Transport of proteins into and across the thylakoid membrane

Transcription

1 Journal of Experimental Botany, Vol. 51, GMP Special Issue, pp , February 2000 Transport of proteins into and across the thylakoid membrane Colin Robinson1, Cheryl Woolhead and Wayne Edwards Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK Received 26 May 1999; Accepted 29 September 1999 Abstract 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 pro- tein translocases have now been shown to operate in a wide variety of bacterial species, suggesting a wide- spread requirement for the translocation of folded pro- teins across biological membranes. Numerous integral membrane proteins are also targeted into the thylakoid membrane, and these too follow at least two dis- tinct 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. Introduction 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 cyano- bacteria, 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 cyto- 1 To whom correspondence should be sent. Fax: CG@dna.bio.warwick.ac.uk Oxford University Press 2000

2 370 Robinson et al. sol. Most of the abundant thylakoid proteins are synthe- evolving complex proteins (23K, 16K), photosystem I sized in the cytosol and these proteins must therefore be subunit N and photosystem II subunit T. Transport of transported into the organelle and directed across the these proteins requires neither soluble factors nor nucleoside soluble stromal phase to their correct destination. Recent triphosphates but is instead totally dependent on the studies on the biogenesis of thylakoid proteins have DpH across the thylakoid membrane (Mould and pointed to the operation of a surprising variety of mechanisms Robinson, 1991; Cline et al., 1992). Competition studies for the targeting of proteins into and across the showed that this system operates in parallel with the Sec thylakoid membrane. In this article these advances are pathway and, most importantly, the choice of pathway is reviewed and the unexpected pathway complexity is dis- dictated by the type of presequence present (Cline et al., cussed in terms of the evolution of the pathways and the 1993; Robinson et al., 1994; Henry et al., 1994). Studies properties of the substrate proteins. on the targeting signals have shown that both types contain the three domains described above, but DpHdependent routing is dependent on the presence of (at Biogenesis of thylakoid lumen proteins: two very different targeting pathways least) two particular determinants: a twin-arginine motif immediately before the hydrophobic domain and a highly Particular interest has centred on the biogenesis of hydrophobic residue two or three residues thereafter thylakoid lumen proteins because these must also be (Chaddock et al., 1995; Brink et al., 1998). Typical signals transported across the thylakoid membrane, an especially for the Sec- and DpH-dependent pathways are shown complex targeting pathway. In fact, these studies have in Fig. 1. had a greater impact than envisaged and have paved the The requirements of the DpH-dependent system are way for new insights into the export of bacterial proteins. highly unusual because all other known protein transport Initial studies on the biogenesis of thylakoid lumen pro- systems rely on nucleoside triphosphates, and soluble teins gave no clues as to the actual complexity involved. factors also play an important role in many cases. All of the known lumenal proteins are synthesized in the However, recent mechanistic studies have shown that this cytosol with superfically similar bipartite presequences system exhibits even more unusual properties. Proteins comprising two targeting signals in tandem: an aminoter- are known to be transported in a largely unfolded state minal envelope transit signal followed by a thylakoid- by most protein translocases, including those in the targeting signal. The envelope transit signal functions to chloroplast envelopes (Guéra et al., 1993; America et al., transport the protein into the stroma, where it is usually 1994), the mitochondrial envelopes and the endoplasmic removed by a stromal processing peptidase (SPP). reticulum. The bacterial and thylakoidal Sec systems 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 Fig. 1. Thylakoid-targeting signals for imported lumenal proteins. The is also involved (Laidler et al., 1995; Roy and Barkan, figure shows the targeting peptides for representative substrates of 1998) although its precise function remains to be elucid- the DpH-dependent translocase: from top to bottom the sequences ated. It is now generally assumed that the thylakoidal Sec are for spinach/wheat 23K, spinach/maize 16K, barley PSI-N and cotton/arabidopsis PSII-T. Important features include a conserved twinpathway will turn out to be similar in most respects to Arg motif immediately upstream of the H-domains together with a bacterial Sec pathways. highly hydrophobic amino acid (for example, leucine, isoleucine or The major suprise in the last few years has been the methionine) two or three residues thereafter. Underneath are the signal peptides for known Sec substrates including those of wheat/spinach identification of a parallel pathway in chloroplasts for 33K, spinach/barley PSI-F and spinach/barley PC. H-domains are other lumenal proteins such as the 23 and 16 kda oxygen- underlined and charged residues given in bold.

