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Vol. 20, Issue 17, 3965-3973, September 1, 2009
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Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125
Submitted October 2, 2008;
Revised June 23, 2009;
Accepted June 25, 2009
Monitoring Editor: Reid Gilmore
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Keenan et al., 2001). Cotranslational protein targeting begins with recognition of the cargo—ribosomes translating nascent polypeptides containing signal sequences—by the SRP (Walter et al., 1981). The cargo is brought to the vicinity of the target membrane via the interaction between the SRP and SRP receptor (FtsY in bacteria) (Gilmore et al., 1982a). On arrival at the membrane, SRP unloads its cargo to the protein-conducting channel, composed of the sec61p complex in eukaryotic cells or secYEG complex in bacteria and archaea (Simon and Blobel, 1991; Gorlich et al., 1992; Halic et al., 2006). The SRP and SRP receptor also reciprocally stimulate each other's GTPase activity (Powers and Walter, 1995). Thus, after cargo unloading, guanosine triphosphate (GTP) hydrolysis drives disassembly of the SRP 
SR complex, returning the components into the cytosol for the next round of protein targeting (Connolly et al., 1991).
The SRP pathway is conserved throughout all three kingdoms of life. Although the protein components of SRP and SR vary across species, the functional core of SRP is a highly conserved ribonucleoprotein complex, composed of a 54-kDa SRP GTPase (SRP54 in eukaryotes or Ffh in bacteria) and an SRP RNA (Keenan et al., 2001). The SRP receptor also contains a conserved GTPase domain that is highly homologous to the GTPase domain in SRP54, and together the GTPase domains of SRP and SR form a unique subgroup in the GTPase superfamily (Keenan et al., 2001). Both proteins contain a central GTPase G-domain that adopts the classical Ras-type GTPase fold (Freymann et al., 1997; Montoya et al., 1997). Unique to the SRP family of GTPases is an N-terminal extension, termed the N-domain, that forms a four-helix bundle (Freymann et al., 1997; Montoya et al., 1997). The N- and G-domains form a structural and functional unit called the NG-domain. In addition to the GTPase domains, the SRP and SR proteins contain unique effector domains that allow them to carry out their biological functions. SRP has a C-terminal extension, a methionine-rich M-domain, which interacts with the SRP RNA (Batey et al., 2000) and with the signal sequence of the cargo (Zopf et al., 1990). SR has an N-terminal extension, an acidic A-domain, which interacts with the target membrane (Parlitz et al., 2007) and potentially with the sec translocon (Angelini et al., 2005).
SRP and SR form a complex with one another directly through their GTPase domains and reciprocally activate each other's GTPase activity within the complex (Powers and Walter, 1995). Both structural and biochemical analyses suggested that these GTPases undergo major structural rearrangements during complex formation (Shan and Walter, 2003; Focia et al., 2004). One of the important conformational changes involves the intramolecular rearrangement at the interface between the N- and the G-domains (Shan and Walter, 2003; Egea et al., 2004; Focia et al., 2004). Two conserved motifs at the N–G-domain interface, ALLEADV on the N-domain and DARGG on the G-domain, act as a fulcrum that mediates the repositioning of the N-domain relative to the G-domain in both SRP and SR (Focia et al., 2004). In addition, an inhibitory element from the first helix of the N-domain is removed (Neher et al., 2008). These structural rearrangements bring the two N-domains into proximity with one another, allowing them to make additional interface contacts that stabilize the complex (Egea et al., 2004; Focia et al., 2004). After a stable SRP SR complex is formed, additional conformational rearrangements occur in both GTPase active sites to activate GTP hydrolysis within the complex (Shan et al., 2004).
