Targeting of proteins into and across the thylakoid membrane — a multitude of mechanisms

Plant Molecular Biology 26: 15-24, 1994. © 1994 Kluwer Academic Publishers. Printed in Belgium.

Mini-review

Targeting of proteins into and across the thylakoid membrane - a multitude of mechanisms

Colin Robinson ~ and Ralf B. K16sgen 2 l Department of Biological Sciences, University of Warwick, Coventry CV4 7 AL, UK; 2 Botanisches Institut der Ludwig-Maximilians-Universitiit, Menzinger Strasse 67, 80638 Manchen, Germany

Received 16 May 1994; accepted 16 May 1994

Key words: chloroplast, precursor proteins, protein transport, thylakoid membrane

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Introduction

Within the wider field of protein translocation, the study of thylakoidal protein translocation is still in its infancy. It is nearly 20 years since Blobel and Dobberstein [5] made the seminal observation that protein transport across the endoplasmic reticulum could be reconstituted in vitro, and it was only three years later that protein transport into isolated chloroplasts was demonstrated [9, 24]. The mitochondrial, glyoxysomal, peroxiso- mal and bacterial membranes have also joined the select group of membranes which have been shown in vitro to actively transport proteins, and over the past 20 years a combination of biochemi- cal, molecular and genetic approaches has yielded a phenomenal amount of information about the ways in which proteins are transported across biological membranes. Perhaps not surprisingly, some unifying themes have emerged from these studies. In most cases, proteins are targeted across the appropriate membrane by virtue of specific, often cleavable targeting signals which are recognised by the translocation apparatus in the target membrane. Molecular chaperones fea- ture prominently in many of the translocation mechanisms, usually involved in the unfolding of proteins prior to translocation, the refolding of proteins on the trans side of the membrane, or, in the case of mitochondrial Hsp70, physically pull- ing proteins across the membrane [30]. Finally, it is worth noting that when the protein- translocating membrane also happens to be an

energy-transducing membrane, as is the case with the bacterial plasma membrane and the mitochondrial inner membrane, protein translocation is generally harnessed to the local form of proton electrochemical gradient, although nucleoside triphosphates are almost always required in ad- dition.

It is only relatively recently that the protein- translocating properties of the chloroplast thylakoid membrane have been studied in any detail, after the demonstration in 1989 of protein import by isolated thylakoids [31 ], although several pre- ceding reports had strongly suggested that thylakoid lumen proteins are indeed actively transported across the membrane (rather than arriving in the lumen by vesicle flow from the inner envelope membrane). As a result, many aspects of thylakoidal protein transport are as yet inevitably vague in comparison with the detailed mechanistic information that has emerged from other systems. None of the components of the thylakoidal translocation apparatus have been purified or cloned, no translocation-defective mutants have so far been isolated, and the number of proteins known to be transported across the thylakoid membrane is still in single figures. Nevertheless, the field has already produced interesting results and major surprises. Most other membranes transport a wide range of proteins by a single characteristic pathway, with exceptional mechanisms operating only for a minority of proteins. For example, the available evidence suggests that hundreds of protein species are transported

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across the chloroplast envelope by an essentially common mechanism which is apparently conserved even among the different types of plastids [12, 14, 33]. Yet studies on the translocation of a mere handful of thylakoidal proteins have already unearthed three completely different mechanisms for their translocation, with clear hints of more mechanisms to come. Still more intruigu- ingly, one of these mechanisms has probably been inherited from the chloroplast's prokaryotic an- cestor, whereas another seems to be completely novel among known protein transport systems in terms of operating mechanism.

