©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Monomeric, Tightly Folded Stromal Intermediate on the pH-dependent Thylakoidal Protein Transport Pathway (*)

(Received for publication, August 9, 1994; and in revised form, November 2, 1994)

Alison M. Creighton Andrew Hulford Alexandra Mant David Robinson Colin Robinson (§)

From the Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two distinct mechanisms have been previously identified for the transport of proteins across the chloroplast thylakoid membrane, one of which is unusual in that neither soluble factors nor ATP are required; the system requires only the transthylakoidal DeltapH. We have examined this mechanism by studying the properties of one of its substrates: the extrinsic 23-kDa protein (23K) of photosystem II. Previous work has shown that this protein can be transported into isolated thylakoids as the full-length precursor protein; we show that the stromal import intermediate form of this protein is similarly translocation-competent. Gel filtration tests indicate that the stromal intermediate is probably monomeric. Protease sensitivity tests on both the initial in vitro translation product and the stromal import intermediate show that the presequence is highly susceptible to digestion whereas the mature protein is resistant to high concentrations of trypsin. The mature protein becomes very sensitive to digestion if unfolded in urea, or after heating, and we therefore propose that the natural substrate for this translocation system consists of a relatively unfolded presequence together with a tightly folded passenger protein. The ability of thylakoids to import pre-23K is destroyed by prior treatment of the thylakoids with low concentrations of trypsin, demonstrating the involvement of surface-exposed proteins in the import process. However, we can find no evidence for the binding of pre-23K or i23K to the thylakoid surface, and we therefore propose that the initial interaction of these substrates with the thylakoidal translocase is weak, reversible, and probably DeltapH-independent. In the second phase of the translocation mechanism, the DeltapH drives either the translocation and unfolding of proteins, or the translocation of a fully folded protein.


INTRODUCTION

Recent studies on the biogenesis of cytosolically synthesized thylakoidal proteins in chloroplasts have pointed to the operation of a remarkable variety of mechanisms for their transport into and across the thylakoid membrane. Most integral thylakoid membrane proteins are synthesized in the cytosol with stroma-targeting (or ``envelope transit'') signals, and thus the information specifying integration into the thylakoid membrane is localized in the mature proteins (Viitanen et al., 1988; Lamppa, 1988; Cai et al., 1993). In contrast, proteins destined for the thylakoid lumen are usually synthesized with bipartite presequences containing an envelope transit and a ``thylakoid transfer'' signal in tandem. The envelope transit signal functions to target the precursor protein into the stroma and is usually removed by a stromal processing peptidase; the transfer signal then directs translocation across the thylakoid membrane, after which complete maturation is carried out by a thylakoidal processing peptidase (Smeekens et al., 1986; Hageman et al., 1986; James et al., 1989; Ko and Cashmore, 1989).

The properties of thylakoid transfer signals have elicited a great deal of interest because they share key features with ``signal'' sequences which mediate protein transport across the bacterial plasma membrane, namely the presence of a hydrophobic core region and short chain residues at the -3 and -1 positions, relative to the terminal cleavage site (von Heijne et al., 1989; Bassham et al., 1991). These observations led to the proposal that the thylakoidal protein transport apparatus evolved from a translocation system in a cyanobacterial-type progenitor of the chloroplast, and functional similarities between bacterial and thylakoidal translocation systems have emerged from the demonstration that thylakoid transfer signals can direct protein export in Escherichia coli (Seidler and Michel, 1990) and from the finding that E. coli signal peptidase and pea thylakoidal processing peptidase have very similar reaction specificities (Halpin et al., 1989).

Because all thylakoid transfer signals contain apparently similar hydrophobic domains and processing sites, it was widely assumed for some time that they were transported across the thylakoid membrane by a common mechanism. Remarkably, several recent studies have shown that lumenal proteins are transported by two completely different translocation mechanisms and almost certainly by two distinct translocases. The advent of in vitro assays for the import of proteins by isolated thylakoids has shown that the transport of a subset of lumenal proteins, including the extrinsic 33-kDa photosystem II (PSII) (^1)protein (33K) and plastocyanin (PC), requires nucleoside triphosphates (NTPs) and a stromal protein factor (Hulford et al., 1994; Robinson et al., 1994). The transthylakoidal DeltapH is not a prerequisite for the translocation of these proteins, although it may stimulate the translocation rate (Theg et al., 1989; Cline et al., 1992). In contrast, other lumenal proteins, including the extrinsic 23- and 16-kDa photosystem II proteins (23K, 16K), photosystem I (PSI) subunit N, and photosystem II subunit T, do not require either NTPs or stromal factors for their transport across the thylakoid membrane; these proteins are transported by a mechanism which appears only to require the thylakoidal DeltapH (Mould and Robinson, 1991; Klösgen et al., 1992; Cline et al., 1992; Nielsen et al., 1994; Henry et al., 1994).