A novel SRP-dependent protein targeting pathway has been found in chloroplast (Schuenemann et al., 1998). A unique feature of the cpSRP pathway is that it uses a posttranslational mode of targeting. Instead of recognizing ribosome nascent chain complexes as cargo, the cpSRP recognizes light-harvesting chlorophyll-binding proteins (LHCPs) that are imported into the chloroplast as fully synthesized proteins and delivers LHCPs from the chloroplast stroma to the thylakoid membrane (Delille et al., 2000; Tu et al., 2000). Analogous to the cytosolic SRP pathways, the cpSRP pathway is mediated by two GTPases, cpSRP54 and cpFtsY, that are close homologues of the cytosolic SRP54 and SR GTPases, respectively. Intriguingly, the other strictly conserved component of the cytosolic SRP pathway, the SRP RNA, has not been found in the cpSRP pathway. Instead, a novel 43-kDa protein, cpSRP43, binds to a unique C-terminal extension in cpSRP54, and together the cpSRP43 cpSRP54 complex constitutes the chloroplast SRP (Groves et al., 2001). Although early models suggested that cpSRP43 might act as a functional homologue of the SRP RNA to regulate the GTPase activity of the chloroplast SRP and SRP receptor (see below; Goforth et al., 2004), kinetic analyses showed that cpSRP43 does not considerably affect either the complex formation or GTP hydrolysis rates of cpSRP54 and cpFtsY (Jaru-Ampornpan et al., 2007). Instead, cpSRP43 interacts specifically with the cargo, the LHCPs, to facilitate substrate recognition (Delille et al., 2000).
In cytosolic SRP pathways, complex formation between the SRP and SR GTPases is extremely slow, presumably because it is limited by the extensive conformational changes required to form a stable complex (Peluso et al., 2001; Zhang et al., 2008). The SRP RNA overcomes this problem by enhancing the association rate between the two GTPases 200-fold, bringing the SRP–SR interaction rate to a range appropriate for their biological function (Peluso et al., 2000). Moreover, the SRP RNA accelerates the rate at which the SRP SR complex hydrolyzes GTP 5- to 10-fold (Peluso et al., 2001; Siu et al., 2007). Many reports have suggested that the SRP RNA may play a regulatory role by bridging the communication between cargo binding and the GTPase cycle (Batey et al., 2000; Bradshaw and Walter, 2007; Bradshaw et al., 2009). The SRP RNA therefore plays a crucial role in the SRP pathway, explaining why it is highly conserved from bacteria to archaea to eukaryotes.
How does the chloroplast SRP bypass such a key component? Previous kinetic analyses revealed that in the absence of the SRP RNA, the association kinetics between cpSRP54 and cpFtsY is 200-fold faster than that of their Escherichia coli homologues and matches the rate of the RNA-stimulated interaction between bacterial SRP and SR (Jaru-Ampornpan et al., 2007). This provides a simple explanation for the absence of the SRP RNA in the cpSRP pathway, but also raises additional questions. What governs the kinetics of interaction between the SRP and SR GTPases? How can the chloroplast GTPases interact much more efficiently than their bacterial homologues despite their high sequence homology? The crystal structure of apo-cpFtsY shows that, compared with free bacterial FtsY, the conformation of apo-cpFtsY is closer to that observed in the Ffh FtsY complex, especially with regard to the relative position of the G- and N-domains (Chandrasekar et al., 2008). This and additional biochemical results led to a model in which cpFtsY is preorganized in a conformation that is more conducive to interaction with its binding partner and thus bypasses some of the conformational changes that limit the rate of association between the bacterial SRP and SR GTPases.
In this work, we present additional evidence for this model by showing that cpFtsY is intrinsically 5- to 10-fold more efficient at interacting with the SRP GTPase. More importantly, we found that the M-domain of cpSRP54, without the help from the SRP RNA, provides an additional 
100-fold stimulation in complex formation between the cpSRP and cpFtsY GTPases. Both of these factors allow the chloroplast SRP and SR GTPases to achieve the same efficiency of interaction as the RNA-catalyzed interaction between their bacterial homologues. The stimulatory effects of the SRP RNA and the M-domain of cpSRP54 are specific to their homologous binding partners and not interchangeable across species, suggesting that the classical and the cpSRP pathways have diverged to use different molecular mechanisms to mediate the communication between the M-domains and the GTPase modules.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The coding sequence of E. coli Ffh NG (1-295) was cloned into pET 28b (Novagen, Madison, WI) between NcoI and XhoI restriction sites. The recombinant protein, with a His6 tag at the C terminus, was expressed in BL21 DE3* (Invitrogen, Carlsbad, CA) and purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity column (QIAGEN, Valencia, CA). E. coli Ffh NG (1-295) was further purified by cation exchange over a MonoS column (GE Healthcare, Chalfont St. Giles, Buckinghamshire, United Kingdom) using a linear gradient of 150–600 mM NaCl. FtsY (47-497) interacts with Ffh with the same kinetics as either full-length FtsY or FtsY-NG (Supplemental Figure 1); thus the large A-domain in E. coli FtsY does not affect the interaction between the SRP and SR GTPases.
