Regulation of the GTPase Cycle in Post-translational Signal Recognition Particle-based Protein Targeting Involves cpSRP43*

The chloroplast signal recognition particle consists of a conserved 54-kDa GTPase and a novel 43-kDa chromodomain protein (cpSRP43) that together bind light-harvesting chlorophyll a/b-binding protein (LHCP) to form a soluble targeting complex that is subsequently directed to the thylakoid membrane. Homology-based modeling of cpSRP43 indicates the presence of two previously identified chromodomains along with a third N-terminal chromodomain. Chromodomain deletion constructs were used to examine the role of each chromodomain in mediating distinct steps in the LHCP localization mechanism. The C-terminal chromodomain is completely dispensable for LHCP targeting/integration in vitro. The central chromodomain is essential for both targeting complex formation and integration because of its role in binding the M domain of cpSRP54. The N-terminal chromodomain (CD1) is unnecessary for targeting complex formation but is required for integration. This correlates with the ability of CD1 along with the ankyrin repeat region of cpSRP43 to regulate the GTPase cycle of the cpSRP-receptor complex.

Signal recognition particle (SRP) 1 and its receptor are components of a ubiquitous mechanism for cotranslational protein targeting to the endoplasmic reticulum in eukaryotes and to the cytosolic membrane of prokaryotes (for review see Refs. 1 and 2). An SRP-like protein targeting pathway in chloroplasts mediates the post-translational localization of a family of lightharvesting chlorophyll a/b-binding proteins (LHCPs) to the thylakoid membrane. LHCPs are nuclear encoded thylakoid proteins that are synthesized in the cytoplasm and post-translationally imported into the chloroplast. Following import into the chloroplast stroma, LHCPs bind chloroplast SRP (cpSRP) to form a soluble targeting complex (for review see Refs. 3 and 4). cpSRP contains a conserved 54-kDa GTPase (cpSRP54) (5,6) but is structurally unique from cytosolic SRPs in that it lacks an RNA moiety (7) and contains a novel 43-kDa protein (cpSRP43) (8,9). The cpSRP⅐LHCP targeting complex, termed transit complex, is directed to a thylakoid translocase containing Albino3 (ALB3) (10,11) by a mechanism that requires cpFtsY (11)(12)(13)(14), a homolog of pro-and eukaryotic SRP receptors, FtsY and SR␣, respectively. Consistent with the GTPase activity of cpFtsY and cpSRP54, GTP is required for LHCP integration (6). It is noteworthy that GTP is not required for transit complex formation (14,15). Rather, binding of GTP by cpSRP54 and cpFtsY appears to take place upon their interaction at the thylakoid membrane (11), which closely resembles GTP binding by SRP54 and its receptor in protein targeting to the ER (16,17).
The unique ability of cpSRP to bind substrates post-translationally is attributed to cpSRP43, which binds both cpSRP54 and a charged 18-amino acid element in LHCPs (L18) to bring about the formation of transit complex (18,19). In the absence of L18 binding, the transit complex fails to form, and LHCPs do not properly integrate into the thylakoid membrane (18). These adaptor functions associated with cpSRP43 are likely mediated by the putative ankyrin (Ank) domains and chromodomains (chromosome organization modifier) present throughout the protein (see Fig. 1 for domain organization) (9). Ank domains are repetitive ϳ33-amino acid motifs that together form a characteristic and stable structure with surfaces that are tailored for many different macromolecular interactions (for review see Ref. 20). Chromodomains (CDs) are a family of highly conserved ϳ44-amino acid motifs first discovered in the Drosophila Polycomb gene silencing protein (21). CD-containing proteins have been found in diverse eukaryotic organisms ranging from Schizosaccharomyces pombe to humans, where CDs mediate protein-protein interactions within the nucleus (for review see Ref. 22).
Yeast two-hybrid studies have been used to examine the protein interaction targets of Ank and CDs in cpSRP43 (23). Results from this work suggest that the first domain of a four-Ank domain repeat binds to the L18 motif of LHCPs, whereas the third and fourth Ank domains appear to support homodimerization of cpSRP43. However, the relevance of homodimerization domains is not clear because cpSRP appears to function as a cpSRP43/cpSRP54 heterodimer (7). In the same two-hybrid study, binding to cpSRP54 was attributed to the combined effects of the two CDs at the C terminus of cpSRP43. Whether CD-mediated binding of cpSRP43 to cpSRP54 is needed for any step of the LHCP localization mechanism is not currently known.