Integration of nuclear-encoded thylakoid membrane proteins

The primary aim of this article is to examine the mechanisms by which proteins are translocated across the thylakoid membrane, but it is also important to consider the integration of proteins into this membrane so that similarities and differences will become apparent. With the interesting exception of CFoII (see below), it seems that nuclear-encoded hydrophobic proteins are generally inserted into the thylakoid membrane by means of uncleaved, internal signals; many PSII and PSI proteins containing one or more membrane-spanning helices appear to be synthesised only with stroma-targeting presequences. In two cases this has been confirmed experimen- tally: the light-harvesting chlorophyll-binding protein of PSII (LHCPII) and the CP24 apoprotein of the same complex have been shown to integrate by means of information which is present in the mature protein [21, 38, 54, 7]. The nature of this 'information', however, is unclear. LHCPII is believed to contain three membrane-spanning e-helices, and deletion of any one was found to block integration into the thylakoid membrane [26], suggesting that integration may depend on amino acid stretches distributed throughout the entire molecule.

Mechanistic studies on the LHCPII integration process have been carried using in vitro assays for the insertion of LHCPII into isolated thyla-

koids, and these studies have shown that the presence of both a stromal protein factor and ATP is essential [8, 10]. The precise role of the stromal factor remains to be determined, but there are indications that it may function as a chaperone molecule to maintain the hydrophobic LH- CPII protein in a soluble, integration-competent form [46]. Whether this holds true for other integral thylakoid membrane proteins is not known, since very few have been studied in any detail. The exact contribution of the thylakoid membrane to the integration process for LHCPII (and other proteins) is also obscure at present. Efficient integration of LHCPII depends upon the thylakoidal ApH [ 11 ] but it is not yet known whether any thylakoidal proteins are directly involved in me- diating integration (for example, whether there is a form of receptor for integral membrane proteins).

The two-phase import pathway for nuclear- encoded thylakoid lumen proteins

Early studies on the biogenesis of cytosolically synthesised lumenal proteins have pointed out major differences to the LHCPII-type integration pathway, but gave no hint of the complexity of protein traffic actually involved. Initial work on plastocyanin (PC), followed by similar types of study on the 33, 23 and 16 kDa proteins of the oxygen-evolving complex (33K, 23K and 16K), revealed the existence of an apparently similar two-phase pathway for all four proteins. In each case, the protein is synthesised in the cytosol with a bipartite presequence containing two targeting signals in tandem. The first 'envelope transit' signal targets the protein into the chloroplast stroma, where it is removed by the stromal processing peptidase, SPP [19, 28]; these signals are structurally and functionally similar to the presequences of stromal and integral thylakoid membrane proteins [18, 35]. The intermediate forms are subsequently targeted across the thylakoid membrane by means of the remaining 'thylakoid transfer' signals and are processsed to the mature forms by a membrane-bound thylakoidal

processing peptidase, TPP [19, 28]. This two- phase import model was formulated after the observation of transient stromal intermediate forms during the transport of several lumenal proteins into intact chloroplasts [28, 36, 51 ], together with demonstrations that the maturation sequence could be reconstructed using partially purified preparations of SPP and TPP [19, 28]. More recently, pulse-labelling studies in Chlamy- domonas reinhardtii have demonstrated the presence of transient intermediate forms of lumenal proteins during transport to the thylakoid lumen [25]. Consistent with this import pathway, the presequences of all four proteins contain two distinct domains which have been shown by mu- tagenesis studies to specify translocation across the envelope and thylakoid membranes, respectively [ 18, 35]. The envelope transit domains thus resemble the presequences of stromal proteins in being hydrophilic, basic and rich in hydroxylated residues. Thylakoid transfer signals, on the other hand, share key features with 'signal' peptides which direct protein translocation across the endoplasmic reticulum and the bacterial plasma membrane; both types of peptide contain a hydrophobic stretch of residues (h-region) and a motif of short chain residues at the -3 and -1 positions which is known to be important for the proteolytic processing of signal peptides [55]. Further work showed these similarities to be functionally significant. Comparisons of the reaction specificities of pea TPP with Escherichia coli signal peptidase confirmed that TPP is a signal- type peptidase, albeit with more stringent sub- strate requirements at the -3 and -1 positions than the bacterial enzyme [20], and E. coli has been shown to efficiently export the intermediate forms of 33K and plastocyanin into the periplasm [17, 50].