cpSRP54 and cpFtsY were expressed and purified as described (Jaru-Ampornpan et al., 2007). Mutations of cpFtsY were introduced using the QuickChange Mutagenesis protocol (Stratagene, La Jolla, CA). cpFtsY G288W was purified using the same procedure as that for the wild-type protein. cpFtsY F71V and F71A were purified from inclusion bodies as described previously (Chandrasekar et al., 2008). The coding sequence of cpSRP54 NG (1-294 of the mature protein) and a His6 tag at the C terminus was cloned into pAcUW51 (BD Biosciences, San Jose, CA) between BamHI and HindIII restriction sites. The resulting plasmid was then used for protein expression from baculovirus at the Protein Expression Center of Caltech (Pasadena, CA). The recombinant cpSRP54 NG-His6 was purified by affinity chromatography using Ni-NTA twice.
To construct the domain swap mutant proteins, pDMF6 encoding E. coli Ffh (Freymann et al., 1997) was modified to contain an EcoRI site before the start of the Ffh M-domain. The plasmid encoding FfhNG-cpSRP54M was constructed by replacing the sequence of FfhM (residues 296-453) with a polymerase chain reaction (PCR) fragment encoding the cpSRP54M (residues 296-488) using the EcoRI and BamHI restriction sites. The chimeric protein was expressed in Rosetta competent cells (Novagen, Madison, WI) and purified using the same procedure as that for the wild-type Ffh protein (Peluso et al., 2001).
Kinetics
All GTPase assays were performed at 25°C in assay buffer [50 mM KHEPES, pH 7.5, 150 mM KOAc, 2 mM Mg(OAc)2, 2 mM dithiothreitol (DTT), 0.01% Nikkol, and 10% glycerol]. GTP hydrolysis reactions were followed and analyzed as described previously (Peluso et al., 2001). The reciprocally stimulated GTPase reaction between SRP and SR was determined in multiple turnover reactions ([GTP] >> [E]). The concentration dependence of the observed rate constant (kobsd) is fit to the equation below, in which kcat is the rate constant at saturating SR concentrations, and Km is the concentration of SR that gives half the maximal rate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cpFtsY Is Intrinsically Faster than E. coli FtsY at Interacting with the SRP GTPase
We first asked how well the core GTPase domains from the E. coli and chloroplast pathways are conserved. To this end, we tested whether the SRP and SRP receptor GTPases can interact with one another across different species. The SRP and SRP receptor reciprocally stimulate the GTPase activity of each other, providing a convenient assay to monitor complex formation between the two GTPases (Peluso et al., 2001). In this assay, the observed rate constant of GTP hydrolysis is monitored as a function of SR concentration. The slope of the initial linear portion of the concentration dependence represents the rate constant of the reaction: GTP SRP + SR GTP 
products (kcat/Km), and the rate at saturating SR concentrations (kcat) represents the GTP hydrolysis rate once the complex is formed. For the E. coli GTPases, kcat/Km is equal to the association rate constant between SRP and SR during complex formation (Peluso et al., 2001). For the chloroplast GTPases, this rate constant provides a lower limit for the association rate constant between cpSRP54 and cpFtsY to form an active complex (Jaru-Ampornpan et al., 2007). In situations where the values of kcat are comparable, the differences in kcat/Km reflect differences in either the rate or stability of complex formation. Therefore for the analyses below, we used the kcat/Km values as indices to compare the relative ability of the SRP and SR GTPases to form a complex with their binding partners.