Recent progress in the production of functional recombinant cpSRP (7) and cpFtsY (11,14) has provided the tools to examine the relationship between protein interaction activities and the role of these activities in mediating distinct steps in the LHCP localization mechanism. In this context, we have examined CD-specific mutants of cpSRP43 in assays that reconstitute cpSRP formation, cpSRP⅐LHCP targeting complex formation, and LHCP integration into isolated thylakoids. Our analyses extend to a previously unexamined third CD near the N terminus identified here by structural modeling. Results of these studies also led us to examine the influence of CD mutants on GTP hydrolysis by cpSRP54 and cpFtsY. Binding of SRP54 to SR␣ (Ffh and FtsY in bacteria) reciprocally stimulates GTP hydrolysis by each protein in cotranslational SRP targeting (24), an activity promoted by the 60 S ribosomal subunit in targeting to the ER (17). Most importantly, our data suggest that each CD functions at distinct steps in the LHCP targeting/ insertion mechanism and that in post-translational targeting, cpSRP43 may functionally replace the ribosome as an activator of the cpSRP54/cpFtsY GTPase cycle.

EXPERIMENTAL PROCEDURES
All reagents, enzymes, and standards were purchased commercially. A peptide corresponding to the L18 region of pLHCP (VDPLYPGGS-FDPLGLASS) was described previously (18). Precursor LHCP template used for radiolabeled in vitro transcription/translation was described previously (25). L18 fused to the endoplasmic reticulum-targeted protein preprolactin (L18-PPL) was described previously (18). Primers for DNA amplification using PCR were purchased from Integrated DNA Technologies. PCR amplifications were performed with PfuTurbo DNA polymerase (Stratagene), and PCR products were restricted with enzymes purchased from New England Biolabs. All cloned sequences were verified by DNA sequencing (Molecular Resource Laboratory, University of Arkansas for Medical Sciences, Little Rock).
Cloning, Expression, and Purification of Recombinant Proteins-Recombinant, purified cpSRP54-his and Trx-cpFtsY were produced and isolated as described previously (11,14). Mature cpSRP43 in pGEX-6P-2 (14) was used to express GST-cpSRP43 from which mature cpSRP43 was obtained following proteolytic removal of the GST fusion partner. GST-cpSRP43 from the soluble bacterial fraction was initially purified over glutathione-Sepharose TM (Amersham Biosciences) and further purified by anion exchange chromatography using a Resource Q column (Amersham Biosciences) with bis-Tris, pH 6, and elution with a linear gradient of KCl followed by desalting into 10 mM HEPES-KOH, pH 8.0, 10 mM MgCl 2 (HKM) prior to use. For the production of cpSRP43, purified GST-cpSRP43 was desalted into 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, pH 7.0, and incubated overnight with PreScission Protease TM (Amersham Biosciences) at 4°C to cleave the GST tag. Cleaved cpSRP43 was then desalted into phosphate-buffered saline and passed over glutathione-Sepharose TM to remove the cleaved GST tag and PreScission Protease TM . cpSRP43 was further purified by anion exchange by using a Resource Q column (Amersham Biosciences) followed by desalting into HKM buffer before use.
All cpSRP43 deletion clones were constructed by PCR (see below) and cloned into BamHI-and EcoRI-restricted pGEX-6P-2 by using the same restriction sites introduced into the forward and reverse primers, respectively. Coding sequences for the construction of cpSRP43 deletion constructs were based on the alignment of the CDs of cpSRP43 and mouse modifier protein 1 (mM-HP1b) ( Fig. 2A). The forward primer used to construct mcpSRP43 lacking the N-terminal CD (CD1) contained the coding sequence for the N-terminal 24 amino acids of mcpSRP43 followed by the coding sequence of the Ank1 region. The reverse primer corresponded to the 3Ј end of cpSRP43 and the neighboring stop codon. Hence, the resulting plasmid (p-cpSRP⌬CD1) codes for mature cpSRP43 lacking CD1 amino acids SSYG . . . EYET. Plasmid coding for cpSRP43 that lacks the second chromodomain (CD2) of cpSRP43 (p-cpSRP⌬CD2) was produced by overlap extension (26) by using internal primers that overlap at the 5Ј ends and bind immediately outside of CD2. The same exact outside primers used to construct mcpSRP43-6P-2 (14), when paired with each appropriate internal primer, resulted in two separate overlapping PCR products that were subsequently spliced by extension of the overlapping ends to produce the coding sequence for mcpSRP43 that begins AAVQRN and lacks the CD2 amino acids FEYA . . . EDGL. Plasmid coding for cpSRP43 that lacks the C-terminal (CD3) chromodomain (p-cpSRP⌬CD3) was produced by using an exact forward primer and a reverse primer that binds immediately upstream of the CD3 coding region and introduces a stop codon immediately following CD2 such that the protein begins AAVQRN and ends GLEYE. Plasmid coding for cpSRP-A1-CD2 utilized the same reverse primer as was used to generate cpSRP⌬CD3 along with an exact forward primer that binds at the beginning of the Ank region such that the protein begins YETP and ends GLEY. All deletion and domain expression constructs were transformed into Escherichia coli strain BL21 Star (Invitrogen) for isopropyl-1-thio-␤-D-galactopyranoside-induced protein expression. GST-cpSRP⌬CD1, GST-cpSRP⌬CD2, GST-cpSRP⌬CD3, and GST-cpSRP-A1-CD2 were produced and purified as described above for GST-cpSRP43. Cleaved constructs were produced and purified as described for cpSRP43.