Several further approaches have been used to confirm the operation of separate translocation pathways for lumenal proteins. In studies on the biogenesis of four lumenal proteins, Cline et al.(1993) have shown that 23K competes only with 16K for transport across the thylakoid membrane, and 33K competes only with PC. This study provided strong evidence for the presence of parallel translocation pathways, and there are now clear indications that these lumenal proteins are indeed synthesized with two distinct types of thylakoid transfer signal; Robinson et al.(1994) have shown that PC is quantitatively diverted onto the DeltapH-dependent pathway if its presequence is replaced with that of 23K or 16K. Finally, it has been found that azide, a known inhibitor of Sec-dependent protein export in bacteria, also inhibits the transport of 33K and PC (but not 23K or 16K) across the chloroplast thylakoid membrane (Knott and Robinson, 1994; Henry et al., 1994). It therefore appears increasingly likely that 33K and PC are translocated by a Sec-related mechanism, whereas 23K, 16K, PSI-N, and PSII-T are transported by a mechanism with very different operational requirements.

An intriguing evolutionary twist to the story emerges from a simple analysis of the substrates for the two translocation mechanisms. Of the lumenal proteins listed above, only 33K and PC are present in cyanobacteria (the likely progenitors of chloroplasts), and in these prokaryotes they are synthesized with presequences which resemble both classical signal sequences and the thylakoid transfer signals of their higher plant counterparts (Kuwabara et al., 1987; Briggs et al., 1990). 23K, 16K, and PSI-N, on the other hand, are all absent in cyanobacteria (the situation with respect to PSII-T is currently unclear). Thus, the evolutionary development of these proteins in chloroplasts may well have been accompanied by the emergence of a novel, DeltapH-driven system for their transport across the thylakoid membrane, although it is also possible that this mechanism is in fact present in both cyanobacteria and chloroplasts, in which case the chloroplast-specific proteins may have ``chosen'' this system in preference to a Sec-related one.

Although the basic requirements for the 23K-type system have been mapped out (i.e. a continuous DeltapH is required but not stromal factors or ATP), very little is known about the actual translocation mechanism. In this study we have analyzed the conformation of the 23K substrate for the thylakoidal protein translocase, and we provide evidence that the stromal intermediate form of this protein is translocation-competent, monomeric, and comprises a relatively unfolded presequence and a tightly folded mature protein moiety. The results imply that the thylakoidal DeltapH drives either the translocation and concomitant unfolding of thylakoidal proteins or the translocation of fully folded proteins.


EXPERIMENTAL PROCEDURES

Chloroplast Isolation and Import Assays

Chloroplasts were isolated from 8-9-day-old pea seedlings (Pisum sativum, var. Feltham First) by Percoll pad centrifugation as described by Brock et al.(1993). Precursors of wheat 23K and 33K were synthesized by in vitro transcription of cDNA clones followed by translation in a wheat germ cell-free system in the presence of [S]methionine (James et al., 1989). Assays for chloroplast protein import were as described by Mould and Robinson(1991). After import incubations, stromal extracts were prepared by pelleting the organelles and lysing them in 10 mM Hepes-KOH, pH 8.0, 5 mM MgCl(2) (HM) for 10 min on ice, after which the lysate was centrifuged for 20 min at 30,000 times g to generate a stromal supernatant fraction.

Chromatography

The apparent molecular mass of i23K was estimated by calibrated gel filtration through a column (65 times 1.7 cm) of Sephacryl S300 (Pharmacia). Pre-23K was incubated with intact chloroplasts (1.2 ml total incubation volume) in the presence of 2 µM nigericin, after which the chloroplasts were treated with trypsin (50 µg/ml for 40 min on ice) and then an equivalent concentration of soybean trypsin inhibitor and 1 mM [4-(2-aminoethyl)-benzenesulfonylfluoride, HCl] (Calbiochem). The chloroplasts were pelleted and lysed in 250 µl of HM buffer, after which the sample was centrifuged at 13,000 times g for 30 min to generate the stromal fraction. 170 µl of extract was mixed with NaCl (75 mM), dithiothreitol (1 mM), and Pharmacia low molecular weight markers and loaded onto the column. 1.5-ml fractions were collected and concentrated by precipitation with trichloroacetic acid.

DeltapH Measurements and Protease Treatment of Thylakoids

DeltapH values were estimated by 9-aminoacridine fluorescence quenching as detailed in Brock et al.(1995). DeltapHs were set up in the dark by reverse action of the ATP synthase, using a method modified from that of Mills(1986). Intact pea chloroplasts were prepared at 1 mg/ml chlorophyll as described in Brock et al.(1993) and then diluted with 4 volumes of 30 mM Hepes-KOH, pH 8.2, 25 mM KCl, 6 mM MgCl(2), 10 mM dithiothreitol. The suspension was illuminated for 8 min at 4 °C with 70 watts/m^2 white light to activate the CF(1) ATPase activity, and the thylakoids were pelleted (5 min in a microcentrifuge), resuspended in HM buffer, and washed once in HM before being resuspended in HM to 0.2 mg/ml chlorophyll. Trypsin was added to the concentrations shown in Fig. 6, and the thylakoids were incubated in the dark, on ice, for 15 min. The thylakoids were then diluted with 20 volumes of HM containing 50 µg/ml soybean trypsin inhibitor, after which they were pelleted and resuspended in the same buffer to 0.2 mg/ml chlorophyll. The suspension was mixed with 0.1 volumes of pre-23K translation mix, and import reactions were carried out in the dark for 15 min at 24 °C following the addition of 0.5 mM MgATP; samples were processed as in Brock et al.(1993). For DeltapH measurements, the thylakoid/translation mix was diluted to 50 µg/ml chlorophyll, 0.5 mM MgATP was added, and the 9-aminoacridine fluorescence quenching monitored as in Brock et al.(1995).


