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- Materials and methods
A series of thylakoid membrane proteins, including PsbX, PsbY and PsbW, are synthesized with cleavable signal peptides yet inserted using none of the known Sec/SRP/Tat/Oxa1-type insertion machineries. Here, we show that, although superficially similar to Sec-type signal peptides, these thylakoidal signal peptides contain very different determinants. First, we show that basic residues in the N-terminal domain are not important, ruling out electrostatic interactions as an essential element of the insertion mechanism, and implying a fundamentally different targeting mechanism when compared with the structurally similar M13 procoat. Second, we show that acidic residues in the C-domain are essential for the efficient maturation of the PsbX and PsbY-A1 peptides, and that even a single substitution of the −5 Glu by Val in the PsbX signal peptide abolishes maturation in the thylakoid. Processing efficiency is restored to an extent, but not completely, by the highly hydrophilic Asn, implying that this domain is required to be hydrophilic, but preferably negatively charged, in order to present the cleavage site in an optimal manner. We show that substitution of the PsbX C-domain Glu residues by Val leads to a burial of the cleavage site within the bilayer although insertion is unaffected. Finally, we show that substitution of the Glu residues in the lumenal A2 loop of the PsbY polyprotein leads to a block in cleavage on the stromal side of the membrane, and present evidence that the PsbY-A2 signal peptide is required to be relatively hydrophilic and unable to adopt a transmembrane conformation on its own. These data indicate that, rather than being merely additional hydrophobic regions to promote insertion, the signal peptides of these thylakoid proteins are complex domains with uniquely stringent requirements in the C-domain and/or translocated loop regions.
Most chloroplast thylakoid proteins are nuclear-encoded in plants and are therefore inserted into the membrane post-translationally after import from the cytosol (reviewed in [1,2]). Many of these proteins are synthesized with an N-terminal ‘transit’ peptide that mediates interaction with the envelope-localized import machinery and transport into the stroma, after which this presequence is removed by the stromal processing peptidase. In these cases, insertion into the thylakoid membrane involves targeting determinants located in the mature protein, and this has been experimentally confirmed for one thylakoid protein, the major light-harvesting chlorophyll-binding protein, Lhcb1 [3,4]. Further studies on this protein have shown it to integrate using a complex pathway involving stromal signal recognition particle (SRP), FtsY, GTP and membrane-bound translocation machinery that includes the protein Albino3 [5–9]. The Alb3 protein is related to the Oxa1p and YidC proteins that play important roles in the insertion of proteins into the bacterial plasma membrane and mitochondrial inner membrane, respectively [10,11]. In general, this insertion pathway resembles that used by at least some plasma membrane proteins in bacteria, which also involves SRP and FtsY (reviewed in ). This is perhaps unsurprising as chloroplasts are widely accepted to have evolved from endosymbiotic cyanobacteria.
Other thylakoid membrane proteins are inserted by a different pathway that contrasts markedly with the highly complex SRP-dependent pathway. The proteins PsbX, PsbW, CFoII and PsbY are synthesized with N-terminal bipartite presequences in which the first domain specifies import from the cytoplasm across the chloroplast envelope, after which it is removed by a stromal processing peptidase. The second domain resembles typical ‘signal’ peptides, containing three distinct domains: an N-terminal charged region (N-domain), hydrophobic core region (H-domain) and more polar carboxyterminal region (C-domain) ending with an Ala-Xaa-Ala consensus region. Signal peptides usually specify translocation by Sec-type translocation systems in the endoplasmic reticulum, bacterial plasma membrane or thylakoid membrane, and have been studied in detail in these systems (reviewed in [13,14]). However, these thylakoid proteins have been shown to insert into the thylakoid membrane in the absence of SRP, SecA, nucleoside triphosphates or (δpH, thereby excluding all the ‘assisted’ modes of insertion into thylakoids reported to date [15–17]. Furthermore, proteolysis of thylakoids destroys the membrane-bound Sec and twin-arginine translocase machineries but has no effect on the insertion of these proteins . In the absence of identifiable translocation factors it has been suggested that these proteins insert spontaneously into the thylakoid membrane. A similar mechanism was originally proposed for M13 procoat, which is also synthesized with a cleavable signal peptide and which inserts into the Escherichia coli plasma membrane by an SRP/Sec-independent pathway. However, this protein is now known to be highly dependent on YidC for insertion , whereas recent studies have shown that PsbX, PsbW and PsbY do not rely at all on the thylakoidal YidC homolog, Alb3 . Because none of the known thylakoidal protein transport machinery is required for the insertion of these proteins, it has been suggested that they may insert spontaneously into the thylakoid membrane.