Coding sequences for cpSRP43 domains CD1, CD2, and CD3 were amplified by PCR using forward primers corresponding to the coding sequences of the desired amino acids indicated in Fig. 1. A BamHI restriction site was incorporated at the 5Ј end of the forward primer to allow ligation of the restricted PCR products into pGEX-4T-2 (Amersham Biosciences) restricted with BamHI and SmaI. CD1, CD2, and CD3 expression constructs were transformed into E. coli strain BL21 Star for isopropyl-1-thio-␤-D-galactopyranoside-induced protein expression. Following bacterial lysis, expressed proteins were affinity-purified by using glutathione-Sepharose affinity chromatography followed by purification by anion exchange chromatography before being desalted into HKM buffer.
The nucleotide sequence coding for cpSRP54 lacking the C-terminal 26 amino acids was amplified by using an exact forward primer that introduced a KpnI site and a reverse primer that bound immediately upstream of the 26 amino acids to be deleted. The reverse primer also incorporated an in-frame 6-histidine tag plus a HindIII site such that the protein ends ARRKHHHHHH. The purified PCR product was restricted and ligated into KpnI/HindIII-restricted pGEM-4Z prior to subcloning into pPROLar.A122 by using the same restriction sites. Expression and purification of cpSRP⌬C26 -54-his were performed as described for cpSRP54-his.
The nucleotide sequence coding for the M domain of cpSRP54, beginning MGDVLS and ending GSGN, was amplified from pNH2 (5) by using XbaI and SmaI sites to create pGEM-3Z-M domain-cpSRP54 for in vitro transcription/translation. The nucleotide sequence coding for the NG domain of cpSRP54, beginning MFGQLTG and ending GRIL, was similarly amplified by using a forward primer that incorporated an XbaI site and added the additional amino acids MG immediately preceding the NG domain. The reverse primer incorporated a stop immediately following the coding sequence for the NG domain and provided a blunt end for subsequent ligation into SmaI-and XbaI-restricted pGEM 3Z to create the in vitro transcription/translation clone pGEM-3Z-NG domain-cpSRP54.
Transit Complex and pLHCP Transport Assays-Transit complex was formed in 30-l assays essentially as described (18) with the following changes. Thirty picomoles of recombinant cpSRP43 or appropriate deletion construct and an equimolar amount of recombinant cpSRP54-his or cpSRP⌬C26 -54-his were substituted for stromal extract (SE) and added to 5 l of 35 S-labeled pLHCP translation product (15) containing 1.7 fmol of LHCP diluted 1:3 in HK buffer (10 mM HEPES-KOH, pH 8.0). Each assay was brought to 30 l final volume with HK buffer and final concentrations of 0.2 mM GTP and 5 mM ATP and incubated for 30 min at 25°C before the addition of 5 l of cold 50% glycerol. Control assays were conducted as above but included either no protein components or 20 l of SE. For each assay, 10 l of sample was analyzed by 6% nondenaturing PAGE and imaged on a Typhoon Phos-phorImager (Amersham Biosciences) to distinguish cpSRP⅐LHCP transit complex from aggregated LHCP. For assays in which L18 synthetic peptide was used to compete with LHCP for binding to cpSRP, transit complex was formed by adding 40 pmol each of cpSRP54-his and either cpSRP43 or cpSRP-A1-CD2 that had been preincubated for 10 min with varying (from 0 to 500 M) amounts of synthetic L18 peptide in HKM buffer. Protein mixtures were allowed to incubate for 10 min at 25°C, and from this point the assay was performed as described above in the absence of additional GTP and ATP.