The SRP and SRP receptors from both systems can cross-react with their heterologous binding partners. The chloroplast SRP receptor cpFtsY can interact with the E. coli SRP GTPase Ffh (Figure 1A, closed circles) and with the isolated NG-domain of Ffh (Ffh NG; Figure 1B, closed circles), with rate constants similar to those with its homologous partner, the NG-domain of cpSRP54 (cpSRP54 NG; Figure 1C, closed circles; and Table 1). Analogously, in the absence of the SRP RNA, the E. coli SRP receptor FtsY can interact with its heterologous partner cpSRP54 NG (Figure 1C, open circles) with rates similar to those with its homologous partners Ffh and Ffh NG (Figure 1, A and B, open circles). Therefore, the core GTPase modules of SRP and SRP receptor from the two pathways are largely conserved and interchangeable.
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10-fold faster kcat/Km over that of E. coli FtsY (Figure 1, A and B, and Table 1). Because the GTPase rates at saturating FtsY concentrations (i.e., kcat) are within twofold of each other for FtsY compared with cpFtsY, these differences in kcat/Km values stem primarily from differences in complex formation. Furthermore, the basal GTPase rates of cpFtsY and FtsY are similar to one another and are at least 200-fold slower than the stimulated reaction rates (Supplemental Table 1), indicating that the higher reactivity of cpFtsY than FtsY observed in Figure 1 reflects a higher efficiency of complex assembly with cpFtsY. These results provide independent evidence for the previously proposed model that cpFtsY is preorganized in a conformation that is more conducive to interaction with the SRP GTPases than bacterial FtsY. This effect partly explains why cpSRP54 and cpFtsY can efficiently interact with one another in the absence of the SRP RNA (Jaru-Ampornpan et al., 2007; Chandrasekar et al., 2008).
What are the molecular features in cpFtsY that allow it to interact more efficiently with the SRP GTPases? Complex formation requires the rearrangement of the N-domain relative to the G-domain. Previous crystallographic analyses suggest that, compared with bacterial FtsY, the relative position of the G- and N-domains in cpFtsY is more similar to that in the structure of the Ffh FtsY complex (Chandrasekar et al., 2008). This may arise, in part, from the tighter packing interactions at the N–G-domain interface, especially between the conserved ALLVSDF and SARGG motifs (highlighted in green and blue, respectively, in Figure 2A). In cpFtsY, the aromatic ring of Phe71 from the ALLVSDF motif inserts into the core of the N-domain and packs against the SARGG motif (Figure 2A). Phe71 is uniquely conserved among chloroplast FtsYs and is replaced by smaller residues in other species. We probed the importance of this packing by mutagenesis. Mutation of cpFtsY Phe71 to valine, its corresponding residue in E. coli FtsY, reduces the interaction rate of cpFtsY with cpSRP54 sixfold (Figure 2B, green circles). Mutating this residue to Ala reduces the rate even further (eightfold; Figure 2B, green squares). The conserved SARGG motif also contributes significantly in the domain–domain packing interaction, because mutation of the universally conserved Gly288 to a bulky tryptophan is detrimental, reducing the value of kcat/Km 76-fold (Figure 2B, blue). None of these mutations significantly reduce the basal GTPase activity of cpFtsY (Supplemental Table 1), indicating that the observed defects are specific to the interaction of cpSRP54 with cpFtsY.
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). With wild-type cpFtsY efficient complex formation with cpSRP54 was observed, whereas with mutant cpFtsY G288W no detectable complex formation could be found during gel filtration chromatography analysis (Figure 2C). Mutant cpFtsY F71V also exhibits a defect in complex formation (Figure 2D); the smaller defect of cpFtsY F71V than cpFtsY G288W in the gel filtration analysis is consistent with the less severe reduction of this mutant in kcat/Km in the GTPase assay. Together, these results demonstrate that the packing interaction at the N–G-domain interface is important for the formation of the SRP SR complex and possibly gives rise to the advantage of cpFtsY in interacting with the SRP GTPases.