The initial stages of this spontaneous insertion mechanism involve binding of the intermediate-size protein to the membrane, after which both hydrophobic domains (one in the signal peptide, the other in the mature protein) insert into the membrane and form a transmembrane loop intermediate . As a result, the hydrophilic, negatively charged domain is translocated across the membrane to the lumen where further processing by a thylakoid processing peptidase (TPP) removes the remaining presequence leaving the mature protein inserted in the membrane with a lumenal N-terminus and a stromal C-terminus. The presence of the hydrophobic signal peptide has been shown to be essential in the case of CFoII  but the important features within this class of signal peptide have not been studied in any systematic manner and, because Sec- and Tat-type signal peptides interact with proteinaceous binding sites, it is possible that these thylakoid signal peptides may possess unique characteristics that are essential for their correct functioning. In this study we have analyzed the importance of charged residues in the insertion and proteolytic processing of PsbX, PsbW and PsbY. We show that basic residues in the N-domain are not important for either process whereas acidic residues in the C-domains of several of the signal peptides play important roles in the processing of precursor forms to the mature size. These requirements are completely unlike those of M13 procoat, which also bears a signal peptide, or the Sec-type signal peptides of translocated proteins.
- Top of page
- Materials and methods
Previous studies have shown that a series of thylakoid membrane proteins are synthesized with cleavable signal peptides, yet are inserted by mechanisms that do not rely on any of the known translocation machinery, either in the soluble phase or at the membrane surface. It has been suggested that these signal peptides provide an additional hydrophobic region that helps to drive the insertion process, perhaps through the formation of a ‘helical hairpin’ that might provide the required driving force to flip the N-terminus of the mature protein across the thylakoid membrane. Intriguingly, these signal peptides resemble those of Sec-dependent lumenal proteins to a marked degree, and one of this class of signals can even function as a Sec-type signal for a lumenal passenger protein in chloroplasts . However, the data shown here point to defining features in some of these peptides that are essential for their correct functioning and which are not apparent in other forms of signal peptide. Our data also lead to new ideas on the biogenesis of the unique PsbY polyprotein.
The studies on the PsbW truncations focused on the role of basic residues in the N-domain, because previous work on M13 procoat and Sec-type signal peptides has shown that basic residues in the N-domain play essential roles in insertion/translocation [12,13,32]. In fact, our data indicate quite clearly that these play no important function in the insertion of PsbW, because their removal inhibits insertion to only a minor extent. When considered in conjunction with other data on this group of thylakoid proteins, it is now very interesting to compare and contrast their insertion mechanism with that of procoat. Initial models for the insertion of these thylakoid proteins were based heavily on that of procoat insertion. M13 procoat and pPsbW are very similar indeed in structural terms, in that they possess a single transmembrane span in the mature protein, are synthesized with rather similar cleavable signal peptides and the intervening loop regions (which are flipped across the membrane) are of similar lengths and overall charge. Both proteins form loop intermediates prior to cleavage by signal peptidase but it is now clear that their insertion requirements are completely different in almost every sense. Previous work has shown procoat to rely heavily on the protonmotive force (reviewed in ) whereas pPsbW is ΔµH+- independent, as are the other thylakoid proteins in this group [15,16]. Procoat is also totally dependent on YidC for efficient insertion  whereas the thylakoid proteins do not require the homologous Alb3 protein . We have now shown that these proteins differ in the means by which they initiate insertion; electrostatic forces play a central role in the early stages of the procoat insertion mechanism  whereas pPsbW contains no basic residues in the C-terminal region and our data show that basic residues in the N-terminal region are not important for insertion into thylakoids. pPsbW must therefore interact with the thylakoid membrane by other means. Basic residues in the N-domain are also highly important for the functioning of Sec-type signal peptides, possibly to promote interaction with anionic phospholipids or SecA [13,32,33], and it therefore appears that the signal peptides of these Sec-independent thylakoid proteins function in fundamentally different ways, despite the superficial similarities.