On the basis of the above findings it was widely assumed that lumenal proteins were transported across the thylakoid membrane by a system which had been inherited from the cyanobacterial-type progenitor of the chloroplast, and, consistent with this idea, 33K and PC are targeted across the thylakoid membrane in cyanobacteria by peptides which resemble both signal peptides and the thy-

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lakoid transfer signals of their higher plant coun- terparts [6, 37]. Figure 1 summarises the basic import pathways for proteins destined for the stroma, thylakoid membrane and thylakoid lumen.

Distinct mechanisms of thylakoidal protein translocation

Although thylakoid transfer signals vary in some cases from the 'consensus' bacterial signal peptide, in that the amino-terminal sections are sometimes longer and more highly charged (see Fig. 2 and below) there are obvious shared similarities. It was therefore natural to assume at this point that thylakoidal protein translocation could be described in simple terms: the operation of a prokaryotic-type mechanism which would almost

Fig. 1. Basic pathways for the import of proteins into the chloroplast stroma, thylakoid membrane and thylakoid lumen. The diagram depicts the basic import pathways for a stromal protein, Rubisco small subunit (SSU), a thylakoid membrane protein, LHCPII, and a thylakoid lumen protein, 23K. SSU and LHCPII are synthesised with stroma-targeting presequences (black ovals) which mediate binding to receptors on the chloroplast surface and transport across the envelope membranes. 23K is synthesised with a bipartite presequence containing a similar stroma-targeting signal followed by a thylakoid-transfer signal (hatched oval). The stroma-targeting signals of all three precursors are removed by the stromal processing peptidase (SPP). LHCPII inserts into the thylakoid membrane by means of signals in the mature protein whereas the cleavable thylakoid transfer signal of 23K mediates translocation across the thylakoid membrane. Insertion of LHCPII requires ATP and a stromal protein factor; the requirements for thylakoidal protein translocation are shown in greater detail in Fig. 3.

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certainly turn out to resemble the sec-dependent export mechanism, involving the participation of a soluble chaperone (SecB) and an ATP- dependent translocation factor (SecA) in E. coli (reviewed in [29]). This simple view was only challenged after assays were developed for the import of proteins by isolated thylakoids. Thyla- koidal protein import was first demonstrated by Kirwin et al. [31 ], who showed that 33K could be imported by isolated pea thylakoids with moder- ate efficiency in an ATP-driven assay. Later work [32, 42, 43] showed that 33K, 23K and 16K were imported with high efficiency in a light-driven assay, and these studies provided the first indications of separate translocation mechanisms: import of 33K was strictly dependent on the presence of soluble stromal factors whereas 23K and 16K could be imported with very high efficiency in the complete absence of stromal extracts. A similar import assay was developed by Cline et al. [11], who also showed that the import of 23K and 16K by thylakoids is unusual among protein transport mechanisms in being ATP-independent. More recent work on the import of 33K and PC, and on the lumenal photosystem I protein, PSI-N, has confirmed that lumenal proteins fall into two clear groups in terms of requirements for translocation across the thylakoid membrane. 23K, 16K and PSI-N are transported by a

Fig. 2. Structures of thylakoid transfer signals. The thylakoid transfer signals of wheat 33K (w33K), Silene pratensis PC (silPC), and wheat 23K (w23K) are shown, together with a possible structure for the spinach 16K signal (the SPP cleavage site has not been determined for this precursor). SPP cleavage sites are denoted by arrows. Also shown is the presequence of 33K from the cyanobacterium Anacystis nidulans (A.nid). The signals contain three domains: a charged N-terminal domain (charged residues shown in bold), a hydrophobic domain (H-region; shown boxed in grey) and a signal peptidase-type cleavage site for TPP in which short- chain residues are present at the -3 and -1 positions.

mechanism which is absolutely dependent on the thylakoidal ApH, but which is unique among known transport mechanisms in that soluble protein factors and ATP are not required [ 11, 32, 41, 42]. 33K and PC, on the other hand, have completely different requirements: the ApH is not a prerequisite for translocation, though it may stim- ulate transport [11, 52] but the presence of stromal protein factors and ATP is obligatory [27, 44, 47]. The two groups of proteins thus have translocation requirements which differ diametrically in three fundamental respects.

