Journal of Experimental Botany, Vol. 51, No. 90001, pp. 369-374,
February 2000
© 2000 Oxford University Press
Transport of proteins into and across the thylakoid membrane
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Received 26 May 1999; Accepted 29 September 1999
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Abstract |
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The biogenesis of thylakoid proteins is a complex issue that requires the operation of at least four pathways within the chloroplast. Two of the pathways are used for soluble lumenal proteins, where the proteins bear cleavable targeting signals that are recognized by one of two distinct translocases. These pathways differ in fundamental respects. A subset of lumenal proteins are transported in an unfolded state by a typical Sec system, whereas others are transported by a novel class of translocase that appears to function primarily in the transport of fully-folded proteins. Related protein translocases have now been shown to operate in a wide variety of bacterial species, suggesting a widespread requirement for the translocation of folded proteins across biological membranes. Numerous integral membrane proteins are also targeted into the thylakoid membrane, and these too follow at least two distinct routes. Some proteins use a signal recognition particle-dependent pathway that requires GTP and unidentified apparatus in the thylakoid membrane. Others, however, require none of the known targeting factors and may insert spontaneously into the membrane. In this article, the rationale behind this pathway complexity is discussed in relation to the properties of the substrate proteins and the evolutionary origins of the chloroplast.
Key words: Thylakoid, proteins, chloroplast, Sec system, translocase.
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Introduction |
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TopAbstractIntroductionReferences |
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Transport of proteins into or across membrane bilayers occurs on a large scale in almost every type of prokaryotic and eukaryotic cell. The underlying mechanisms have attracted a great deal of attention over the last 2–3 decades, primarily because the overall process of protein translocation represents such a major feat of biochemistry (Jungnickel et al., 1994). An entire range of proteins, differing widely in size, shape and hydrophobicity, must be recognized and then transported across membranes which are usually designed to be anything but permeable. Energy-transducing membranes, such as the bacterial plasma membrane, mitochondrial inner membrane and the thylakoid membranes of chloroplasts and cyanobacteria, should not even permit leakage of protons. To achieve this, cells have needed to overcome three major hurdles. First, the substrate proteins have to be recognized as being scheduled for transport. This usually means that they are synthesized with a built-in targeting signal, which in many cases is removed once translocation is completed. Secondly, the proteins must be quantitatively translocated across the appropriate membrane. This is important; some proteins would be very toxic, even lethal, if active on the wrong side of the membrane. Thirdly, the entire process often has to occur without allowing significant leakage of ions at the same time.
In the case of the chloroplast, an additional hurdle has to be overcome: that of intraorganellar sorting. The chloroplast is a structurally complex organelle comprising numerous distinct compartments (Robinson et al., 1998). The organelle is bounded by a double-membrane envelope inside which is found the soluble stromal phase and a major, interconnecting thylakoid membrane network. It is widely accepted that chloroplasts arose from endosymbiotic cyanobacteria and they still contain their own prokaryotic-type genetic system. However, in the course of evolution, most of the original genes have been transferred to the nucleus and chloroplast biogenesis thus requires the import of numerous proteins from the cytosol. Most of the abundant thylakoid proteins are synthesized in the cytosol and these proteins must therefore be transported into the organelle and directed across the soluble stromal phase to their correct destination. Recent studies on the biogenesis of thylakoid proteins have pointed to the operation of a surprising variety of mechanisms for the targeting of proteins into and across the thylakoid membrane. In this article these advances are reviewed and the unexpected pathway complexity is discussed in terms of the evolution of the pathways and the properties of the substrate proteins.
Biogenesis of thylakoid lumen proteins: two very different targeting pathways
Particular interest has centred on the biogenesis of thylakoid lumen proteins because these must also be transported across the thylakoid membrane, an especially complex targeting pathway. In fact, these studies have had a greater impact than envisaged and have paved the way for new insights into the export of bacterial proteins. Initial studies on the biogenesis of thylakoid lumen proteins gave no clues as to the actual complexity involved. All of the known lumenal proteins are synthesized in the cytosol with superfically similar bipartite presequences comprising two targeting signals in tandem: an aminoterminal ‘envelope transit’ signal followed by a thylakoid-targeting signal. The envelope transit signal functions to transport the protein into the stroma, where it is usually removed by a stromal processing peptidase (SPP). Thereafter, the thylakoid-targeting signal directs translocation into the lumen (reviewed in Robinson et al., 1998). All known thylakoid-targeting signals strongly resemble bacterial ‘signal’ peptides in containing three characteristic domains: an amino-terminal charged domain, hydrophobic core domain and a more polar carboxy-terminal domain. In bacteria, signal peptides have long been known to promote export to the periplasmic space by the general secretory pathway (the Sec pathway), and the expectation was that these proteins must be translocated by a Sec-related system in the thylakoid membrane. A Sec-type system has indeed been identified in chloroplasts in recent years and a stromal SecA homologue and ATP have been shown to be required for the translocation of several lumenal proteins including plastocyanin and the 33 kDa protein (33K) of the oxygen-evolving complex (Yuan et al., 1994; Nakai et al., 1994). A SecY homologue is also involved (Laidler et al., 1995; Roy and Barkan, 1998) although its precise function remains to be elucidated. It is now generally assumed that the thylakoidal Sec pathway will turn out to be similar in most respects to bacterial Sec pathways.