The M-Domain of cpSRP54 Accelerates cpSRP54–cpFtsY Association
The above-mentioned results demonstrate that the higher reactivity of cpFtsY than E. coli FtsY contributes 5- to 10-fold to the 200-fold more efficient association between cpSRP and cpFtsY in the absence of the SRP RNA (Figure 1). We hypothesized that the remaining 50- to 100-fold effect could arise from cpSPR54, in particular its unique M-domain that interacts with cpSRP43 instead of the SRP RNA.
To test this hypothesis, we compared the interaction rate of cpSRP54 with that of cpSRP54 NG. Remarkably, full-length cpSRP54 exhibits 100-fold faster association kinetics (kcat/Km) compared with the isolated NG-domain of cpSRP54 (Figure 3A, open squares vs. circles). Thus, the M-domain of cpSRP54 can act as a functional mimic of the SRP RNA and accelerates the interaction between the cpSRP54 and cpFtsY GTPase domains. The effect of the M-domain is specific to the interaction between the two chloroplast GTPases, because the basal GTP binding and hydrolysis activity of cpSRP54 NG is indistinguishable, within experimental errors, from that of full-length cpSRP54 (Supplemental Table 1).
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cpFtsY complex is clearly visible as soon as the two proteins are mixed together (Figure 4A, black). Complex formation is close to completion within 5 min, with <40% of cpFtsY remaining in the monomer form (Figure 4A, red). In contrast, complex formation is much slower in the case of cpSRP54 NG (Figure 4B). Only 5% of cpFtsY went into the complex after an hour of incubation (Figure 4B, red). Qualitatively, these results provide additional evidence that the M-domain of cpSRP54 stimulates complex formation between cpSRP54 and cpFtsY.
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; Chandrasekar et al., 2008). The interaction between the E. coli GTPases is only stimulated when the M-domain binds the SRP RNA (Figure 3B; Peluso et al., 2001). The SRP RNA, however, does not affect the kinetics of cpSRP54–cpFtsY association (Figure 3B). In summary, the results in this section demonstrate that in both the bacterial and chloroplast SRP pathways, the cargo-binding M-domain of SRP communicates with the GTPase domains and stimulates the interaction between the SRP and SR GTPases, but each pathway has evolved unique molecular mechanisms (RNA vs. protein) to achieve this communication (see more below).
The M-Domains of SRP Specifically Communicate with Their Homologous Receptors in Each Pathway
The SRP RNA stimulates the association kinetics between bacterial Ffh and FtsY 200-fold. The above-mentioned results showed that the M-domain of cpSRP54 stimulates complex formation between the cpSRP and cpFtsY GTPases. We next asked whether the effects of the SRP RNA and the M-domain of cpSRP54 are interchangeable between the two pathways, as the core NG-domains of these proteins can interact with the heterologous partners (Figure 1). We therefore tested whether the SRP RNA can exert its stimulatory effect in reactions containing cpFtsY, and analogously, whether the M-domain of cpSRP54 can exert its stimulatory effect in reactions containing E. coli FtsY.
Using the GTPase assay in this mix-and-match experiment, we systematically analyzed the effect of the SRP RNA and the M-domain of cpSRP54 on the two different SRP receptors. With E. coli Ffh, the association rate between Ffh and FtsY is stimulated 376-fold by the SRP RNA (Figure 5A, open vs. closed circles; Peluso et al., 2001). In contrast, there is less than twofold difference when the binding partner is cpFtsY instead of E. coli FtsY (Figure 5A, inset, open vs. closed squares; and Table 1). These results suggest that cpFtsY, unlike E. coli FtsY, lacks the ability to respond to the SRP RNA bound to Ffh. Similarly, when cpFtsY interacts with its homologous partner cpSRP54, the SRP RNA does not provide any rate acceleration (Figures 3B and 5B, open vs. closed squares). The SRP RNA has no effect on the interaction of E. coli FtsY either when paired with cpSRP54 (Figure 5B). These results are expected in light of recent work that demonstrates that cpSRP54 does not bind the bacterial SRP RNA (Richter et al., 2008; Jaru-Ampornpan and Shan, data not shown).