Transport of pLHCP into thylakoid membranes was essentially performed as described previously (27) with the following changes. Thylakoid membranes were isolated and washed in buffer containing 1 M potassium acetate and 1 mM dithiothreitol in Import Buffer (IB: 50 mM HEPES-KOH, pH 8, 330 mM sorbitol). Each 75-l reaction containing 100 pmol of cpSRP54-his (or cpSRP⌬C26 -54-his), Trx-cpFtsY, and cpSRP43 construct; 1 mM GTP; and 1 mM ATP was incubated at 25°C for 10 min and then added to 25 l of 2ϫ salt-washed thylakoids (1ϫ thylakoids ϭ 0.5 mg/ml chlorophyll) in IBM (IB ϩ 10 mM MgCl 2 ). After a 5-min incubation, 5 l of 35 S-labeled pLHCP translation product diluted 1:3 in HK buffer was added, and the reaction was allowed to proceed for 30 min at 25°C in light. Following collection of thylakoids by centrifugation and protease treatment with thermolysin (28), the thylakoids were solubilized with 50 l of SDS-PAGE solubilization buffer and analyzed on 12.5% SDS-PAGE by loading 10 l of each assay per lane. Integrated LHCP, as noted by the formation of a correctly sized degradation product, was visualized by PhosphorImaging.
Protein Binding Assays-cpSRP54-his and cpSRP⌬C26 -54-his binding assays were performed by combining 100 pmol of each GST-fused cpSRP43 deletion or domain expression construct with 100 pmol of cpSRP54-his or cpSRP⌬C26 -54-his and 70 l of a 50% glutathione-Sepharose slurry in 10 mM HK, 50 mM potassium acetate, and 10 mM MgCl 2 , pH 8.0, in a final volume of 270 l. Samples were allowed to mix end-over-end for 1 h at 4°C and then were washed as described (19) by utilizing PSU 6.5-mm centrifuge filters (Whatman). Proteins were eluted in 50 l of 40 mM glutathione in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl. Eluted proteins were separated by 12.5% SDS-PAGE and visualized directly by staining with Coomassie Blue. Interactions with pLHCP, L18-PPL, cpSRP54-M domain, and cpSRP54-NG domain were performed by incubation of 100 pmol of each GST fusion protein with 20 l of 35 S-labeled, in vitro translated product produced as for pLHCP (15). Incubation, washing, and elution were performed as above, and eluates were visualized by 12.5% SDS-PAGE and PhosphorImaging.
Assay of GTPase Activity-Recombinant cpSRP43 and deletion constructs were assayed for GTPase activity in the presence and absence of cpSRP54-his and cpFtsY as described (29). GTPase activity was measured in solution by determining the amount of inorganic phosphate released by GTP hydrolysis. Assays containing 150 pmol of each indicated protein component and 2 mM GTP in 10 mM HEPES, pH 8.0, and 10 mM MgCl 2 were incubated at 30°C for 1 h. After incubation SDS was added to a final concentration of 6% to denature protein components and prevent subsequent GTPase activity. The addition of ascorbic acid and ammonium molybdate (to 6 and 1%, respectively) was followed by a 5-min incubation, and subsequently each assay was brought to 1% sodium citrate, sodium (meta)arsenite, and acetic acid for a final volume of 1.05 ml. The absorbance of each sample was then measured at 850 nm. Throughout the duration of the assay, the amount of GTP hydrolyzed increased linearly. Furthermore, a standard curve of inorganic phosphate was linear between 2 and 75 nmol of P i and was used to determine the amount of P i released in each assay. A substrate control that lacked protein components and a zero time control with the protein denatured by the addition of 6% SDS prior to the addition of GTP varied from 0.42 to Ϫ0.21 nmol of P i between experiments and were included to correct for nonspecific hydrolysis and background in each experiment.

Domain Analysis Indicates the Presence of Three Chromodomains in cpSRP43-
The unusual function of a chromodomain protein (see Fig. 1 for domain organization) in protein targeting led us to investigate the role of individual CDs at distinct steps of the LHCP localization pathway. We first subjected the cpSRP43 sequence to SMART (Simple Modular Architecture Research Tool) analysis (30), which indicated the presence of a repeating ankyrin domain region and two CDs at the C-terminal region of cpSRP43 as reported previously (23). In addition, an uncharacterized third CD was predicted at the N terminus of cpSRP43. An alignment of the CDs (termed CD1, CD2, and CD3) of cpSRP43 revealed a high level of similarity between all three domains and a CD from a well characterized mouse heterochromatin-associating protein, mouse modifier protein 1 (mM-HP1b) ( Fig. 2A). Fig. 2 shows the core structure of each CD present in cpSRP43, each derived using the homology building program Swiss model (31) and the coordinates from a determined threedimensional structure of a known chromatin binding domain from mouse modifier protein 1 (1AP0) (32). The models suggest that the most highly conserved residues found among the three predicted chromodomains likely stabilize the ␤-sheet structure. The amino acids of the ␤-sheet core exhibit a strikingly invariant conformation, reflecting the canonical CD conformation that has been observed in other systems. For example, the positions of the Tyr and Trp residues indicated by asterisks are identical to those important for function in HP1 of Drosophila and mouse (33). However, the third conserved residue neces-sary for histone binding in HP1 domains, Phe, is changed to a Gly or Met in the CDs of cpSRP43 which may influence target specificity. The most variable region among the CDs is the C-terminal ␣-helix, the function of which is not well characterized in other CD-containing proteins. The structural features of the models are reasonable in terms of what is known about protein structure in general, i.e. a well packed core is formed by hydrophobic residues and surface residues are primarily hydrophilic. Furthermore, main chain conformations are in acceptable regions where bond angles and lengths as well as planarity are all acceptable using PROCHECK (34). Overall, the models indicate reasonable conformations that adhere to known chromodomain structures. As such, these models argue strongly for the presence of a previously unidentified CD, CD1, near the N terminus of cpSRP43.
