membrane targeting domain'' (Dreses-Werringloer et al., 1991) and, indeed, both preproteins can efficiently be targeted to, and inserted into, the inner membrane of mitochondria from fungi in vitro (Brink et al., 1994). Although these results support a role of amphiphilic alpha-helices in recognition of preproteins by mitochondrial surface receptors, the chloroplast import experiments presented here show clearly that, in spite of their structural similarity to mitochondrial import sequences, the two transit peptides only serve to target proteins to the chloroplast stroma but not to the envelope membrane. It should be noted in this context that not all transit peptides of chloroplast envelope proteins necessarily have a structure that is similar to mitochondrial presequences. The precursor of the 2-oxoglutarate/malate translocator, an integral protein of the chloroplast envelope that is even more hydrophobic than the TPT, is synthesized with a transit peptide that closely resembles those of soluble chloroplast proteins (without an amphiphilic alpha-helix) but is nevertheless correctly targeted to the chloroplast envelope (Weber et al., 1995).

The second major outcome of the experiments presented is the finding that the information for the targeting of TPT and the 37-kDa protein to and into the envelope membrane is located in the respective mature parts of the two precursor proteins. Despite the presence of a thylakoid-targeting presequence of CF(0)II that had been attached to the mature proteins, both proteins were exclusively targeted to the envelope membrane. Thus, analogous to the findings for most integral thylakoid proteins, these envelope proteins also integrate apparently independent of their transit peptides via uncleaved targeting signals. A similar conclusion has been drawn from experiments with the maize Bt1 protein, a putative metabolite translocator protein from maize endosperm (Li et al., 1992). This protein can be imported into chloroplasts in vitro, and LHCP, when fused to the transit peptide of the Bt1 protein, was found associated with the thylakoids. Likewise, after fusion to the stroma-targeting transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase, the mature Bt1 protein was found after import in the envelope membrane. Although in this instance a putative envelope membrane protein from non-green plastids was analyzed with chloroplasts, the results obtained are in line with our observations that transit peptides of envelope membrane proteins often have a stroma-targeting function and that signals for integration of proteins into the inner envelope membrane of chloroplasts reside in the mature parts of these proteins.

The question arises by what mechanism the insertion of these proteins into the inner envelope membrane is achieved. For mitochondrial proteins, two models have been proposed, a ``conservative sorting pathway,'' i.e. the proteins are first completely imported into the organelle and then redirected into or across the membrane, and a ``nonconservative (stop-transfer) pathway'' in which the proteins are arrested in the membrane during the translocation process. A clear example for the conservative pathway is the Rieske Fe-S protein of complex III of the mitochondrial respiratory chain, a protein located on the outer surface of the inner mitochondrial membrane. The precursor is imported into mitochondria, processed to its mature-sized form which is then re-translocated across the inner membrane (Hartl et al., 1986). It is still a matter of controversy to what extent this mechanism also applies for intermembrane sorting to other proteins, cytochrome b(2) and cytochrome c(1). The hydrophobic segments contained in the bipartite transit sequences of these proteins were supposed to serve as stop-transfer signals resulting in the arrest of the translocated protein in the membrane and its subsequent release (van Loon and Schatz, 1987; Glick et al., 1992). Alternatively, these sequences could act as topogenic signals for redirecting the (not necessarily completely) imported protein across the inner mitochondrial membrane (Hartl et al., 1986, 1987; Gruhler et al., 1995).

The 37-kDa protein of the chloroplast envelope membrane structurally resembles the subunit Va of the cytochrome c oxidase of the inner mitochondrial membrane. Both proteins contain an organelle targeting presequence and a hydrophobic sorting sequence at the C terminus. It has been proposed that, during import, a yet unidentified component of the inner mitochondrial membrane binds to the subunit Va of the cytochrome c oxidase sorting sequence to direct it to the inner membrane (Gärtner et al., 1995). We have removed the C-terminal hydrophobic segment of the 37-kDa protein and analyzed the sorting of the truncated protein (37). It turned out that the truncated protein was indeed partially missorted to the stroma and the thylakoids, suggesting an important role of this hydrophobic segment in the envelope-targeting process. A fraction of the truncated protein was still found associated with the envelope membranes after import indicating that removal of the hydrophobic domain obviously did not cause the protein to be generally soluble. However, alkaline treatment of the membranes showed that the truncated protein was not correctly integrated into the envelope membrane. It can be concluded that the terminal hydrophobic region of the 37-kDa protein plays a crucial role also in its anchoring in the membrane although additional signals within the mature protein appear to be required for specific targeting to the envelope membrane. Analogous to the mitochondrial subunit Va of the cytochrome c oxidase protein, we assume a direct insertion mechanism for the chloroplast 37-kDa protein although the conservative sorting pathway can not be excluded by our experiments.

Hydrophobic proteins of the inner mitochondrial membrane, e.g. the ADP/ATP translocator, appear to be inserted into the inner membrane by a ``stop-transfer''-type of import mechanism. Complete translocation of the protein is prevented due to interaction of sorting signals with the components of the import apparatus, and the arrested protein leaves the import site by lateral diffusion without passing the matrix (Hartl and Neupert, 1990; Wachter et al., 1992; Glick et al., 1992). This mechanism might also hold true for the envelope membrane integration of hydrophobic chloroplast proteins like the TPT. The question then arises how specific sorting of nuclear-encoded hydrophobic proteins to either the envelope or the thylakoid membrane is achieved. It appears from hydrophobicity analyses (Kyte and Doolittle, 1982) of plastidial membrane proteins that, in general, the most hydrophobic thylakoid membrane proteins are coded for in the plastom (e.g. subunits I, III, and IV of the thylakoid ATPase; Hennig and Herrmann(1986)), suggesting that a high hydrophobicity might inhibit envelope translocation. On the other hand, the 22-kDa protein of photosystem II possesses at least three hydrophobic membrane-spanning segments (Wedel et al., 1992) and shows a higher hydrophobicity compared with that of the 37-kDa envelope membrane protein, but is nevertheless transported across the envelope membrane. Thus, although highly hydrophobic membrane proteins such as the TPT or the 2-oxoglutarate/malate translocator might not easily be transported across the envelope membrane, the degree in hydrophobicity cannot be the only determinant for the correct sorting to the different membranes in the chloroplast. Work is now in progress to elucidate the additional signals within the mature proteins that are involved in the membrane specificity of protein targeting within chloroplasts.


FOOTNOTES

*
This work was supported by Deutsche Forschungsgemeinschaft Grant Fl 126/3-3, SFB 184 and the Fonds der Chemischen Industrie. 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.

This work is dedicated to Prof. Dr. Johannes Willenbrink on the occasion of his 65th birthday.

§
Present address: Dept. of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.

To whom correspondence and reprint requests should be addressed. Tel.: 49-221-470-2484. Fax: 49-221-470-5039.

(^1)
The abbreviations used are: TPT, triose phosphate-3-phosphoglycerate-phosphate translocator from spinach chloroplasts; CF(0)II, subunit CF(0)-II of the ATP synthase from spinach chloroplasts; LHCP, light-harvesting chlorophyll a/b binding protein from pea chloroplasts; PAGE, polyacrylamide gel electrophoresis.

(^2)
D. Michl, D. Cai, E. Benitez, R. G. Herrmann, and R. B. Klösgen, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank D. Michl for the gene cassettes of the transit peptide and the mature part of CF(0)II.


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