©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The pH-driven, ATP-independent Protein Translocation Mechanism in the Chloroplast Thylakoid Membrane
KINETICS AND ENERGETICS (*)

(Received for publication, August 1, 1994)

Ian W. Brock (1) John D. Mills (2) David Robinson (1) Colin Robinson (1)(§)

From the  (1)Department of Biological Sciences, University of Warwick, Coventry CV4 7AL and the (2)Department of Biological Sciences, Keele University, Staffs ST5 5BG, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous studies have shown that proteins are transported across the chloroplast thylakoid membrane by two very different mechanisms, one of which requires stromal factors and ATP, whereas the other mechanism is ATP independent but completely reliant on the thylakoidal DeltapH. We have examined the role of the DeltapH in the latter mechanism by simultaneously monitoring the magnitude of DeltapH (by 9-aminoacridine fluorescence quenching) and the rate of import of the 23-kDa photosystem II protein into isolated pea thylakoids. We show that protein import can take place, at low but significant rates, at very low values of DeltapH (in the region of 1.2-1.4), and that plots of the rate of protein import against proton concentration gradient are probably hyperbolic in nature. There is no evidence for a threshold level of DeltapH which is required to drive translocation of the 23-kDa protein. Addition of uncouplers midway during import incubations results in a rapid and complete inhibition of translocation, showing that the continuous presence of the DeltapH is required for translocation to take place. During import into intact chloroplasts, the intermediate-size 23-kDa protein substrate for the thylakoidal protein transport machinery is found only in the stromal fraction at all values of DeltapH, suggesting that the initial interaction with the machinery is relatively weak, reversible and DeltapH-independent. We therefore propose that the DeltapH is required for both the initiation and completion of translocation; these roles are in marked contrast to the roles of protonmotive force in mitochondrial and sec-dependent bacterial protein transport.


INTRODUCTION

Extensive efforts have been made to understand the mechanisms by which proteins are translocated across biological membranes, and in vitro translocation assays have been developed for the analysis of most of the known protein-translocating membranes. Several unifying features have emerged from comparisons of the different mechanisms, among which is the observation that some form of energy input is always required to drive the translocation process. Studies on the transport of proteins across the endoplasmic reticulum, chloroplast and mitochondrial envelopes, and bacterial cytoplasmic membranes have shown that ATP (or GTP) is critical for the operation of each of these translocation mechanisms, other than in exceptional circumstances (Theg et al., 1989; Wickner, 1988; Meyer, 1988; Deshaies et al., 1988). In addition, protein transport tends to be coupled to a transmembrane protonmotive force (Deltap) if one is available; mitochondrial protein import is heavily dependent on the electrical potential (Delta) across the inner membrane (Pfanner and Neupert, 1985) and bacterial protein export by the Sec-dependent pathway requires the Deltap across the cytoplasmic membrane (reviewed by Driessen, 1992).

The thylakoid membrane has proved to be an interesting, and surprisingly complex, system for the study of protein translocation. Initially, it was thought that proteins were transported across the chloroplast thylakoid membrane by a mechanism akin to the Sec-dependent pathway because virtually all imported lumenal proteins are synthesized with bipartite presequences containing an ``envelope transit'' domain which targets the precursor into the stroma, and a ``thylakoid transfer'' signal which resembles bacterial export (or signal) peptides in several respects (von Heijne et al., 1989; Bassham et al., 1991). More recent studies using assays for the import of proteins by isolated thylakoids suggest that a subset of proteins are indeed probably transported by a sec-type mechanism: the import of plastocyanin and the extrinsic 33-kDa photosystem II protein (33K) (^1)into isolated thylakoids requires a stromal protein factor and ATP (Hulford et al., 1994; Robinson et al., 1994), just as bacterial protein export requires a soluble factor (SecB) and ATP for the function of the translocation ATPase, SecA (reviewed in Wickner et al., 1991). Furthermore, the SecA inhibitor azide has been shown to block the translocation of plastocyanin and 33K across the thylakoid membrane (Knott and Robinson, 1994). Definitive evidence for the operation of a sec-related pathway in chloroplasts is still lacking, but the circumstantial evidence is fairly convincing at the present time.