The other studies on PsbX and PsbY focused on the C-domain, prompted by the presence of acidic residues in this region. Acidic residues are not important in any of the domains within Sec-type signal peptides and are generally uncommon, especially in the C-domain which is generally five or six residues in length and polar but uncharged . In contrast, our results point to an important function for acidic residues in the translocated regions of these Sec/SRP/Alb3-independent thylakoid membrane proteins. In some cases (e.g. CFoII) the extreme N-terminus of the mature protein is highly negatively charged, and we believe that additional acidic residues in the signal peptide are probably unnecessary. In other cases (for example PsbX and PsbY-A1), acidic residues are not present in the N-terminus of the mature protein and in these cases the signal peptides contain conserved acidic residues in the C-domain. Our data indicate that these residues are very important for the correct maturation of the inserted protein. Substitution of the −5 and −2 Glu residues by Val leads to a complete block in the maturation of PsbX, although insertion appears not to be affected. The −5 Glu, in particular, appears to be important because the presence of Val at this position alone is equally detrimental. Processing efficiency is restored to some extent by Asn at the −5 position. For reasons that are presently unclear, the translocated loop region is cleaved most efficiently when carrying negative charges although other hydrophilic residues can substitute to some extent.
On the basis of these data we propose that the translocated region needs to bear a negative charge in order for the TPP cleavage site to be presented in an optimal manner. A model for the effects of these mutations on the PsbX insertion mechanism is as follows. Insertion of the wild-type protein leads to the formation of a loop intermediate  and the hydrophilic nature of the loop region is essential for correct presentation to TPP. We believe that the presence of negative charges close to the TPP site serves to distance the site from the membrane interior and enable processing to occur. The presence of Val at the −5 site leads to a lengthening of the hydrophobic region which then becomes buried in the membrane interior. This premise is supported by studies on the PsbY-A1V mutant, which contains no negative charges in the TPP cleavage site region. Processing of this mutant is again significantly impaired although not to the same extent as in some of the PsbX mutants.
These studies are reminiscent of some observations made with Sec-type signal peptides [34–36], where alteration of the C-domain or H/C boundary can also affect processing by signal peptidase. However, in the vast majority of these cases, processing was not blocked but rather occurred elsewhere, or the mutations made were far more drastic than those generated in PsbX. It should be emphasized that a near-complete block in processing occurred after only a single substitution (−5 Glu to Val) and processing is drastically affected in the PsbX/NN mutant despite the presence of a highly polar C-domain of the correct length. Overall, these mutations have far more drastic consequences than similar mutations made in Sec-type signal peptides, and we conclude that this may be due to one or both of the following reasons: (a) our studies are on membrane proteins rather than hydrophilic translocated proteins, and the cleavage site region may therefore be more highly constrained in the membrane because the mature protein is not pulled across the bilayer; and/or (b) the unusual lipid composition of the thylakoid membrane (primarily galactolipid rather than phospholipid ) may require that the translocated loop is more effectively presented to the signal peptidase when acidic residues are present, for unknown reasons.
The third aspect of this study concerned the PsbY-A2 signal peptide, but very different results were obtained in this case. Here, the substitution of two Glu residues in the translocated loop by Val does not block cleavage by TPP, indicating that the Glu in the C-domain of this signal peptide is not as important. Possibly, the presence of three helix-breaking proline residues upstream (see Fig. 7) is sufficient to maintain the TPP site away from the membrane, or other effects may operate in this case. However, these mutations do have dramatic effects and in this case it is cleavage at the A1-SP2 site on the stromal surface that is completely inhibited. In fact, the studies on this mutant fortuitously provide important information on the biogenesis of the PsbY polyprotein. In previous work on PsbY , we noted that blockage of the TPP cleavage reaction at either the A1 or A2 sites led to the accumulation of a three-membrane-span intermediate, indicating that cleavage on the stromal side of the membrane had failed to occur in each case. Inhibition of TPP cleavage at both sites led to the stable accumulation of a four-span intermediate. Clearly, cleavage at the stromal site occurs at a late stage prompting the question: why is this protease unable to recognize this site until both cleavages by TPP have occurred? The present study provides further information on this issue; lengthening the H-domain of the A2 signal peptide leads to the stable accumulation of a two-span intermediate containing A1 and the A2 signal peptide (SP2). Our interpretation is that the unidentified protease on the stromal surface can only cleave when SP2 is released from a transmembrane state to adopt a flexible orientation in the membrane. Our model for the overall process is as follows (see Fig. 10).