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If the M-domain of cpSRP54 can act as an independent structural unit to stimulate complex formation with cpFtsY, then fusion of the cpSRP54 M-domain to the NG-domain of Ffh should stimulate the interaction of Ffh NG with cpFtsY. To test this possibility, we constructed a chimeric protein, FfhNG-cpSRP54M, by replacing the M-domain of Ffh (including the linker between the G- and M-domains) with that of cpSRP54. As predicted, the chimeric protein containing the M-domain from cpSRP54 forms an active complex with cpFtsY with a rate constant (kcat/Km) that is 15-fold faster than Ffh NG (Figure 6A, circles vs. squares). This stimulation is specific to the interaction between the two GTPases, because the basal GTPase activity of the fusion protein is similar to those of Ffh NG or Ffh (Supplemental Table 1). This is in contrast to E. coli Ffh in which the Ffh M-domain does not appreciably affect the interaction of its NG-domain with cpFtsY (Table 1). Unfortunately, the effect of the SRP RNA could not be tested in the reciprocal fusion protein, cpSRP54NG-FfhM, because the RNA binding motif in the Ffh M-domain of this chimeric protein does not seem to be well formed and the chimeric protein has lost the ability to bind the SRP RNA (Kd 
10 µM; Jaru-Ampornpan and Shan, data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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nascent chain complexes bearing SRP signal sequences (Walter et al., 1981; Schaffitzel et al., 2006), whereas the cpSRP binds to its substrates, LHCPs, as fully translated proteins (Tu et al., 2000; Delille et al., 2000). Second, the cpSRP lacks the SRP RNA, which is otherwise universally conserved in all the other SRP pathways. Instead, the cpSRP consists of the cpSRP54 GTPase and a novel protein only found in chloroplast, cpSRP43 (Schuenemann et al., 1998). Previously, we showed that cpSRP54 and cpFtsY can form a complex with one another at rates 200-fold faster than that of their bacterial homologues, thereby bypassing the requirement for the SRP RNA (Jaru-Ampornpan et al., 2007). Here, we underscored the molecular mechanisms underlying the large difference in interaction rates between the bacterial and chloroplast SRP and SR GTPases.
Previous biochemical and structural works have suggested a model in which the conformational rearrangement at the N–G-domain interface required for SRP SR complex formation is partly achieved in free cpFtsY, thus allowing it to interact more efficiently with its binding partner cpSRP54 (Jaru-Ampornpan et al., 2007; Chandrasekar et al., 2008). In this work, we provide independent biochemical support for this model by showing that cpFtsY is 5- to 10-fold more efficient at interacting with the GTPase domain of SRP, even when the binding partner is the heterologous E. coli Ffh (Figure 1). Mutational analyses further supported the importance of the domain arrangement in cpFtsY, especially at the N–G domain interface, to the formation of the cpSRP54 cpFtsY complex (Chandrasekar et al., 2008; this work). These results, along with the previous work, support the model that cpFtsY is preorganized in a conformation that allows it to better interact with the GTPase domain of SRP.
Even with the higher reactivity of cpFtsY, the isolated GTPase domains of SRP and SR interact very slowly. For the E. coli SRP and SR GTPases, their interaction rate is accelerated 200-fold by the SRP RNA. Intriguingly, we found here that the M-domain of cpSRP54 acts as a functional mimic of the SRP RNA, stimulating the interaction between cpSRP54 and cpFtsY 100-fold. This, together with the higher reactivity of cpFtsY, allows cpSRP54 and cpFtsY to achieve the same interaction rate as the RNA-catalyzed interaction between the bacterial SRP and FtsY, and alleviates the otherwise strict requirement for the SRP RNA in cytosolic SRP pathways. These results, together with previous work, provide strong evidence that the cargo-responding domains of the SRPs from both bacterial and chloroplast systems communicate with the GTPase domains and kinetically regulate complex formation between the SRP and SR GTPases (Jagath et al., 2001).