3 likewise thread proteins through a relatively narrow pore (Jungnickel et al., 1994). In contrast, it has been found that the DpH-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. A related protein export pathway in bacteria Transport of proteins 371 and functionally similar to the twin arginine-containing targeting signals of DpH-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 corre- sponding, DpH-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 origins of the DpH-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 Fig. 2. Pathways for the targeting of thylakoid lumen proteins in chloroplasts. The figure illustrates the differing characteristics of the DpH- and Sec-dependent pathways. Both types of protein are synthe- sized with bipartite presequences containing envelope transit signals (black boxes) and thylakoid-targeting signals in tandem. The thylakoidtargeting signals of 23K and plastocyanin (DpH- and Sec-dependent substrates, respectively), are depicted by hatched or gray boxes. The envelope transit peptides of both precursors are recognized by a protein transport system in the envelope membranes which facilitates translocation into the stroma. The envelope transit signals are usually removed at this point and the resultant intermediate forms are directed along two distinct routes. Proteins such as 23K are believed to refold in the stroma before being transported in a folded form by a DpH-driven translocase; other lumenal proteins, such as 33K and plastocyanin, are transported by the Sec route. Translocation of such proteins involves stromal SecA and a membrane-bound SecYEG complex. It is presently unclear whether these proteins fold in the stroma, but the later stages involve SecA/ATP-driven translocation of the intermediate in an unfolded state through the membrane-bound Sec apparatus. After translocation, substrates on both pathways are processed to the mature forms by the thylakoidal processing peptidase. The insertion of thylakoid membrane proteins two further pathways The thylakoid membrane houses numerous integral mem- brane 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

4 372 Robinson et al. 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 (CF II) and photosystem II subunits W and X (PsbW, Fig. 3. Distinct routes for the insertion of thylakoid membrane proteins. o Most multispanning proteins, such as the major light-harvesting PsbX ) are synthesized with bipartite presequences that chlorophyll-binding protein (Lhcb1) are synthesized only with stromavery much resemble those of imported lumenal proteins targeting signals (black rectangles); a series of single-span proteins such such as plastocyanin. However, these proteins insert into as PsbW are synthesized with bipartite presequences containing a stroma-targeting signal followed by a hydrophobic, cleavable signal thylakoids in the absence of nucleoside triphosphates, peptide. Signal peptides are represented by hatched rectangles and stromal factors or a DpH, and their insertion is not membrane-spanning regions of the mature proteins by white rectangles. affected by prior protease-treatments of thylakoids that Pre-Lhcb1 is imported, processed to the mature size in the stroma and bound by signal recognition particle (SRP), a complex of 54 kda and totally abolish the Sec, SRP- and DpH-dependent translo- 43 kda subunits. This factor then mediates the GTP-dependent insertion cation processes (Michl et al., 1994; Lorkovic et al., 1995; into the thylakoid membrane, probably via the SecYEG complex used Kim et al., 1998). Because insertion is not dependent on for a subset of lumenal proteins. By analogy with bacterial systems, an additional factor, FtsY, probably functions as a soluble signal peptide any of the known translocation apparatus, it has been receptor mediating transfer to SecYEG. Pre-PsbW, on the other hand, proposed that these proteins insert spontaneously into is imported into the stroma and processed to an intermediate form, the thylakoid membrane, and the role of the signal peptide after which the protein inserts into the thylakoid membrane by an apparently spontaneous mechanism. In this process, the hydrophobic may be simply to provide an additional hydrophobic regions in the signal peptide and mature protein insert co-ordinately to region to assist insertion of the transmembrane section in drive translocation of the intervening region; cleavage by the thylakoidal the mature protein. These distinct insertion pathways are processing peptidase then yields the mature protein (Chaal and summarized in Fig. 3. Howe, 1998). Whereas the Sec-, SRP- and DpH-dependent pathways all show clear signs of having been inherited from the cyanobacterial-type progenitors of higher plant chloroefficiently in the complete absence of this targeting factor, phobic proteins but, since some such proteins insert plasts, the spontaneous insertion pathway used by CF II, o PsbW and PsbX is different. Genes encoding PsbX and it is clear that other features of these membrane proteins CF II are present in cyanobacteria and in the plastid must dictate pathway choice. This aspect of membrane o genomes of several eukaryotic algae, where they are protein insertion is very poorly understood, as is the invariably synthesized without signal-type presequences. mechanism by which multi-spanning proteins are actually Apparently, these have been acquired after the transfer inserted into the bilayer, and further work is clearly of the genes to the nucleus and the insertion mechanism required for an understanding of each stage of the thus differs markedly in this respect. Since the mature targeting pathways. proteins appear very similar in structural terms, it is unclear why the imported proteins need cleavable Concluding remarks signals whereas the plastid-encoded/cyanobacterial pro- It is now abundantly clear that the biogenesis of thylakoids teins do not. Possibly, the latter variants are inserted is a highly complex process involving the operation co-translationally and are able to simply slip into the of multiple targeting pathways. Quite possibly, more will thylakoid membrane more easily. emerge in future studies since only a fraction of the Finally, very recent evidence has shown that the spon- known protein complement has been analysed in any taneous pathway is not simply used by relatively simple detail. In the case of the lumenal proteins, the combined single-span proteins such as PsbW. Several multi- biochemical and genetic approaches have led to the spanning proteins have been shown to insert into thylakoids identification of a novel protein translocase with unpre- without the aid of either SRP or the Sec machinery cedented properties, and the discovery of related systems ( Kim et al., 1999; Thompson et al., 1999), strongly in numerous bacteria has forever changed the ways in suggesting a spontaneous mode of insertion (although the which protein transport mechanisms are viewed. The possible involvement of other, as yet undiscovered transport rationale for the existence of the two parallel pathways, apparatus can not yet be ruled out). SRP has been in the sense that the DpH-dependent pathway appears to suggested to interact preferentially with highly hydro- be used for difficult proteins that present folding-related