We have used these models to construct plasmids that either express cpSRP43 deletions lacking individual CDs or express only the individual CDs (refer to Fig. 1). The proteins were expressed in E. coli as GST fusion proteins, purified to at least 85% homogeneity (Fig. 3), and were used in assays that reconstitute distinct steps of the LHCP localization process.
Chromodomains Exhibit Distinct Functions in Two Stages of LHCP Localization-In order to examine the functional role of CDs in post-translational cpSRP-based protein targeting, purified CD deletion constructs of cpSRP43 were mixed with recombinant cpSRP54-his, radiolabeled LHCP, and assayed for transit complex formation. A soluble cpSRP⅐LHCP transit complex migrates as a single band when subjected to nondenaturing PAGE (Fig. 4) (15,35). Whereas deletion of either CD1 or CD3 had little or no effect on the efficiency of transit complex formation (Fig. 4A, compare lanes 2 and 4 with lane 1), deletion of both the first and third CDs typically reduced the efficiency by 40 -50% (Fig. 4A, compare lane 5 with lane 1). Notably, deletion of CD2 nearly eliminated transit complex formation (Fig. 4A, lane 3). Together, these data indicate that the central CD of cpSRP43 is critical for transit complex formation. In addition, it appears that neither the N-terminal nor the C- FIG. 1. cpSRP43 is composed of ankyrin and chromodomains. A schematic diagram of cpSRP43 illustrates the domain organization predicted by SMART analysis as described under "Experimental Procedures." Three chromodomains (chromo1, chromo2, and chromo3) are represented by triangles and four ankyrin domains (Ank1-Ank4) by cylinders. Numbers corresponding to the amino acid positions of these domains are based on the precursor polypeptide, which is predicted to contain a 60-amino acid chloroplast targeting domain that is processed in the stroma (23). Deletion mutations and complementary expressed regions are indicated below the full-length cpSRP43 with names to the left that denote each protein construct.
terminal CDs play any significant individual role in the formation of transit complex, although simultaneous removal of both, leaving only the Ank-CD2 region of cpSPR43, decreases the efficiency of transit complex formation.
In order to confirm that the interaction between the Ank1-CD2 region of cpSRP43, LHCP, and cpSRP54 occurs in a manner fundamentally similar to cpSRP, we sought to determine the binding specificity of this core region for the L18 motif in LHCP, a charged region of LHCP that binds cpSRP43 (19) and is required for transit complex formation (18). Separate transit complex assays were conducted with full-length cpSRP43 and cpSRP-A1-CD2 in the presence of increasing concentrations of L18 synthetic peptide, which competes with LHCP for binding endogenous cpSRP (18). Although the transit complex forming activity of cpSRP-A1-CD2 is somewhat lower overall, the specificity of the protein for L18 mirrors that of the full-length protein as judged by the ability of the L18 peptide to compete with LHCP for binding to cpSRP (Fig. 4C). These findings indicate that the interactions within the Ank1-CD2 core region of cpSRP43 are necessary and sufficient for formation of a soluble cpSRP⅐LHCP transit complex.
To examine further the role of CDs in LHCP integration, full-length cpSRP43 or each of the CD deletion constructs was incubated with recombinant cpSRP54-his before addition of radiolabeled LHCP, purified recombinant cpFtsY, and saltwashed thylakoids. Because salt-washed thylakoids lack appreciable levels of cpFtsY, integration of LHCP requires the addition of both recombinant cpSRP and cpFtsY (14). Fig. 4B shows that deletion of CD3 had no deleterious influence on integration whereas deletion of CD2 abolished integration, effects that mirror the influence of these CD deletions on transit complex formation (Fig. 4A). However, cpSRP⌬CD1, which supported transit complex formation, exhibited a severe inability to support integration. The integration activity in assays containing cpSRP⌬CD1 was typically reduced 70 -90% relative to assays containing cpSRP43. Likewise, integration with cpSRP-A1-CD2 was greatly reduced. Taken together, these data indicate that CD1 functions downstream from transit complex formation at a step that is critical for efficient LHCP integration into thylakoids.