Figure 6: Import of pre-23K into isolated thylakoids requires surface-exposed proteins. A, the ATPase activity of the CF(1) ATP synthase was activated using light and dithiothreitol as detailed under ``Experimental Procedures.'' Immediately after the activation process, the thylakoids were washed and incubated with trypsin at the indicated concentrations for 15 min on ice. The thylakoids were then incubated in the dark with 0.5 mM MgATP and the resulting DeltapH estimated by 9-aminoacridine fluorescence quenching. B, the thylakoidal ATPase activity was activated as in A, and thylakoids were treated with the indicated concentrations of trypsin for 15 min on ice. The thylakoids were then incubated with pre-23K in the presence of 0.5 mM MgATP as detailed under ``Experimental Procedures.'' Import efficiency was calculated by laser densitometry of the precursor and mature size bands on the fluorograms.



Other methods

Western blots were carried out using the enhanced chemiluminescence method (Amersham International) and the manufacturer's protocol. A fraction containing wheat mature 23K was prepared by isolating chloroplasts from 10-day-old wheat seedlings, generating thylakoid membranes by lysis and subsequent centrifugation, and sonicating the thylakoids for 60 s. The sonicate was then centrifuged at 40,000 times g for 60 min and the supernatant fraction used in Western blotting experiments. Pea thylakoidal processing peptidase was partially purified as described by James et al.(1989).


RESULTS

The Stromal i23K Polypeptide Accumulates in the Presence of Uncouplers in a Translocation-competent Form

In order to understand the DeltapH-driven mechanism in greater detail, we set out to determine the conformation and aggregation state of one of the substrates for the thylakoidal protein translocase. This can be achieved simply by examining the appropriate in vitro synthesized precursor proteins, since isolated thylakoids are capable of efficiently importing the full-length substrates for the DeltapH-driven mechanism. However, we considered it equally important to analyze the stromal intermediate forms following import into intact chloroplasts, because these forms are presumably the natural substrates for the translocase in vivo. Previous studies have shown that stromal factors are not required for the import of in vitro synthesized pre-23K or pre-16K into isolated thylakoids (Mould et al., 1991; Klösgen et al., 1992; Cline et al., 1992), but these studies did not rule out the possibility that stromal factors might bind to the intermediate forms in vivo. More importantly, none of the previous studies have examined the conformations of either the stromal intermediate forms or the in vitro synthesized full-length substrates for the translocase.

We addressed these points by analyzing the properties of pre-23K (i.e. full-length) and the stromal intermediate form of 23K (i23K) which accumulates in the presence of uncouplers, but before undertaking these experiments we addressed the question: is this accumulated stromal i23K form a bona fide intermediate on the translocation pathway? Wheat pre-23K was imported into intact pea chloroplasts for 20 min in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), and the chloroplasts were then washed to remove unbound precursor molecules. Fig. 1shows that the i23K polypeptide effectively accumulates over a 20-min period under these conditions, with no evidence for the presence of the mature size protein. Some pre-23K remains bound to the chloroplasts under these conditions but the quantities were insignificant relative to the amount of stromal i23K (data not shown). The light-driven proton motive force was then restored by the addition of bovine serum albumin (which has a high affinity for CCCP), and the remaining lanes show that the i23K form is rapidly and quantitatively chased into the mature size form. Control tests (not shown) confirmed that the i23K and mature size 23K were indeed in the stroma and thylakoid lumen, respectively, and we therefore conclude that the accumulated i23K is on the correct import pathway. Konishi and Watanabe(1993) have similarly shown that the stromal i23K polypeptide is a substrate for the transport apparatus, although in their study the DeltapH was generated by reverse action of the ATP synthase in the dark. These data indicate that both the pre-23K translation product, and the stromal i23K polypeptide are competent for translocation across the thylakoid membrane, and further tests were aimed at analyzing the properties of each of these polypeptides.


Figure 1: The stromal i23K protein which accumulates in the presence of uncouplers is competent for translocation across the thylakoid membrane. Pre-23K was incubated with pea chloroplasts in the presence of 3 µM CCCP, and samples were analyzed after 10 and 20 min. The chloroplasts were then re-isolated and bovine serum albumin (BSA; 1 mM) was added to the incubation mixture (at the time indicated by the arrow) and further samples were analyzed at time points (in minutes) indicated above the lanes. Samples of the chloroplasts were analyzed after protease treatment. Lane T, translation product. i23K and 23K, intermediate and mature forms of 23K, respectively.