The major surprise to emerge from recent studies is that another mainstream thylakoidal protein transport mechanism operates in chloroplasts, which is fundamentally different in several respects to the putative sec-type mechanism. The extrinsic 23- and 16-kDa photosystem II subunits (23K and 16K), photosystem II subunit T (PSII-T), and photosystem I subunit N (PSI-N) are transported by a mechanism in which stromal factors and ATP are completely unnecessary, and which appears instead to be completely dependent on the thylakoidal DeltapH (Mould and Robinson, 1991; Mould et al., 1991; Cline et al., 1992; Klösgen et al., 1992; Henry et al., 1994; Nielsen et al., 1994). The DeltapH is not a prerequisite for the transport of plastocyanin or 33K by the putative sec-type mechanism, although it may stimulate translocation (Theg et al., 1989; Cline et al., 1992). Competition studies (Cline et al., 1993) and studies on the translocation properties of fusion proteins (Robinson et al., 1994) have provided further compelling evidence for the existence of distinct translocation systems for thylakoid proteins.

It is far from clear why proteins are transported across the thylakoid membrane by two such different mechanisms, but there are some clues as to the possible origins of the two mechanisms. It appears likely that the putative sec-related mechanism was inherited from the cyanobacterial-type progenitor of the chloroplasts, since both 33K and plastocyanin are present in cyanobacteria, where they are probably transported across the thylakoid membrane by such a mechanism (Kuwabara et al., 1987; Briggs et al., 1990). In contrast, 23K, 16K, and PSI-N are all absent from cyanobacteria (the situation regarding PSII-T is unclear) suggesting that the development of these proteins has occurred relatively recently in phylogenetic terms. The implications for the evolution of the chloroplast thylakoidal protein transport mechanisms are fascinating: there is a good chance that the emergence of these proteins in chloroplasts has been accompanied by the emergence of a novel system for their transport across the thylakoid membrane. Alternatively, the DeltapH-driven system may in fact be present in cyanobacteria, in which case the above proteins may have been preferentially transported by this system, for unknown reasons.

The mechanism of action of the DeltapH-driven system is of particular interest because it is unique among known translocation mechanisms in being ATP independent, and hence represents an excellent (and relatively simple) system for studying the energetics of protein translocation. In this report we describe kinetic data on the role of the DeltapH as a driving force for the translocation of 23K across the thylakoid membrane.


MATERIALS AND METHODS

Thylakoids were isolated from 8 to 9-day-old pea seedlings as described (Brock et al., 1993). The thylakoids were washed twice in 10 mM HEPES-KOH, pH 8.0, 5 mM MgCl(2) and resuspended in the same buffer to a concentration of 50 µg/ml chlorophyll. Wheat pre-23K was synthesized by in vitro transcription of a full-length cDNA clone followed by translation in a wheat germ cell-free system (Promega) in the presence of [S]methionine. Thylakoid import assays were basically as described in Brock et al.(1993) except that incubation mixtures contained 940 µl of thylakoid suspension and 60 µl of translation product. Under these conditions, the concentration of K is approximately 10 mM and that of dithiothreitol is 0.6 mM. Mixtures were incubated at 25 °C for periods indicated in the figure legends, after which samples were analyzed directly or after incubation of the thylakoids with 0.2 mg/ml thermolysin for 30 min on ice. DeltapH was estimated by 9-aminoacridine fluorescence quenching as described by Mills(1986) except that the 9-aminoacridine concentration was 5 µM. The intensity of the measuring beam was 0.1-0.2 watts/m^2, and the DeltapH was driven by a quartz halogen lamp filtered through a Corning 2-62 glass filter. DeltapH was calculated from the fractional quenching, q, according to DeltapH = log [q/(1 - q)] + log (V/v) where V is total reaction volume and v is the internal volume of the thylakoids (assumed to be 10 µl/mg chlorophyll). The magnitude of DeltapH was altered by changing the intensity of the incident light, and import reactions were carried out in a random order to obviate any effects due to changes in the import competence of the thylakoids during storage on ice.