Defining a Functional cpSRP43/cpSRP54 Interface-The results in Fig. 4 suggest that CD3 is not required for functional interactions with either soluble or membrane proteins required for LHCP integration into isolated thylakoids. In contrast, protein interactions mediated by CD2 appear necessary at all stages of the localization mechanism. Previous results from yeast two-hybrid assays indicated a critical role for both CD2 and CD3 in binding cpSRP54 (23); hence, we employed the use of GST fusion proteins to examine CD interactions with cpSRP54-his in protein binding assays. GST fused to CD deletion constructs or fused to individual CDs were incubated with cpSRP54-his and re-purified using glutathione-Sepharose TM beads. The proteins were then eluted from the beads with buffer containing glutathione (see "Experimental Procedures").

FIG. 2. Conserved structural features of cpSRP43 CDs.
A, alignment of the three CDs of cpSRP43 with the CD of mouse modifier protein (mM-HP1b) by SMART analysis (30) reveals strong amino acid conservation within the first two predicted ␤-sheets as shown in the schematic above the alignment. Residues demonstrated to be necessary for HP1 chromodomain function are highlighted in red and green; highly conserved residues are shaded in gray. A logo of consensus amino acid properties below the alignment is indicated for each amino acid position as follows: H for hydrophobic, C for charged, P for polar, and G for glycine. The height of each letter indicates the relative level of similarity at each position. B, the three CDs of cpSRP43 were modeled by using the homology building program Swiss model (31), and the coordinates are from an NMR structure of 1APO, a binding domain from mouse modifier protein 1 (32). Representations were generated using Pymol (Delano Scientific).

FIG. 3. SDS-PAGE analysis of purified GST fusion proteins, cpSRP43 deletion constructs, cpSRP54-his, and Trx-cpFtsY.
After expression and purification, 1 g of each indicated component was analyzed on a 12.5% SDSpolyacrylamide gel and stained with Coomassie Blue.
Eluted proteins were separated by SDS-PAGE and visualized directly by staining with Coomassie Blue. Fig. 5, A and B, shows that cpSRP54-his bound to CD2 alone and to deletion constructs that contain CD2. Neither CD1 nor CD3, whether present in the cpSRP⌬CD2 deletion construct or as individual domains, showed any affinity for cpSRP54-his (Fig. 5, A and B). When equimolar amounts of GST-cpSRP43 and GST-CD2 are combined with cpSRP54-his, equivalent amounts of cpSRP54his are copurified (Fig. 5B, top panel), suggesting that CD2 is solely responsible for any interaction with cpSRP54-his. These experiments were repeated using stromal extract as a source of cpSRP54 with identical results, further demonstrating that CD2 alone is responsible for interactions with cpSRP54 rather than the combination of CD2 and CD3 as has been suggested previously. In order to examine the possibility that loss of cpSRP54 binding is attributable to misfolding of the cpSRP43 constructs that lack CD2, we examined their ability to bind L18, the cpSRP43-binding motif in LHCP. When the same coprecipitation assays were performed with L18-PPL (18), no loss of L18 binding was observed with any of the single domain deletions. This suggests that the loss of cpSRP54 binding by cpSRP⌬CD2 did not result from severe misfolding of cpSRP43, but rather that CD2 is the primary binding site for cpSRP54.
To analyze further the interaction between cpSRP43 and cpSRP54, in vitro transcription/translation clones of the M domain and the NG domain of cpSRP54 were used to define further the site of interaction between cpSRP43 and cpSRP54. Neither cpSRP43 nor any cpSRP43 construct tested showed any interaction with the NG domain of cpSRP54 above background levels (Fig. 5A). In contrast, the M domain of cpSRP54 interacted strongly with cpSRP43 and cpSRP43 constructs that contained CD2. Removal of CD1 and CD3 had no observable effect on the amount of coprecipitated M domain, whereas removal of CD2 abolished the ability of cpSRP43 to coprecipitate the M domain of cpSRP54. Similarly, only CD2 as an individual domain showed any interaction with the M domain (Fig. 5B); no individual domain showed any interaction with the NG domain of cpSRP54 above background levels. 2 Taken together, these data indicate that cpSRP54 and cpSRP43 must be in a heterodimer mediated by the CD2/M domain interaction to form transit complex and support LHCP integration.