The i23K Polypeptide Is Monomeric

Although several studies have shown that stromal extracts are not required for the import of 23K into isolated thylakoids (Mould et al., 1991; Cline et al., 1992), these studies did not rule out the possibility that the stromal i23K form binds to stromal proteins in the intact chloroplast. The two types of assay are not strictly comparable because, by presenting isolated thylakoids with in vitro synthesized pre-23K, any requirement for stromal factors may easily have been bypassed. We therefore examined the apparent native molecular mass of accumulated stromal i23K by subjecting it to calibrated gel filtration, and the results (shown in Fig. 2) indicate that the i23K elutes with an apparent molecular mass of about 36 kDa. The actual molecular mass of wheat i23K is about 25 kDa, and given that this technique is rarely completely accurate, we consider these data to strongly suggest that the stromal i23K is monomeric. Given that the Stokes radius of the i23K is slightly larger than expected, it remains possible that the i23K is bound to a stromal component of only low molecular weight, but we consider this to be unlikely. All of the obvious candidates for potential i23K-binding proteins (for example, Hsp 60, Hsp 70, SecA, SecB) are large molecules which would certainly shift the elution volume of the i23K by a very considerable extent, and Cline et al.(1993) reported that saturation of the thylakoidal protein transport system leads to the accumulation of a pea i23K form with a molecular mass of about 20 kDa, although the data were not shown in this report.


Figure 2: The stromal i23K polypeptide is probably monomeric. Stromal i23K was generated using CCCP in an import incubation as described under ``Experimental Procedures.'' The stromal fraction was mixed with molecular mass markers (Pharmacia low molecular weight) and subjected to gel filtration chromatography as detailed under ``Experimental Procedures.'' Fractions were collected and analyzed by SDS-polyacrylamide gel electrophoresis for the elution of i23K and the marker proteins. Peak elution volume was determined by laser densitometry of the fluorograms (for i23K) or stained gels (for markers). The graph shows the K plotted against log of the molecular mass of each of the four marker proteins (in kDa). K = (V - V(0))/(V - V(0)), where V = elution volume, V(0) = void volume, and V = total bed volume. The molecular mass of i23K is calculated to be 36 kDa.



23K Is Tightly Folded Prior to Translocation across the Thylakoid Membrane

Definitive structural and conformational studies are not possible with the in vitro synthesized 23K used in this study due to the low amounts present. However, studies on globular proteins in general have shown that protease resistance is often a good indicator of tightness of folding. A good case in point is dihydrofolate reductase, which is known to fold extremely tightly and which is accordingly very resistant to proteolysis; in the presence of the substrate analog, methotrexate, dihydrofolate reductase folds even more tightly and becomes correspondingly more resistant to proteolysis (Eilers and Schatz, 1986). Similar studies suggest that 23K is also a protein which folds tightly. Fig. 3A shows that when in vitro synthesized pre-23K is incubated with increasing concentrations of trypsin, the precursor is very easily cleaved to a polypeptide (sometimes a close doublet of polypeptides) with a mobility slightly greater than that of mature size 23K. This cleavage fragment (denoted F) has an apparent molecular mass of 22 kDa. However, even fairly high concentrations of trypsin (up to 7 µg/ml) fail to cleave 23K to lower molecular weight forms, and other tests have shown that the majority of pre-23K is still cleaved only to the 22-kDa form when incubated with 30 µg/ml trypsin under these conditions (data not shown). When trypsin is incubated with authentic mature wheat 23K, the protein is again cleaved to the 22-kDa form(s) (Fig. 3B). We interpret these results to indicate that the 23K presequence is relatively unfolded and hence easily digested by trypsin, whereas the mature protein is highly folded and thus resistant to digestion, with the exception of a short, 1-2-kDa section. The wheat 23K mature protein contains many potential trypsin cleavage sites (21 basic residues are present in the mature sequence; James and Robinson, 1991), and further tests were carried out to verify that these are indeed inaccessible because the protein is tightly folded. Pre-23K was unfolded in 8 M urea, and the mixture was then diluted to 2 M urea (the highest concentration at which we found trypsin to retain activity). Under these conditions the 23K is clearly at least partially unfolded, because it is extremely sensitive to proteolysis and is quantitatively digested even by low concentrations of protease (2.5 µg/ml; Fig. 4A). The control tests carried out in the absence of urea confirm that, as shown in Fig. 3, trypsin at up to 5 µg/ml is incapable of digesting pre-23K beyond the 22-kDa polypeptide. We have also found 23K to be highly sensitive to trypsin digestion after heating to 70 °C for 10 min (not shown), presumably because the protein has again been at least partially unfolded.