Chloroplast import assays and fractionation procedures were as described in Nielsen et al.(1994). Nigericin was added from a 100 times stock in ethanol, together with 10 mM KCl; all incubation mixtures contained identical concentrations of ethanol and KCl. ATP synthesis was measured by the luciferin/luciferase method as described by Mills(1986). Fluorograms were quantitated by laser densitometry using a Molecular Dynamics Imagequant v. 3.0.


RESULTS

The Experimental System

The primary aim in this study was to determine the relationship between DeltapH and rate of protein transport, by simultaneously monitoring the thylakoidal DeltapH and the ongoing rate of 23K import. We used an in vitro assay for the import of pre-23K by isolated thylakoids which has been found to be both efficient and reproducible (Brock et al., 1993). The magnitude of the prevailing DeltapH was estimated by 9-aminoacridine (9-AA) fluorescence quenching (Schuldiner et al., 1972). It was originally suggested that quenching was due to the uptake and entrapment of protonated dye in the thylakoid lumen (Schuldiner et al., 1972). According to this model, DeltapH could be calculated from the fractional quenching, q, as described under ``Materials and Methods.'' Although this mechanism of quenching has been challenged (Kraayenhof and Fiolet, 1974; Grzesiek and Dencher, 1985) it has been shown that DeltapH is linear with respect to log [q/(1 - q)] (Casadio and Melandri, 1985).

As with all techniques for monitoring the thylakoidal DeltapH, this technique in practice gives more of an estimate, than an accurate measure, of the DeltapH. However, the advantage of this method is that it allows real-time monitoring of the DeltapH over the entire import incubation period, during which samples were taken at given time points in order to determine the rate of import. The magnitude of the DeltapH was altered by changing the intensity of the incident light. A number of tests (not shown) indicated that the import competence of the isolated pea thylakoids did not change during a period of 60 min after isolation, that import time course analyses over 8-min periods were suitable for determining the rate of import, and that the magnitude of DeltapH did not change during these time courses to a significant extent. In practice, up to six import reactions were possible with each preparation of thylakoids.

The electrical potential, Delta, was not measured during these experiments, and it is possible that it may have contributed to the overall protonmotive force. However, under these steady-state conditions the thylakoidal Delta is very low indeed (in the order of 20-30 mV), and it is most unlikely that this level could influence the rate of protein import to a significant extent. The Delta can be effectively collapsed by the addition of valinomycin, and we have shown previously that this compound does not significantly inhibit the import of 23K by isolated thylakoids (Mould and Robinson, 1991).

The Rate of Import of 23K as a Function of DeltapH

Fig. 1shows the effects on 23K import of varying the light intensity over a wide range of values. At the highest light intensity (160 watts/m^2; DeltapH = 2.6) import of pre-23K proceeds rapidly as evident by the appearance of mature size, protease-resistant 23K; at the end of the 8-min time course, a considerable proportion of available precursor has been imported into the lumen. Decreasing the light intensity to 95 watts/m^2 (DeltapH = 2.4) leads to a small decrease in import rate, with further decreases in light intensity resulting in progressively lower rates of import. Importantly, import still takes place even at very low light intensities, at which the estimated DeltapH is in the region of only 1.3. It must be emphasized that DeltapH estimates at such low values are subject to a great deal of experimental error, but there is no doubt about the main conclusion from this experiment, which is that protein import can take place at extremely low values of DeltapH. This conclusion is reinforced by the results shown in Fig. 2, in which more attention was focused on the rates of import at low light intensities. The data show that lowering the light intensity from 200 to 1 watts/m^2 (DeltapH values 2.5 and 1.7, respectively) again results in a relatively minor change in the rate of import. Further small decreases in light intensity have a much more significant effect on the rate of import, although it is again evident that very low DeltapH values (in the region of 1.25) can support translocation. Import is completely inhibited at high light intensity by the presence of the uncoupler nigericin, emphasizing the critical role of the DeltapH in this translocation mechanism. However, it should be pointed out that a very low basal rate of import can sometimes be observed in the complete absence of detectable fluorescence quenching, and we do not know whether this corresponds to import in the absence of a DeltapH, or whether the import is in response to a DeltapH which is simply too low to be detected.