What is the basis for the observed differences in translocation requirements? One obvious possibility is that these results reflect the differing characteristics of the mature proteins being transported, but a persuasive argument against this possibility is the fact that the two types of mechanism are so comprehensively different. One can easily imagine that some passenger proteins might be able to bypass a stromal element of the translocation apparatus, or an ATP-requiring step, and it is equally possible that some proteins may require a ApH more than others, but it is surely more than coincidental that all five proteins fall squarely into one group or the other. This correlation alone raised the clear possibility of dual thylakoidal protein translocases, and this model has recently received compelling support from two different experimental approaches. In the first, Cline et aI. [ 12] used saturating concentrations ofE. coli-expressed pre-33K and pre-23K to carry out an elegant competition study of thylakoidal protein translocation. They found that 33 K competed with PC for transport across the thylakoid membrane, and 23K competed with 16K, but the two groups did not compete with each other, strongly suggesting that the two groups of proteins follow separate pathways once inside the chloroplast. The integration of LHCPII into the thylakoid membrane was not affected by the presence of either pre-23K or pre-33K, suggesting that LHCPII (and probably other integral membrane proteins) may follow yet another pathway once inside the chloroplast. However, it should be em- phasised that further work is required for an un- derstanding of the relationship, if any, between

lumenal protein transport and LHCPII integration. The studies described above have not ruled out the possibility that common stromal factors mediate LHCPII integration and 33K/PC translocation, and it is as yet unclear whether thylakoidal proteins are in fact involved in the LHCPII integration process.

The second study [47] directly identified the element within a lumenal protein precursor (presequence or mature protein) which dictates the requirements for translocation across the thylakoid membrane, by examining the transport of a chimeric protein comprising the 23K presequence linked to mature PC. It was found that the translocation mechanism was indistinguishable from that of pre-23K, ruling out the possibility that stromal factors and ATP are required specifically for the transport of the plastocyanin mature protein across the thylakoid membrane. The conclu- sion from these studies seems obvious: lumenal proteins are translocated across the thylakoid membrane by (at least) two separate systems with completely different mechanisms. There are, however, some important outstanding questions. How do the systems recognise their cognate sub- strates when the thylakoid transfer signals of these proteins are all so similar? Figure 2 illustrates the thylakoid transfer signals of 33K, 23K, and PC, determined as described in [4], together with a likely structure for the 16K signal; these signals are compared with the signal-type sequence pre- ceding 33K in the cyanobacterium Anacystis nidulans [37]. It is clear that all of the thylakoid transfer signals share common features, especially the presence of the h-region and signal peptidase- type cleavage site, and there are no really clear- cut differences between the 33K/PC and 23K/ 16K groups, although some differences in charge distribution are evident. However, there is as yet no evidence that these differences are functionally significant, though this problem will hopefully be clarified in time through a systematic analysis of the critical features of the two types of transfer signal.

Why have two systems in the first place? There is no obvious rationale for this arrangement; the sec-dependent export mechanism in bacteria has

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been shown to be capable of exporting a vast range of foreign proteins, and it would be unex- pected if a related system were unable to transport three small, soluble proteins such as 23K, 16K and PSI-N. Thus, for the moment, the bal- ance of evidence certainly points to the presence of at least two parallel translocation pathways for lumenal proteins, but it is still possible that a general translocase (or protein pore) in the thylakoid membrane performs the actual membrane transfer of all the hydrophilic lumenal proteins. Thus, the differing transport requirements out- lined above may only reflect the operation of distinct pathways of forwarding proteins to such a general translocase. There is as yet no evidence to support this possibility, but this key topic requires further study before this issue can be regarded as settled.