Figure 3: In vitro synthesized pre-23K consists of a protease-sensitive presequence and a protease-resistant mature protein. A, wheat pre-23K was synthesized in a wheat germ cell-free system, and equal aliquots of translation product were incubated with trypsin (concentrations indicated above the lanes) for 40 min on ice. Mature size 23K was generated by incubation of pre-23K with partially purified thylakoidal processing peptidase (TPP); the mobility of mature size 23K is indicated. F denotes a 22-kDa digestion fragment. B, a fraction containing authentic mature wheat 23K was prepared as described under ``Experimental Procedures,'' and trypsin was incubated with the fraction (at concentrations indicated above the lanes) for 40 min on ice. Wheat germ translation mix components were added so that the incubation conditions were similar to those in A.




Figure 4: Urea converts the 22-kDa polypeptide to a protease-sensitive conformation. A, pre-23K (lanes T) was incubated with trypsin (concentrations in µg/ml are given above the lanes) for 40 min on ice in the absence of urea, or after incubating pre-23K with 8 M urea for 30 min on ice, followed by dilution of the mixture 4-fold with 20 mM Tris-HCl, pH 8.0 (panel denoted + urea). Incubation conditions in the two mixtures were identical apart from the presence/absence of urea. B, pre-23K was incubated with trypsin at the indicated concentrations for 40 min at 25 °C. Symbols are the same as in Fig. 3.



Most of our tests on the protease sensitivity of 23K were carried out on ice. However, similar tests were carried out to exclude the possibility that 23K is partially unfolded at the temperatures used for import assays; Fig. 4B shows that the 22-kDa fragment is resistant to digestion with 15 µg/ml trypsin at 25 °C for 40 min. In other tests (not shown) we have found that equally high concentrations of thermolysin also generate a fragment of 22 kDa, and we therefore conclude that the 22-kDa polypeptide is highly resistant to proteolysis, and hence tightly folded, at both 0 and 25 °C.

Similar studies were carried out to assess the folding of the stromal i23K form which accumulates in the presence of uncoupler. Fig. 5shows the outcome of an experiment in which stromal i23K was incubated with increasing concentrations of trypsin (Fig. 5A) and in which the same concentrations of trypsin were incubated with a mixture of in vitro synthesized pre-23K and stromal extract as a control (Fig. 5B). The data show that given concentrations of trypsin cleave the two forms of 23K to a very similar extent, and high concentrations again generate only the 22-kDa form. The patterns of digestion products are furthermore almost identical (bearing in mind that no pre-23K band is present in the fluorogram shown in Fig. 5A; only i23K is incubated with trypsin). In this experiment it was not possible to confirm that the i23K becomes susceptible to digestion after heating or urea treatment, because both treatments led to extensive aggregation of the bulk stromal protein (not shown). Nevertheless, the data provide strong evidence that the mature 23K protein is tightly folded in both the initial in vitro translation product and in the authentic stromal intermediate form. A second experiment shown in Fig. 5C shows that a substantial proportion of i23K is converted to the 22-kDa polypeptide during digestion with as much as 30 µg/ml trypsin.


Figure 5: The stromal i23K form is protease-resistant. A, pre-23K was imported into intact chloroplasts in the presence of CCCP; the chloroplasts were then protease-treated, lysed, and centrifuged to generate a stromal fraction. Aliquots of the stromal fraction were incubated with trypsin at the given concentrations for 40 min on ice. An aliquot of the membrane fraction from an import assay containing no uncoupler was loaded to provide a mature size marker (lane m). B, in vitro synthesized pre-23K was mixed with stromal extract at a protein concentration identical to that in A, and trypsin added at the indicated concentrations. C, pre-23K was imported into chloroplasts in the presence of CCCP and the stromal fraction containing i23K (lane i) was incubated with trypsin at concentrations indicated above the lanes (in µg/ml). Lane m, mature size marker as in A. Symbols are the same as in Fig. 3and Fig. 4.



Translocation of Pre-23K Requires Surface-exposed Thylakoidal Proteins

Recent work by Brock et al.(1995) has shown that the 23K substrate for the DeltapH-driven system is soluble in the stroma at all values of DeltapH, with no evidence for even weak binding of i23K to receptors on the thylakoid surface in intact chloroplasts. This finding is surprising in view of the efficiency with which the DeltapH-driven system translocates proteins and raises the possibility that typical receptor proteins are not present on the thylakoid surface. In fact, the only evidence to date for the presence of translocase proteins as such is the observation by Cline et al.(1993) that the translocation mechanism is saturable. This finding, however, represents rather indirect evidence for the existence of translocase proteins and, more importantly, gives no information as to the location of the putative proteins in the thylakoid membrane. We therefore addressed this problem directly by testing whether the putative translocase is sensitive to treatment of thylakoids with low concentrations of protease. Preliminary tests (not shown) indicated that after treatment of thylakoids with even low concentrations of trypsin or thermolysin (10 µg/ml), the thylakoids were completely unable to generate a DeltapH by photosynthetic electron transport; this treatment is therefore unsuitable for examining the role of translocator proteins. However, a DeltapH can also be generated by reverse action of the ATP synthase in the dark, and we have found that this protein is much more resistant to proteolysis. Fig. 6A shows that low concentrations of trypsin have no significant effect on the ATPase activity, and DeltapH values over 2.0 are still generated after treatment with as much as 70 µg/ml trypsin. When generated by light, this magnitude of DeltapH is sufficient to drive import of pre-23K at high rates (Brock et al., 1995), and Fig. 6B demonstrates that import into thylakoids is also efficient when driven in the dark by this procedure. The figure also shows that import is totally abolished by pretreatment of the thylakoids with low concentrations of trypsin, with even 20 µg/ml sufficient to block import. Similar concentrations of thermolysin are equally effective in blocking import (data not shown). Since the DeltapH remains high under these conditions, we conclude that translocation is absolutely dependent on the presence of surface-exposed components of the translocase. In turn, these data strongly suggest that the initial interaction of i23K with these components is weak, reversible, and probably DeltapH-independent.