Figure 1: Effects of varying DeltapH on the import of pre-23K into isolated thylakoids. Isolated pea thylakoids were incubated with wheat pre-23K under varying light intensities as described under ``Materials and Methods.'' The light intensities and estimated DeltapH values are given on the left of the panels. In the bottom panel, the import assay was carried out at a light intensity of 160 watts/m^2 in the presence of 2 µM nigericin. Samples were removed at the indicated time points and analyzed directly (- protease) or after protease treatment of the thylakoids (+ protease). Lane T, pre-23K translation product; 23K, mature-size 23K.




Figure 2: Import of 23K at low DeltapH values. Import assays were carried out as detailed in the legend to Fig. 1under the indicated light intensities. Bottom panel, import was carried out at a light intensity of 200 watts/m^2 in the presence of 2 µM nigericin. Symbols are as in Fig. 1.



The mature size 23K bands in the fluorograms were quantified by laser densitometry, and Fig. 3shows the results of a typical experiment. The data show that the rate of import is essentially constant at each light intensity over the 8-min time period and that initial rates of import can therefore be derived from this type of experiment. Fig. 4shows the combined data from four different experiments, with import rates this time plotted against the proton concentration gradient across the thylakoid membrane (rather than the DeltapH which is a logarithmic function). The graphs show the same general trend in each case, with import rates measurable at low values of DeltapH but tending to saturate (with respect to proton gradient) at higher values. The overall shape of the curve is hyperbolic in every case, but the variation between experiments (and the inaccuracies inherent in this technique) are such that it is not possible to interpret the shape of the curve in great detail. Nevertheless, it is worth pointing out that the shapes of the curves are consistent with the involvement of a component which saturates at about DeltapH = 2, although the gradual increases in import rate at higher values of proton gradient are also consistent with the involvement of another component which does not saturate at all.


Figure 3: Initial rates of 23K import at varying values of DeltapH. Import time courses were carried out under varying light intensities as detailed in Fig. 1, and the mature size, protease-protected 23K bands in the fluorograms were quantitated by laser densitometry. The graphs correspond to light intensities of 160, 95, 40, 2.2, and 0.05 watts/m^2 (filled squares, open diamonds, open squares, circles, and filled diamonds, respectively).




Figure 4: Rate of import of 23K into thylakoids as a function of proton concentration gradient. Four separate import time course experiments were carried out as detailed in Fig. 1and Fig. 2, and the rates of import were calculated as in Fig. 3. The graph shows the import rates from the four experiments plotted against the proton concentration gradient (antilog of DeltapH) across the thylakoid membrane (acid, inside).