It is noteworthy that removal of the C-terminal 26 amino acids from cpSRP54, described previously as essential for cpSRP43 binding (7), did not eliminate interaction of cpSRP54 with GST-cpSRP43. The remaining interaction activity (40%; Fig. 6A, compare 2nd and 4th lanes) supported integration of LHCP at levels similar to full-length cpSRP54 (Fig. 6B, compare 2nd and 3rd lanes).
cpSRP43 Regulates the GTPase Activity of cpSRP54/ cpFtsY-Because of the role of GTP binding and hydrolysis by SRP54 and SR (FtsY in E. coli) in modulating events at the target membrane, we explored the possibility that CD1 function is associated with the GTPase cycle, which occurs during targeting/integration events. In protein targeting to the ER, protein interactions between membrane and soluble components are regulated through GTP binding and hydrolysis to ensure that the substrate is released only in the presence of an open translocase (36). It has been shown recently that interactions between the ribosome and SRP as well as between SR␤ and the ribosome are involved in the regulation of GTP hydrolysis (17,36). However, the cpSRP system lacks a ribosomal 2 R. L. Goforth and R. L. Henry unpublished data.

FIG. 4. CDs in cpSRP43 exhibit distinct functions in targeting complex formation and integration.
A, in vitro translated, radiolabeled LHCP was incubated with cpSRP54-his along with cpSRP43 or a cpSRP43 deletion construct as shown above each lane. Control transit complex samples contain either no recombinant proteins (none) or stromal extract (SE). Reactions were resolved on a 6% nondenaturing polyacrylamide gel and visualized by PhosphorImaging as described under "Experimental Procedures." The position of the cpSRP⅐LHCP transit complex is indicated as is the position of aggregated LHCP that remains in the well. B, integration assays were conducted with thylakoids that were salt-washed to remove endogenous proteins, including the peripheral membrane protein cpFtsY, as described under "Experimental Procedures." Subsequently, thylakoids were incubated with recombinant Trx-cpFtsY, cpSRP54-his, and cpSRP43 or an equimolar amount of one of the domain deletions. In vitro translated, radiolabeled pLHCP (TP) was added, and the thylakoids were protease-treated after incubation to remove nonintegrated pLHCP. The formation of a characteristic degradation product (LHCP-DP) indicates properly integrated LHCP. Assays conducted with cpSRP43 resulted in integration of 9.1% of the LHCP translation product and were set to 100% for calculation of the average and standard deviation by using data from three experiments for the deletion constructs. C, in vitro translated, radiolabeled LHCP was incubated with cpSRP54-his, cpSRP43, or cpSRP-A1-CD2 and increasing amounts of L18 peptide. Reactions were resolved on a 6% nondenaturing polyacrylamide gel and visualized by PhosphorImaging as described under "Experimental Procedures." The position of the cpSRP⅐LHCP transit complex is indicated as is the position of aggregated LHCP that remains in the well. component, and no SR␤ sequence homolog has been identified. Therefore, it seems likely that proteins unique to the cpSRP system may act to replace the regulatory function of the ribosome and/or SR␤.
To examine the possibility that cpSRP43 may play a role in regulating GTPase activity of cpSRP54 and cpFtsY, we utilized a colorimetric assay that measures release of inorganic phosphate by GTP hydrolysis (29), a method made possible by the availability of chemical quantities of purified cpSRP targeting components (see Fig. 3). Comparison of the amounts of inorganic phosphate generated by equimolar amounts of constituent proteins indicates that little GTP is hydrolyzed when any single protein component (e.g. cpSRP43, cpSRP54-his, and Trx-cpFtsY) is present (Fig. 7A, lanes 4, 8, 12, 16, 20, 23, and 24). When cpSRP54-his and Trx-cpFtsY are both present, GTP hydrolysis is slightly more than what would be expected from additive GTP hydrolysis (Fig. 7A, compare lane 22 with lanes  23 and 24). Notably, when cpSRP43 is added, GTP hydrolysis is increased (Fig. 7A, compare lane 1 with lane 22), suggesting that cpSRP43 may play a role in the regulation of GTP hydrolysis by cpSRP54 and cpFtsY. Further support for such a role is provided by results of GTP hydrolysis assays conducted with CD deletions in place of cpSRP43. Removal of either the second or the third CD (Fig. 7A, compare lanes 9 and 13 with lane 1) had little effect on the overall amount of P i generated when compared with full-length cpSRP43. However, GTP hydrolysis by cpSRP54-his/Trx-cpFtsY was greatly increased by removal of CD1 (Fig. 7A, compare lane 5 with lanes 1 and 22), demonstrating that cpSRP43 has the ability to increase GTP hydrolysis by cpSR54-his/Trx-cpFtsY 4-fold in the absence of CD1. Similarly, the addition of cpSRP-A1-CD2, which lacks both CD1 and CD3, to cpSRP54 and cpFtsY