DISCUSSION

In this work we have sought to examine in greater detail the DeltapH-driven mechanism by which proteins are transported across the thylakoid membrane. This translocation system is an interesting experimental system because the data from previous studies have suggested that its mechanism of action may be unique among known protein translocation systems. Neither soluble factors nor ATP are required for translocation of proteins such as 23K into isolated thylakoids (Mould et al., 1991; Cline et al., 1992; Klösgen et al., 1992), and the thylakoidal DeltapH appears to be the only requirement. Surprisingly, even relatively low magnitudes of DeltapH are sufficient to support high rates of translocation (Brock et al., 1995). Clearly, a detailed understanding of the biogenesis of this group of lumenal proteins is totally dependent on understanding how the DeltapH drives translocation, and within this context a knowledge of the conformation of the pre-23K or i23K forms prior to transport across the thylakoid membrane is essential. Our data strongly indicate that the soluble 23K substrates for the thylakoidal protein transport machinery are highly folded, and we therefore propose that the unfolding of these proteins is likely to take place during translocation across the thylakoid membrane.

Several lines of investigation support the above proposal. First, the lack of requirement for stromal factors suggests that the pre-23K presented to isolated thylakoids consists of a folded mature 23K moiety with a relatively unfolded presequence. There is abundant evidence that chloroplast protein presequences are substantially, if not completely unfolded (von Heijne and Nishikawa, 1991; Theg and Geske, 1992; Pilon et al., 1992), and it is therefore to be expected that they are very susceptible to proteolysis. On the other hand, there is no evidence for the presence of a factor in cell-free translation systems which maintains chloroplast protein precursors in an unfolded conformation. On the contrary, the precursor of 5-enolpyruvylshikimate-3-phosphate synthase was found to be catalytically active, and thus presumably correctly folded, after synthesis in a reticulocyte lysate (Della-Cioppa et al., 1986). In a similar vein, an artificial construct comprising a chloroplast protein presequence followed by dihydrofolate reductase was shown to bind substrate analogs after synthesis in a wheat germ lysate, again indicating the presence of a correctly folded passenger protein (America et al., 1994). In the case of 23K, there is no assay for biological activity, but the protease sensitivity data show that the mature 23K protein is far more sensitive to digestion by trypsin after incubation with urea or at high temperatures. In the absence of these treatments, mature size 23K is resistant to concentrations of trypsin which would normally digest a wide variety of precursor proteins to completion. Strictly speaking, this observation indicates that 23K adopts a different conformation after heat or urea treatment, but obviously the most likely interpretation of these results is that mature 23K folds tightly after synthesis in cell-free translation systems. In our experience, dihydrofolate reductase is the only other mature size protein that can withstand incubation with 30 µg/ml trypsin for 40 min; this protein is known to fold very tightly, and the evidence indicates that 23K is a protein which likewise folds particularly tightly.

The data obtained from studies on the stromal i23K polypeptide are entirely consistent with this hypothesis. Fractionation data indicate that the protein is almost certainly monomeric, which would in itself suggest that the 23K has probably refolded after transport across the envelope membranes. Furthermore, the protease sensitivity patterns closely resemble those obtained in studies on the in vitro synthesized precursor protein; the mature 23K moiety is likewise particularly resistant to digestion over a similar range of protease concentrations, strongly suggesting that it has adopted a similar conformation. Since the i23K polypeptide is competent for translocation across the thylakoid membrane, we propose that the natural substrate for the DeltapH-driven thylakoidal protein transport machinery consists of a folded 23K mature protein together with an extended thylakoid transfer signal. The conformation of 23K during translocation across the thylakoid membrane is not known, but studies on every other protein-translocating membrane analyzed to date have shown that the proteins are transported in a substantially unfolded state.

As yet, we do not know how protein translocation is harnessed to the DeltapH, but it is clear that surface-exposed thylakoidal proteins are involved in the translocation process. Perhaps surprisingly, however, there is no evidence for the binding of i23K to receptors on the thylakoid surface; recent studies using intact chloroplasts (Brock et al., 1995) have shown that i23K is soluble in the stroma at all values of DeltapH tested. Similarly, we have failed to detect any binding of pre-23K to receptors in studies with isolated thylakoids. Following incubation of pre-23K with thylakoids in the presence of nigericin, a small proportion of precursor was in fact found to be associated with the thylakoids, but an identical amount of binding was observed with protease-treated thylakoids, and we therefore concluded that the binding was entirely nonspecific (data not shown). In view of these results, we propose a basic two-step model for the translocation mechanism. The first step involves the binding of i23K or pre-23K to the translocase, and we believe that this binding is weak, reversible, and probably DeltapH-independent. The second step involves the translocation of the protein across the membrane, in which the DeltapH most likely provides the driving force for both translocation and unfolding of the 23K mature protein.