Although the measurements of DeltapH are only approximate for values under about DeltapH = 2 (proton gradient of 100:1, lumen/stroma), the data clearly show that high rates of import take place at or around this value and that import proceeds at significant rates at proton gradients well below this figure. To put these figures into perspective, the combined data in Fig. 4are shown as a ``best fit'' plot in Fig. 5, together with the values of proton gradient required to drive the synthesis of ATP under these light intensities. The results show that ATP synthesis only occurs at significant rates at values of DeltapH of about 3 and greater, in good agreement with previous work (Rumberg and Becher, 1984; Junesche and Gräber, 1987) which showed ATP synthesis having a threshold of DeltapH = 2.7 and saturating at DeltapH = 4, depending on DeltaGp. In fact, in our experimental system ATP synthesis was barely detectable even at the highest light intensities (not shown) because no electron acceptors are present, and the maximum values of DeltapH obtained (about 2.6) are thus in fact relatively low; values of over 3.0 can be routinely measured if electron acceptors are present to exploit the full potential of the thylakoidal electron transport machinery. The results shown in Fig. 5Bwere obtained in the presence of the electron acceptor methyl viologen, but this compound was found to inhibit thylakoidal protein import (not shown), and hence the maximum values of DeltapH in Fig. 5A are significantly lower.


Figure 5: Comparison of the dependence of 23K import and ATP synthesis on magnitude of DeltapH. A, the data from the four experiments shown in Fig. 4are shown plotted as a best-fit curve. B, the rate of ATP synthesis was determined under the same light intensities and experimental conditions, except that the electron acceptor methyl viologen was included at 10 µM.



The results shown in Fig. 5are useful in two respects. First, the ATP synthesis data serve as an internal control for the DeltapH measurements because the plot shown in Fig. 5B is similar to those obtained in other studies using a variety of techniques (including 9-AA fluorescence quenching) for the DeltapH estimations (for example, Quick and Mills, 1987; Bizouarn et al., 1987). The calculated values for DeltapH are not, therefore, gross under- or over-estimates. Second, the data show that the levels of DeltapH required to drive protein translocation are much lower than those required for ATP synthesis. A proton concentration gradient of 100 can drive 23K translocation at near maximal rates whereas a gradient of over 1000 is required for efficient ATP synthesis.

The DeltapH Does Not Promote Binding to the Thylakoid Membrane in the Absence of Translocation and Is Continuously Required for the Transport Process

One of the surprising findings of previous studies (Mould and Robinson, 1991) is that in the absence of a DeltapH, the 23K intermediate form (i23K) accumulates almost exclusively in the stroma; there is no evidence for the stable binding of the intermediate form to receptor proteins on the thylakoid surface. In this respect the transport of 23K across the thylakoid membrane differs radically from its transport across the envelope membranes, where, in the absence of sufficient ATP for the translocation step, pre-23K (and other precursors) bind extremely tightly to the import receptors on the chloroplast surface (Olsen et al., 1989). (^2)One possible interpretation of this observation is that the initial interactions with the thylakoidal import machinery are weak and reversible, but we considered it equally possible that the DeltapH might serve a dual purpose by promoting both the translocation of proteins across the thylakoid membrane and the initial binding of the proteins to the translocation apparatus. If this were the case, it was deemed likely that binding of intermediate forms to the thylakoid membrane might be detected at lower levels of DeltapH. This possibility was tested by localizing i23K after import of pre-23K into intact chloroplasts in the presence of increasing concentrations of nigericin. The results (Fig. 6) show that in the presence of 2 µM nigericin the vast majority of imported protein is found as the intermediate form in the stromal fraction. Lowering the concentration of nigericin leads to a progressive increase in the efficiency of transport across the thylakoid membrane, and the majority of 23K is found in the thylakoid fraction as mature size protein in the absence of uncoupler. Interestingly, in all cases the i23K form is found exclusively in the stromal fraction, and we conclude from these data that low levels of DeltapH do not promote extensive binding to the thylakoid membrane. We cannot exclude the possibility that the DeltapH promotes binding to the translocation apparatus, with translocation across the membrane then taking place very rapidly thereafter. However, we have no evidence to support this possibility, and the most likely explanation of the results is that the initial interaction of i23K with the thylakoidal transport machinery is reversible, relatively weak, and DeltapH-independent.