5 Transport of proteins 373 problems to the more constrained Sec system, is now precursor chloroplast proteins to chloroplast envelopes. Plant appreciated. Molecular Biology 23, Henry R, Kapazoglou A, McCaffery M, Cline K Membrane proteins likewise use a variety of pathways Differences between lumen targeting domains of chloroplast for their insertion into the thylakoid network, but in this transit peptides determine pathway specificity for thylakoid case the choice of pathway is dictated by more unknown transport. Journal of Biological Chemistry 269, factors. Some proteins appear simply to insert into the Hoffman NE, Franklin AE Evidence for a stromal GTP thylakoid membrane without the aid of any targeting requirement for the integration of a chlorophyll a/b binding polypeptide into thylakoid membranes. Plant Physiology apparatus (although this point certainly bears further 105, examination) whereas others enter a tortuous pathway Hynds PJ, Robinson D, Robinson C The Sec-independent involving interaction with SRP (and probably FtsY ) in twin-arginine translocation system can transport both tightly the stroma, followed by a GTP-dependent release into folded and malfolded proteins across the thylakoid membrane. thylakoid-bound translocation machinery and, finally, Journal of Biological Chemistry 273, Jungnickel B, Rapoport T, Hartman E Protein translateral exit into the bilayer. How this process is location: common themes from bacteria to man. FEBS co-ordinated is largely unknown, but the availability of Letters 346, effective in vitro assays, together with the powerful recent Kim SJ, Robinson C, Mant A Sec/SRP-independent impact of genetic studies, suggests that this area should insertion of two thylakoid membrane proteins bearing progress rapidly in coming years. cleavable signal peptides. FEBS Letters 424, Kim SJ, Jansson S, Hoffman NE, Robinson C, Mant A Distinct assisted and spontaneous mechanisms for the insertion of polytopic chlorophyll-binding proteins into the References thylakoid membrane. Journal of Biological Chemistry 274, America T, Hageman J, Guéra A, Rook F, Archer K, Keegstra Laidler V, Chaddock AM, Knott TG, Walker D, Robinson C. K, Weisbeek P Methotrexate does not block import of A SecY homolog in Arabidopsis thaliana. Sequence of a DHFR fusion protein into chloroplasts. Plant Molecular a full-length cdna clone and import of the precursor protein Biology 24, into chloroplasts. 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