The major suprise in the last few years has been the identification of a parallel pathway in chloroplasts for other lumenal proteins such as the 23 and 16 kDa oxygen-evolving complex proteins (23K, 16K), photosystem I subunit N and photosystem II subunit T. Transport of these proteins requires neither soluble factors nor nucleoside triphosphates but is instead totally dependent on the 
pH across the thylakoid membrane (Mould and Robinson, 1991; Cline et al., 1992). Competition studies showed that this system operates in parallel with the Sec pathway and, most importantly, the choice of pathway is dictated by the type of presequence present (Cline et al., 1993; Robinson et al., 1994; Henry et al., 1994). Studies on the targeting signals have shown that both types contain the three domains described above, but pH-dependent routing is dependent on the presence of (at least) two particular determinants: a twin-arginine motif immediately before the hydrophobic domain and a highly hydrophobic residue two or three residues thereafter (Chaddock et al., 1995; Brink et al., 1998). Typical signals for the Sec- and pH-dependent pathways are shown in Fig. 1
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The requirements of the
pH-dependent system are highly unusual because all other known protein transport systems rely on nucleoside triphosphates, and soluble factors also play an important role in many cases. However, recent mechanistic studies have shown that this system exhibits even more unusual properties. Proteins are known to be transported in a largely unfolded state by most protein translocases, including those in the chloroplast envelopes (Guéra et al., 1993; America et al., 1994), the mitochondrial envelopes and the endoplasmic reticulum. The bacterial and thylakoidal Sec systems likewise ‘thread’ proteins through a relatively narrow pore (Jungnickel et al., 1994). In contrast, it has been found that the pH-driven system has the ability to transport fully-folded globular proteins across the thylakoid membrane which is, after all, designed to be impermeable even to protons (Clark and Theg, 1997; Hynds et al., 1998). How this impressive feat is achieved remains to be resolved. The basic mechanistic features of the targeting pathways for lumenal proteins are illustrated diagramatically in Fig. 2.
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A related protein export pathway in bacteria
The origins of the pH-dependent pathway were unknown until the recent identification and sequencing of the first component of the translocase. Voelker and Barkan succeeded in isolating a maize mutant in this pathway, termed hcf106 (Voelker and Barkan, 1995), and the sequencing of the gene encoding the Hcf106 protein (Settles et al., 1997) led to the realization that this system was in fact far more widespread than initially supposed. Homologues of the hcf106 gene are present in nearly all of the sequenced bacterial genomes as unassigned open reading frames and it is now clear that a basically similar translocase operates in these organisms. Most of the substrates for this pathway appear to bind redox cofactors such as FeS or molybdopterin centres, and such proteins are synthesized with signal peptides that are structurally and functionally similar to the twin–arginine-containing targeting signals of pH-dependent lumenal proteins (Berks, 1996). Because these cofactors appear only to be inserted in the cytoplasm, folding of the proteins has to take place at this stage and this precludes translocation of this type of protein by the Sec pathway (Santini et al., 1998). The Escherichia coli genome contains several genes that encode Hcf106 homologues, one of which is unlinked whereas the other is the first gene in a four gene operon. These genes have been termed tat genes (for twin-arginine translocation) and it has recently been shown that their disruption leads to a block in export of a range of cofactor-containing proteins (Sargent et al., 1998; Weiner et al., 1998; Bogsch et al., 1998).
Of the proteins known to be targeted by the corresponding, pH-driven thylakoidal system, few if any are believed to bind cofactors. It therefore seems likely that this type of translocase is primarily used for the targeting of two types of protein: those that bind cofactors and which are thus obliged to fold prior to translocation, and those that simply fold too rapidly or tightly for the Sec system to handle.