It is interesting to note that although the GTPase modules (the NG-domains) of SRP and SR can interact with their heterologous binding partners across species, the effects exerted by the M-domains or the SRP RNA are not interchangeable. The stimulatory effect of the SRP RNA or the M-domain of cpSRP54 during complex formation can only be attained when the homologous binding partners are paired together. The SRP RNA can only exert its stimulatory effect during the interaction of E. coli Ffh with E. coli FtsY. Analogously, the M-domain of cpSRP54 can only exert its stimulatory effect during the interaction of cpSRP54 or the chimeric protein (FfhNG-cpSRP54M) with cpFtsY. This specificity implies that the two pathways have evolved distinct mechanisms to mediate communication between the M- and the GTPase domains. In cytosolic SRP pathways, the SRP receptor has evolved to establish a specific communication with the SRP RNA. Conversely, in the cpSRP pathway, the cpFtsY has evolved to establish a specific communication with the M-domain of cpSRP54.
How does the SRP RNA or the cpSRP54 M-domain stimulate complex formation between the SRP and SR GTPases? Although the detailed molecular mechanism remains unclear, three possible models can be envisioned based on previous and this work. First, the SRP RNA helps to preposition the Ffh NG-domain such that it is more active at interacting with FtsY (Jagath et al., 2001). By analogy, the M-domain of cpSRP54 might preposition the NG-domain of cpSRP54. Second, the SRP RNA positions the M-domain of Ffh and allows it to transiently interact with the SRP or SR GTPase during complex formation (Zheng and Gierasch, 1997), whereas in cpSRP54 the M-domain itself is properly positioned to establish these interactions. Third, the two pathways use distinct mechanisms to stimulate complex formation. The SRP RNA may provide a direct tether that holds the cytosolic SRP and SR GTPases together during complex formation (Peluso et al., 2000), whereas cpSRP54 could use its M-domain to provide this tether. Our data seem to favor the third possibility. This is because the E. coli SRP, even though its M- and NG-domains would be pre-positioned by the SRP RNA, cannot efficiently interact with the chloroplast SRP receptor. Analogously cpSRP54, even though its M- and NG-domains would be prepositioned, cannot efficiently interact with the E. coli FtsY. The stimulatory effect of the SRP RNA and the M-domain of cpSRP54 is highly specific to their homologous receptors, arguing against the first two models in which the origin of the stimulatory effect would be more generic.
It was recently shown that in cytosolic SRP pathways, the SRP RNA exerts its stimulatory effect on SRP SR complex assembly only in the presence of cargo or a stimulatory detergent such as Nikkol that partially mimics the effect of the cargo (Bradshaw et al., 2009; Zhang et al., 2009). This led to the proposal that the SRP RNA acts as a molecular linker that turns on the GTPase cycles of SRP and SR in response to signal sequence binding in the M-domain. Similarly, we found that the stimulatory effect of the cpSRP54 M-domain on the cpSRP54–cpFtsY interaction is also dependent on the presence of the stimulatory detergent Nikkol (Supplemental Figure 2). This suggests that, analogous to the cytosolic SRP, the stimulatory effect of the cpSRP54 M-domain on complex formation between the chloroplast SRP and SR GTPases might occur only in response to binding of its cargo LHCP. Thus, the M-domain of cpSRP54 might have also subsumed the function of the SRP RNA as a molecular linker that bridges the communication between cargo binding and SRP SR complex formation.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Shu-ou Shan (sshan{at}caltech.edu)
Abbreviations used: cpSRP, chloroplast signal recognition particle; GppNHp, 5'-guanylylimido-diphosphate; GTP, guanosine triphosphate; LHCP, light-harvesting chlorophyll binding protein; SR, signal recognition particle receptor; SRP, signal recognition particle.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The data were fit to the equation in Materials and Methods and gave a kcat value of 4.4 min–1and a Km value of 2.2 µM for cpFtsY, and a kcat value of 6.0 min–1and a Km value of 39 µM for E. coli FtsY. (B) Reactions of E. coli Ffh NG with cpFtsY (
). The data were fit to the equation in Materials and Methods, and the kcat/Km values are listed in 
) or absence (