Figure 6: The intermediate form of 23K is soluble in the stroma at all values of DeltapH. Pre-23K was imported into intact pea chloroplast in the absence of nigericin or in the presence of nigericin at varying concentrations indicated above the lanes. After import, the chloroplasts were reisolated, lysed in 10 mM HEPES-KOH, pH 8.0, 5 mM MgCl(2) for 10 min on ice, and centrifuged for 5 min at 10,000 times g. Samples of stromal and thylakoidal fractions were analyzed. i23K and m23K, intermediate and mature forms of 23K, respectively.



The combined data from Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6clearly indicate that the magnitude of DeltapH is important in determining the rate of translocation of 23K across the thylakoid membrane, but a final test was carried out to address the question: is the DeltapH the driving force for translocation, or does it somehow activate the translocation machinery? This is an important question because studies on the thylakoidal ATP synthase have clearly shown that the DeltapH activates this enzyme, as well as driving ATP synthesis per se. We therefore carried out parallel time course analyses of the import of 23K by isolated thylakoids and added nigericin midway through one of the import reactions (Fig. 7). The results show that nigericin rapidly and completely inhibits protein import, and we conclude that the thylakoidal DeltapH is continuously required for protein translocation to occur.


Figure 7: The DeltapH is continuously required for import of 23K into thylakoids. Parallel thylakoid import assays were conducted as detailed under ``Materials and Methods,'' and samples analyzed at time points indicated; nigericin (2 µM) was added to one of the reactions after 4 min (filled diamonds). Mature size 23K was quantitated as detailed under ``Materials and Methods.''




DISCUSSION

The unprecedented lack of requirement for ATP or soluble factors in the 23K-type thylakoidal protein translocation mechanism makes this process an interesting area of study but also complicates an experimental analysis of the mechanism, since soluble factors and ATP-requiring reactions are generally much easier to study than the actual processes of membrane transfer (the soluble factors are more amenable to study, and the time scales of the reactions in solution are more protracted).Indeed, the only evidence to date for the presence of proteinaceous translocation apparatus in the thylakoid membrane is the observation that the transport of pre-23K into isolated thylakoids is saturable (Cline et al., 1993). The DeltapH plays an all-important role in the 23K-type translocation mechanism, and it is therefore crucial to understand the bioenergetics of protein transport in this case. In this study we have examined the magnitude of DeltapH required to drive translocation, and we have provided the first kinetic data concerning the role of the DeltapH. Although many details remain to be clarified, several important features of the translocation system have become apparent.

1) There is no evidence for a threshold level of DeltapH which is required to drive 23K transport across the thylakoid membrane. Numerous plots of proton concentration gradient versus rate of 23K import have been made, and in every case the graph shows the same basic characteristics: the rate of import increases steeply at low values of proton gradient and levels off at higher values. In no case have we found evidence for a threshold level of DeltapH at which translocation is suddenly triggered. If such a threshold exists, it must correspond to an extremely low DeltapH and would be of questionable physiological significance.

2) Translocation of 23K can be driven by extremely low values of DeltapH. The simple observation is that 23K transport can be detected, albeit at low rates, if any DeltapH is apparent. The quenching of the 9-AA fluorescence signal at the lowest light intensities used in this study is such that the estimated values of DeltapH are subject to an enormous amount of error, but there is no doubt that a DeltapH of well below 2.0 can support the translocation of 23K across the thylakoid membrane. This may be physiologically important because several of the known lumenal proteins (including 33K, 23K, 16K, and plastocyanin) are present as mature size proteins in dark grown tissue in which the etioplasts are photosynthetically incompetent (Sutton et al., 1987). In these tissues it is unclear whether there is any DeltapH at all across the thylakoid membrane, but we favor the possibility that reverse action of the thylakoidal ATP synthase may generate a small but sufficient proton gradient.