Phylogeny of the translocation mechanisms

Intriguing clues have emerged in the last year or so which, while not answering the question of why two mechanisms operate, offer a possible explanation as to how separate mechanisms might have emerged. Firstly, there are clear indications that a subset of thylakoidal proteins are probably translocated by a prokaryote-type, sec-dependent system. Sec gene homologues have been found in the plastid genomes of glaucophyte, chromophyte and rhodophyte algae [15, 49, 53], and although homologues of these genes have not been found in the plastomes of higher plants, it seems likely that they are present in the nuclear genomes. Ob- viously, the most likely role of the putative Sec proteins in chloroplasts is in thylakoidal protein transport, although it must be stressed that there is no experimental evidence for this assumption at present. Consistent with this possibility, it has been shown that the SecA inhibitor, azide [45], inhibits the transport across the thylakoid membrane of two of the lumenal proteins studied: 33K and plastocyanin [34]. These results agree with the observed requirements for the transport of these proteins across the thylakoid membrane; transport of 33K and PC requires the presence of

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soluble factors and ATP [27, 47], and sec- dependent translocation in bacteria likewise involves a soluble factor (the chaperone, SecB) and requires ATP (for the function of SecA, reviewed in [29]).

The final correlation emerges when the photosynthetic apparatus of higher plants/green algae is compared with that of oxygenic cyanobacteria, which have also been shown to contain Sec proteins or sec gene homologues [2, 43]. Of the six lumenal proteins characterised in higher plants, only three have been found in cyanobacteria: 33K, PC and PSI-F. Recent studies (Mant et al., submitted, Karnauchov et al., submitted) have shown that PSI-F follows the 33K/PC-type translocation pathway, and it is therefore intriguing that the other three proteins (23K, 16K and PSI-N) should be the ones transported by the ApH- driven, sec-independent mechanism. Genes for these proteins have not been found in photosynthetic bacteria, raising the interesting possibility that the development of the higher-plant photosynthetic machinery has involved the acquisition, not only of new lumenal components, but also of a new system for their transport into the lumen. Alternatively, the ApH-driven system may in fact be present in cyanobacteria for the translocation of as yet unknown proteins into the lumen (or the periplasm), in which case the phylogenetically younger lumenal proteins may have been prefer- entially targeted using this system.

Although it seems likely that one of the thylakoidal protein pathways involves a sec-type mechanism, there is another possibility which has emerged from studies by Franklin and Hoffman [16]. These workers have cloned a cytosolically synthesised homologue of the 54 kDa protein of signal recognition particle, a ribonucleoprotein complex which functions in protein transport across the endoplasmic reticulum and in the export of certain proteins in bacteria (reviewed in [39]). It is therefore possible that the stromal translocation factor involved in 33K and plastocyanin translocation is in fact a chloroplast homologue of the signal recognition particle. Alter- natively, such a particle might function in the sorting of chloroplast-encoded proteins into either

the envelope or thylakoid membrane. Apocyto- chrome f, for example, is synthesised in the chloroplast with an apparent thylakoid transfer signal [1, 56] and there is a good chance that a signal recognition particle could mediate the co-translational targeting of the precursor protein into the thylakoid membrane, although when synthesised in E. coli the presequence appears to interact with the sec machinery [48].

CfolI uses a third translocat ion pathway

In terms of biogenesis, subunit II of the ATP synthase complex (CFoII) is unique among known thylakoidal proteins. To date, it is the only integral protein which is targeted into the thylakoid membrane by means of a bipartite presequence similar in overall structural terms to those of lumenal proteins [23]. It was originally imag- ined that CFoII would be imported by one of the two thylakoidal protein transport mechanisms described above for lumenal proteins, with the single membrane-spanning helix near the N- terminus perhaps fulfilling a stop-transfer function. It now appears that this is not the case. In vitro studies on the insertion of CFoII into the thylakoid membrane have shown that, surprisingly, the integration process can proceed efficiently in the complete absence of stromal factors, ATP and the thylakoidal ApH; furthermore, integration is not inhibited by the presence of high concentrations of pre-23K [40]. This apparent absence of any stimulatory influence (as yet unique among integral and lumenal thylakoid proteins) has prompted the suggestion of a spon- taneous integration mechanism in which the two hydrophobic sections within pre-CFoII (one in the thylakoid transfer signal, one in the mature protein) anchor in the thylakoid membrane and allow the intervening region (the N-terminus of the mature protein) to flip across the thylakoid membrane. Further work is required to test this possibility, but whatever the outcome there seems to be little doubt that the CFoII integration mechanism differs from any of the translocation/ integration mechanisms identified to date. In

Fig. 3 we have illustrated the important features of the three known pathways for thylakoidal protein translocation mediated by bipartite presequences.