Whatever the conformation of 23K during transport across the thylakoid membrane, the emerging evidence indicates that the DeltapH-driven protein transport system is increasingly unusual among known protein transport systems. All other known mechanisms require the presence of nucleotide triphosphates, except in certain unusual circumstances (Deshaies et al., 1988; Meyer, 1988; Wickner et al., 1991; Theg et al., 1989; Hulford et al., 1994) and a second recurring theme in other systems is that proteins are generally maintained in a relatively unfolded (and hence translocation-competent) state during passage through soluble phases of the cell or organism. In bacteria, precursor proteins on the Sec-dependent pathway are usually bound by the cytosolic chaperone protein SecB, which prevents folding of the precursor proteins before they reach the export machinery in the plasma membrane (reviewed by Wickner et al.(1991)). Signal recognition particle performs a similar role in the cytosol of eukaryotic cells, by arresting the translation of secretory proteins until co-translational translocation across the endoplasmic reticulum can take place (reviewed by Luirink and Dobberstein(1994)). Against this background, the occurrence of stable, folded intermediates on the DeltapH-dependent thylakoidal protein transport pathway is most unexpected and provides a further indication that this mechanism operates in a different manner.

In some respects the thylakoidal translocation mechanism may more closely resemble that of the chloroplast envelope-localized protein translocase, because the precursor proteins for both systems are likely in some cases to comprise protease-sensitive presequences and folded mature proteins. For example, pre-23K synthesized in a wheat germ lysate can be efficiently imported by both chloroplasts and thylakoids. However, there may be fundamental differences between the two translocation mechanisms. There is evidence that at least some precursor proteins may be unfolded on the chloroplast surface prior to transport across the envelope membranes in an unfolded/partially folded state (America et al., 1994; Schnell et al., 1990), although pre-23K has not been analyzed in this respect. This binding/unfolding process is very stable and ATP-dependent. In contrast, any unfolding process occurs during translocation of 23K by the DeltapH-dependent thylakoidal system (it remains to be determined whether other substrates for this system contain folded mature protein moieties). What is clear is that the substrates for the thylakoidal system do not bind stably, and translocation is ATP-independent. We therefore suggest that the thylakoidal system represents a novel class of protein translocase, in which the DeltapH is harnessed to drive either the translocation and unfolding of proteins or the translocation of fully folded proteins.


FOOTNOTES

*
This work was supported by Agricultural and Food Research Council Grants PG88/517 and PG88/524 and by Science and Engineering Research Council Grant GR/H99387. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44-203-523557; Fax: 44-203-523701.

(^1)
The abbreviations used are: PSI and PSII, photosystems I and II, respectively; PC, plastocyanin; 33K, 23K, 16K, the 33-, 23-, and 16-kDa proteins of the photosystem II oxygen-evolving complex; CCCP, carbonyl cyanide m-chlorophenylhydrazone.


ACKNOWLEDGEMENTS

We are extremely grateful to Steve Theg and Bill Ettinger for providing a protocol for restoring the DeltapH after CCCP treatment.