3) The data support a model in which the DeltapH provides the driving force for the translocation reaction. There is little doubt that the continuous presence of the DeltapH is required for translocation to proceed, and we thus conclude that the DeltapH probably functions in providing the driving force for translocation across the membrane. There is no evidence that the DeltapH promotes binding to the translocation apparatus, although we cannot formally rule out this possibility. Given the soluble nature of the i23K polypeptide during chloroplast import reactions, we believe that the initial interactions with the thylakoidal translocation apparatus are weak, reversible, and probably DeltapH-independent. We therefore propose that the 23K presequence is unlikely to protrude across the thylakoid membrane and ``sense'' the acidic lumenal pH, and we suggest instead that the DeltapH is required at an early stage to initiate, and probably also to maintain, the translocation process once binding has taken place. This situation would contrast markedly with the Sec-dependent translocation mechanism, where the ATP-dependent SecA reaction is proposed to provide the impetus for the early stages of translocation, with the Deltap providing the driving force for the later stages (reviewed in Driessen, 1992). It is also likely that the thylakoidal mechanism differs considerably from that of mitochondria, where the electrical potential, Delta, is required for the transport of proteins across the inner membrane (Pfanner and Neupert, 1985). It has been proposed that the Delta drives the movement of presequences (Martin et al., 1991), probably by an electrophoretic effect, but the main driving force for translocation appears to be ATP hydrolysis. The relative orientation of the Deltap is in any case different from that of the thylakoid, since protons are pumped out of the mitochondrial matrix but into the thylakoid lumen.

If the DeltapH is indeed required for the early stages of thylakoidal protein transport, the above model also implies that the DeltapH plays a dynamic role in the translocation process rather than translocation relying on a pH difference across the membrane (acid, inside). We have no information on how this is achieved, but obvious possibilities are that protein translocation is coupled to the efflux of protons or the influx of hydroxide ions (although many other mechanisms can be envisaged). The graphs of proton gradient versus translocation rate are consistent with this type of model, although further analyses are required in order to obtain more accurate information on the kinetics of translocation at low DeltapH values. If protein translocation is coupled to proton movement, accurate data at low values of DeltapH are absolutely essential for an estimate of the protein-proton stoichiometry because a hyperbolic curve would be indicative of 1 H binding event per transport event, whereas sigmoidal kinetics would suggest a higher ratio. Our data are most consistent with a low proton-protein stoichiometry, but the limited accuracy at the critical low values of DeltapH precludes any firm conclusions.

Two key outstanding issues must be settled before this mechanism can be understood in genuine detail. First, it will be important to determine whether proton/ion movement is in fact coupled to protein translocation; an answer to this question is obviously central to an understanding of any DeltapH-driven mechanism. Second, further work is required to determine the type(s) of protonmotive force which is (are) capable of driving protein transport by this mechanism. We have consistently referred to this mechanism as ``DeltapH-driven'' but it should be emphasized that we do not yet know whether translocation can be driven by the electrical potential Delta. This is because the Delta across the thylakoid membrane is almost non-existent under steady-state conditions, and it would not therefore be surprising if, in the presence of nigericin, such a small Delta were unable to support translocation. It will be important to resolve this point because in bacteria, both components of the total protonmotive force can support protein translocation (Bakker and Randall, 1984; Shiosuka et al., 1990). Further studies in which the Delta is artificially enhanced will hopefully provide important clues concerning the mechanism by which the thylakoidal DeltapH/Deltap drives protein translocation.


FOOTNOTES

*
This work was supported 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.

§
Recipient of a short term fellowship from the European Molecular Biology Organization. To whom correspondence should be addressed. Tel.: +44-203-523557; Fax: +44-203-523701.

(^1)
The abbreviations used are: 33K, 23K, 16K, the 33-, 23-, and 16-kDa proteins of the photosystem II oxygen evolving complex; 9-AA, 9-aminoacridine.

(^2)
I. W. Brock, J. D. Mills, D. Robinson, and C. Robinson, unpublished results.


ACKNOWLEDGEMENTS

We are grateful to Professor J. F. Allen for helpful advice throughout this work.


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