As with the translocation of 23K, 16K and PSI-N, the mechanism of integration of CFoII appears to be a phylogenetically recent development. CFoI, a close relative of CFoII (the genes almost certainly arose by duplication of an an- cestor gene) is encoded by the plastid genome in higher plants and integrates by means of signals in the mature protein (Michl et al., submitted). A similar mechanism probably applies to the integration of the cyanobacterial homologues to CFoI and CFoII (subunits b and b', respectively) since these are likewise synthesised without signal

Fig. 3. Multiple mechanisms of thylakoidal protein translocation. The six lumenal proteins shown, and the integral membrane protein, CFoII, are synthesised with bipartite presequences and imported into the chloroplast, probably by a common mechanism. In the stroma, all of the precursors ex- cept pre-PSI-N and pre-CFoII are cleaved to intermediate forms by SPP. Further targeting into the thylakoids involves the operation of three distinct pathways. Translocation of PSI-N, 23K and 16K across the thylakoid membrane appears to require only the thylakoidal ApH (pathway A) whereas translocation of 33K and PC is dependent on the presence of a stromal factor, ATP and an azide-sensitive factor (AzSF); it is possible that the AzSF corresponds to the stromal translocation factor. The role of stromal factors and ATP in PSI-F translocation is not yet known, but this protein has been grouped with 33K and PC (pathway B) because translocation is sensitive to azide, does not require the thylakoidal ApH, and does not compete with that of 23K. A third pathway (C) is followed by the integral protein, CFoII, whose insertion into the thylakoid membrane does not require stromal factors, ATP or the ApH. After translocation into or across the thylakoid membrane, all of the proteins are apparently cleaved to the mature size by a common thylakoidal processing peptidase, TPP.

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peptide-type presequences [ 13 ]. Taken together, these observations suggest that it was the transfer of the CFoII gene to the nucleus that neces- sitated the acquisition of an additional thylakoid transfer signal and the development of a novel integration mechanism.

Summarising remarks

In this review we have attempted to illustrate the complexity of thylakoidal protein integration/ translocation, and to trace the likely origins of at least some of the mechanisms involved. Many of the most interesting points have only emerged during the last year or so, and this is therefore very much an ongoing story in which a number of aspects remain to be clarified and rationalised. Further studies are of course required to elucidate in detail the various integration and translocation mechanisms, and to compare these mechanisms with those involved in other systems, particularly in bacteria. Of particular interest are the mechanisms which appear to be unique to thylakoids, and in this respect the ApH-driven mechanism is a promising area of study because no other membrane has been shown to transport proteins using a protonmotive force alone. It will also be important to trace the evolution of these mechanisms (and to rationalise their appearance if possible). For example, did the ApH-dependent translocation mechanism suddenly appear de novo during the evolution of the chloroplast, or is the mechanism operating also in cyanobacteria? Or did it perhaps evolve from the sec-dependent mechanism as a slimmed-down, more efficient system able to take full advantage of the thylakoidal protonmotive force? Future studies, perhaps using a greater variety of techniques, should answer these questions and give a better idea of the true complexity of thylakoidal protein translocation. Above all, though, it would be satisfying to be able to relate the extraordinary complexity of thylakoidal protein transport to the needs of the chloroplast during the biogenesis of the thylakoid network. At the moment there is simply no apparent advantage in targeting photosynthetic pro-

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teins by such a variety of mechanisms. The chal- lenge now is to work out whether there is method in the thylakoid's madness, or whether this membrane is simply being perverse.

Acknowledgements

We are extremely grateful to John Gray, Chris Howe and Reinhold Herrmann for critically read- ing this manuscript and providing helpful com- ments.

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Targeting of proteins into and across the thylakoid membrane — a multitude of mechanisms