REFERENCES

  1. America, T., Hageman, J., Guera, A., Rook, F., Archer, K.. Keegstra, K., and Weisbeek, P. (1994) Plant Mol. Biol. 24, 283-294 [Medline] [Order article via Infotrieve]
  2. Bassham, D. C., Bartling, D., Mould, R. M., Dunbar, B., Weisbeek, P., Herrmann, R. G., and Robinson, C. (1991) J. Biol. Chem. 266, 23606-23610 [Abstract/Free Full Text]
  3. Briggs, L. M., Pecararo, V. L., and McIntosh, L. (1990) Plant Mol. Biol. 15, 633-642 [Medline] [Order article via Infotrieve]
  4. Brock, I. W., Hazell, L., Michl, D., Nielsen, V. S., Møller, B. L., Herrmann, R. G., Klösgen, R. B., and Robinson, C. (1993) Plant Mol. Biol. 23, 717-725 [Medline] [Order article via Infotrieve]
  5. Brock, I. W., Mills, J. D., Robinson, D., and Robinson, C. (1995) J. Biol. Chem. 270, 1657-1662 [Abstract/Free Full Text]
  6. Cai, D., Herrmann, R. G., and Klösgen, R. B. (1993) Plant J. 3, 383-392
  7. Cline, K., Ettinger, W., and Theg, S. M. (1992) J. Biol. Chem. 267, 2688-2696 [Abstract/Free Full Text]
  8. Cline, K., Henry, R., Li, C., and Yuan, J. (1993) EMBO J. 12, 4105-4114 [Abstract]
  9. Della-Cioppa, G., Bauer, S. C., Klein, B., Shah, D. M., Fraley, R. T., and Kishore, G. M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6873-6877 [Abstract]
  10. Deshaies, R. J., Koch, B. D., and Schekman, R. (1988) Trends Biochem. Sci. 13, 384-388 [CrossRef][Medline] [Order article via Infotrieve]
  11. Eilers, M., and Schatz, G. (1986) Nature 322, 228-232 [Medline] [Order article via Infotrieve]
  12. Hageman, J., Robinson, C., Smeekens, S., and Weisbeek, P. (1986) Nature 324, 567-569
  13. Halpin, C., Elderfield, P. D, James, H. E., Dunbar, B., Zimmermann, R., and Robinson, C. (1989) EMBO J. 8, 3917-3921 [Abstract]
  14. Henry, R., Kapazoglou, A., McCaffrey, M., and Cline, K. (1994) J. Biol. Chem. 269, 10189-10192 [Abstract/Free Full Text]
  15. Hulford, A., Hazell, L., Mould, R. M., and Robinson, C. (1994) J. Biol. Chem. 269, 3251-3256 [Abstract/Free Full Text]
  16. James, H. E., and Robinson, C. (1991) Plant Mol. Biol. 17, 179-182 [Medline] [Order article via Infotrieve]
  17. James, H. E., Bartling, D., Musgrove, J. E., Kirwin, P. M., Herrmann, R. G, and Robinson, C. (1989) J. Biol. Chem. 264, 19573-19576 [Abstract/Free Full Text]
  18. Klösgen, R. B., Brock, I. W., Herrmann, R. G., and Robinson, C. (1992) Plant Mol. Biol. 18, 1031-1034 [Medline] [Order article via Infotrieve]
  19. Ko, K., and Cashmore, A. R. (1989) EMBO J. 8, 3187-3194 [Abstract]
  20. Konishi, T., and Watanabe, A. (1993) Plant Cell Physiol. 34, 315-319
  21. Knott, T. G., and Robinson, C. (1994) J. Biol. Chem. 269, 7843-7846 [Abstract/Free Full Text]
  22. Kuwabara, T., Reddy, K. J., and Sherman, L. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8230-8235 [Abstract]
  23. Lamppa, G. K. (1988) J. Biol. Chem. 263, 14996-14999 [Abstract/Free Full Text]
  24. Luirink, J. L., and Dobberstein, B. (1994) Mol. Microbiol. 11, 9-13 [Medline] [Order article via Infotrieve]
  25. Meyer, D. I. (1988) Trends Biochem. Sci. 13, 471-474 [Medline] [Order article via Infotrieve]
  26. Mills, J. D. (1986). in Photosynthesis Energy Transduction: A Practical Approach (Hipkins, M. F., and Baker, N. R., eds) pp. 143-187, IRL Press, Oxford
  27. Mould, R. M., and Robinson, C. (1991) J. Biol. Chem. 266, 12189-12193 [Abstract/Free Full Text]
  28. Mould, R. M., Shackleton, J. B., and Robinson, C. (1991) J. Biol. Chem. 266, 17286-17290 [Abstract/Free Full Text]
  29. Nielsen, V. S., Mant, A., Knoetzel, J., Møller, B. L., and Robinson, C. (1994) J. Biol. Chem. 269, 3762-3766 [Abstract/Free Full Text]
  30. Pilon, M., Rietveld, A. G., Weisbeek, P., and de Kruijff, B. (1992) J. Biol. Chem. 267, 19907-19913 [Abstract/Free Full Text]
  31. Robinson, C., Cai, D., Hulford, A., Hazell, L., Michl, D., Brock, I., Schmidt, I., Herrmann, R. G., and Klösgen, R. B. (1994) EMBO J. 13, 279-285 [Abstract]
  32. Schnell, D. J., Blobel, G., and Pain, D. (1990) J. Cell Biol. 111, 1825-1838 [Abstract]
  33. Seidler, A., and Michel, H. (1990) EMBO J. 9, 1743-1748 [Abstract]
  34. Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375 [Medline] [Order article via Infotrieve]
  35. Theg, S. M., and Geske, F. J. (1992) Biochemistry 31, 5053-5060 [Medline] [Order article via Infotrieve]
  36. Theg, S. M., Bauerle, C., Olsen, L., Selman, B., and Keegstra, K. (1989) J. Biol. Chem. 264, 6730-6736 [Abstract/Free Full Text]
  37. Viitanen, P. V., Doran, E. R., and Dunsmuir, P. (1988) J. Biol. Chem. 263, 15000-15007 [Abstract/Free Full Text]
  38. Von Heijne, G., and Nishikawa, K. (1991) FEBS Lett. 278, 1-3 [CrossRef][Medline] [Order article via Infotrieve]
  39. Von Heijne, G., Steppuhn, J., and Herrmann, R. G. (1989) Eur. J. Biochem. 180, 535-545 [Abstract]
  40. Wickner, W., Driessen, A., and Hartl, F.-U. (1991) Annu. Rev. Biochem. 60, 1165-1172

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.