Figure 10. Model for the maturation of PsbY. 1. After insertion, PsbY forms a double loop intermediate with two signal peptides (SP1, SP2) and two regions (A1 and A2) destined to become single-span mature proteins. 2. TPP cleaves SP2 which is rapidly degraded; SP2 continues to be held in a transmembrane form due to interactions with A2. TPP then cleaves after SP2 yielding the mature A2 protein. 3. SP2 is now more flexible and the A1–SP2 junction on the stromal surface can be accessed by an unknown protease (hence the question mark) completing the maturation process. In the case of the PsbY-A2/VV mutant, SP2 is now more hydrophobic and able to maintain a transmembrane conformation, preventing cleavage on the stromal side.
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Stage 1. The PsbY polyprotein inserts into the membrane in the double loop formation shown in Fig. 10, and TPP cleaves at one of the two sites. Most probably, TPP can cleave at either site first but for simplicity it is shown as cleaving at the SP1-A1 site. This releases SP1 which is rapidly degraded.
Stage 2. TPP cleaves at the SP2-A2 site, releasing A2 as a single-span mature protein and generating the A1-SP2 intermediate (Stage 2).
Stage 3. SP2 is now far more flexible, either because it is no longer tethered at the lumenal face by charged residues or because it is not bound to its partner polypeptide region, A2. The stromal loop region is more accessible and cleavage in this loop can now occur.
One possibility is that this final cleavage can only occur when the A1 and SP2 regions are unconstrained by cognate partner polypeptide regions (A1-SP1, A2-SP2). First, the PsbY-A2V mutant can be cleaved at both positions by TPP but the A1-SP2 intermediate accumulates as a stable species (see lower panel of Fig. 10). In our view, this is most likely because the SP2 H-domain is now significantly longer and is effectively a true membrane-spanning region. The H-domains of the signal peptides of these thylakoid membrane proteins are much shorter and are generally less hydrophobic than true membrane-spanning regions and, we believe, can only adopt transmembrane conformations when tethered to genuine transmembrane spans.
Further evidence for this proposed model comes from considerations of the stabilities of SP1 and SP2. It is notable that the A1-SP2 intermediate is highly stable, as are the PsbX and PsbW loop intermediates generated in a previous study . Clearly, the signal peptides are completely resistant to proteolysis when bound to genuine transmembrane spans. In complete contrast, the signal peptides cannot be detected in even low amounts when released during normal insertion reactions, despite being as large as some of the mature proteins (e.g. PsbX and PsbW are only 4 and 6 kDa, respectively). Tricine gels readily resolve these small mature proteins but the complete absence of the cleaved signal peptides, even immediately after insertion  means that they are degraded very rapidly indeed. We propose that this is due solely to their low hydrophobicity, which precludes the maintenance of transmembrane configurations and instead leads to other positions in the membrane, or even release from the membrane , upon which they are degraded by proteases that perhaps specifically target peptides that are unable to adopt transmembrane conformations.
In summary, we have shown that the signal peptides of these spontaneously-inserting proteins have evolved with specific and unusual properties that are especially important for correct proteolytic cleavage following insertion. In the cases of PsbX and PsbY-A1, the hydrophobicity of the C-domain is critical for correct maturation and negative charges in particular appear to be favored. In the case of PsbY-A2, the negative charge in the translocated loop plays a key role in defining the hydrophobicity of the A2 signal peptide, which is of necessity low in order to facilitate the movements that allow the final cleavage on the stromal surface. In general, these signal peptides are not merely additional hydrophobic regions but are rather exquisitely structured extensions whose properties complement those of the N-terminal regions of the mature proteins.