The insertion of thylakoid membrane proteins—two further pathways
The thylakoid membrane houses numerous integral membrane proteins and, while a proportion are synthesized within the chloroplast, it is now clear that most are imported from the cytosol. As with lumenal proteins, in vitro insertion assays have been widely used in attempts to unravel the insertion mechanisms involved. These studies have demonstrated that at least two further pathways are followed by integral membrane proteins. Most integral membrane proteins are synthesized only with stroma-targeting envelope transit signals, and thus the information specifying insertion must reside in the mature protein (Lamppa, 1988; Viitanen et al., 1988). The major light-harvesting protein of photosystem II, Lhcb1, has been the subject of many of these studies and it is now clear that the insertion of this protein requires targeting factors in both the stromal phase and the thylakoid membrane. Insertion is totally reliant on a stromal form of signal recognition particle (SRP) which comprises a homologue of the 54 kDa subunit of eukaryotic SRPs together with a novel 43 kDa subunit (Li et al., 1995; Schuenemann et al., 1998). This finding comes as no real surprise because there is now good evidence that SRP plays a major role in the targeting of membrane proteins in bacteria (reviewed by de Gier et al., 1997). Some differences are apparent since other SRPs in E. coli and eukaryotes contain an RNA molecule which appears to be absent in the chloroplastic SRP, but otherwise this appears to be another example of a prokaryotic-type pathway that has been inherited from the cyanobacterial progenitor of the chloroplast. Insertion by this route requires nucleoside triphosphates, preferably GTP (Cline et al., 1992; Hoffman and Franklin, 1994) and the presence of targeting machinery in the thylakoid membrane—probably the Sec apparatus, since this has been implicated in bacterial SRP-dependent pathways (Valent et al., 1998).
Rather more surprising is the finding that some proteins require none of the known targeting apparatus for their insertion into thylakoids. A series of single-span membrane proteins, including subunit II of the ATP synthase (CFoII) and photosystem II subunits W and X (PsbW, PsbX) are synthesized with bipartite presequences that very much resemble those of imported lumenal proteins such as plastocyanin. However, these proteins insert into thylakoids in the absence of nucleoside triphosphates, stromal factors or a pH, and their insertion is not affected by prior protease-treatments of thylakoids that totally abolish the Sec, SRP- and pH-dependent translocation processes (Michl et al., 1994; Lorkovic et al., 1995; Kim et al., 1998). Because insertion is not dependent on any of the known translocation apparatus, it has been proposed that these proteins insert spontaneously into the thylakoid membrane, and the role of the signal peptide may be simply to provide an additional hydrophobic region to assist insertion of the transmembrane section in the mature protein. These distinct insertion pathways are summarized in Fig. 3.
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Whereas the Sec-, SRP- and
pH-dependent pathways all show clear signs of having been inherited from the cyanobacterial-type progenitors of higher plant chloroplasts, the ‘spontaneous’ insertion pathway used by CFoII, PsbW and PsbX is different. Genes encoding PsbX and CFoII are present in cyanobacteria and in the plastid genomes of several eukaryotic algae, where they are invariably synthesized without signal-type presequences. Apparently, these have been acquired after the transfer of the genes to the nucleus and the insertion mechanism thus differs markedly in this respect. Since the mature proteins appear very similar in structural terms, it is unclear why the imported proteins need cleavable signals whereas the plastid-encoded/cyanobacterial proteins do not. Possibly, the latter variants are inserted co-translationally and are able to simply slip into the thylakoid membrane more easily. Finally, very recent evidence has shown that the ‘spontaneous’ pathway is not simply used by relatively simple single-span proteins such as PsbW. Several multi-spanning proteins have been shown to insert into thylakoids without the aid of either SRP or the Sec machinery (Kim et al., 1999; Thompson et al., 1999), strongly suggesting a spontaneous mode of insertion (although the possible involvement of other, as yet undiscovered transport apparatus can not yet be ruled out). SRP has been suggested to interact preferentially with highly hydrophobic proteins but, since some such proteins insert efficiently in the complete absence of this targeting factor, it is clear that other features of these membrane proteins must dictate pathway choice. This aspect of membrane protein insertion is very poorly understood, as is the mechanism by which multi-spanning proteins are actually inserted into the bilayer, and further work is clearly required for an understanding of each stage of the targeting pathways.
Concluding remarks
It is now abundantly clear that the biogenesis of thylakoids is a highly complex process involving the operation of multiple targeting pathways. Quite possibly, more will emerge in future studies since only a fraction of the known protein complement has been analysed in any detail. In the case of the lumenal proteins, the combined biochemical and genetic approaches have led to the identification of a novel protein translocase with unprecedented properties, and the discovery of related systems in numerous bacteria has forever changed the ways in which protein transport mechanisms are viewed. The rationale for the existence of the two parallel pathways, in the sense that the pH-dependent pathway appears to be used for ‘difficult’ proteins that present folding-related problems to the more constrained Sec system, is now appreciated.
Membrane proteins likewise use a variety of pathways for their insertion into the thylakoid network, but in this case the choice of pathway is dictated by more unknown factors. Some proteins appear simply to insert into the thylakoid membrane without the aid of any targeting apparatus (although this point certainly bears further examination) whereas others enter a tortuous pathway involving interaction with SRP (and probably FtsY) in the stroma, followed by a GTP-dependent release into thylakoid-bound translocation machinery and, finally, lateral exit into the bilayer. How this process is co-ordinated is largely unknown, but the availability of effective in vitro assays, together with the powerful recent impact of genetic studies, suggests that this area should progress rapidly in coming years.
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Footnotes |
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1 To whom correspondence should be sent. Fax: +44 1293 523701. E-mail:CG{at}dna.bio.warwick.ac.uk
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