also resulted in a dramatic increase in GTP hydrolysis as compared to the addition of cpSRP43 (Fig. 7A, compare lane 17 with lanes 1 and 22). The increased rate of hydrolysis observed when either cpSRP⌬ CD1 or cpSRP-A1-CD2 is added to cpSRP54/cpFtsY was specific for GTP. In equivalent assays where GTP was replaced with ATP, only low levels of nucleotide hydrolysis were observed. The greatest level of inorganic phosphate released from ATP hydrolysis for any mixture of proteins did not exceed 2.2 nmol. 2 To examine whether stimulation of cpSRP54/cpFtsY GTP hydrolysis activity by CD1 deletion constructs (cpSRP⌬CD1 or cpSRP-A1-CD2) stems from CD2 alone or involves the Ank repeat region of cpSRP43, GTP hydrolysis by cpSRP54/cpFtsY was measured in the absence or presence of individual chromodomains. GTP hydrolysis by cpSRP54-his in combination with Trx-cpFtsY was unaffected by addition of CD1, CD2, or CD3 (Fig. 7B). Taken together, these data suggest that the requirement for CD1 in LHCP integration may stem from its participation in events that regulate GTP binding and/or hydrolysis. Moreover, our data are consistent with a model where CD1 acts to negatively modulate the GTPase stimulatory activity of an Ank repeat region in cpSRP43. DISCUSSION In this report, we have used recombinant cpSRP to understand the role of cpSRP43 chromodomains in the LHCP targeting/insertion mechanism. We have combined functional assays were incubated with recombinant cpSRP54-his or cpSRP⌬C26 -54-his and recovered and analyzed as described in Fig. 5. B, integration assays were conducted as described under "Experimental Procedures" with salt-washed thylakoids by incubation of radiolabeled LHCP (TP) with recombinant Trx-cpFtsY, cpSRP43, and either cpSRP54-his or cpSRP⌬C 26-54-his. Thylakoids were protease-treated after incubation and analyzed as described in Fig. 4B. The formation of a characteristic degradation product (LHCP-DP) indicates properly integrated LHCP. Relative levels of integration are shown above each lane. and interaction studies to understand the functional relevance of chromodomain interactions within the LHCP targeting/insertion pathway. Our studies address the role of two previously identified chromodomains (CD2 and CD3) as well as the role of a chromodomain identified by sequence alignment and structural modeling (CD1). In this context, our work establishes the importance of chromodomain interactions in the formation of a cpSRP⅐LHCP transit complex, which serves as the soluble form of LHCP targeted to the membrane. Of the three CDs, only CD2 is required for transit complex formation and for binding of cpSRP43 to cpSRP54. Together, these data show for the first time that cpSRP43 must be in a heterodimer with cpSRP54 in order to support transit complex formation. It is noteworthy that a previous structural model of cpSRP, based largely on results from a yeast two-hybrid approach, supports a structural model in which CD2 and CD3 of cpSRP43 create a single binding site for cpSRP54 (23). However, CD3 alone shows no ability to bind cpSRP54, and deletion of CD3 has no impact on cpSRP43 binding to cpSRP54 (Fig. 4). Hence, our data support a different structural model of cpSRP in which CD3 plays little or no role in cpSRP54-cpSRP43 heterodimer formation. Furthermore, CD3 was found to be dispensable for both transit complex formation and LHCP integration suggesting that its evolutionary preservation may be related to LHCP localization steps not examined by our assays and which precede transit complex formation in the stroma, e.g. import across the envelope membranes mediated by TIC and TOC transporters (reviewed in Refs. 37 and 38). In this context, we are currently examining Arabidopsis mutants for the ability of cpSRP⌬CD3 to compensate for the absence of cpSRP43 expression.
It has been suggested that the cpSRP43 binding region of cpSRP54 lies within the methionine-rich C-terminal M domain (23), and peptide scanning of cpSRP54 indicated that the interaction is contained within the C-terminal 26 amino acids of this domain (7). In contrast, we show that removal of the C-terminal 26 amino acids from recombinant cpSRP54 reduces binding to cpSRP43 by 60% (Fig. 6A) but does not eliminate the interaction, suggesting that additional interaction sites within the cpSRP54 M domain participate in binding to cpSRP43. The remaining ability of cpSRP⌬C26 -54-his to bind cpSRP43 is sufficient for SRP targeting/integration activities; deletion of this region from cpSRP54 did not inhibit LHCP integration (see Fig. 6B). This may point to stabilization of the cpSRP43/ cpSRP54 interaction by association with LHCP, which is known to interact with both SRP components (18,19,35). Additionally, we cannot exclude the possibility that once transit complex is formed, other regions of cpSPR54 and/or cpSRP43 interact that do not function in SRP heterodimerization per se.
In contrast to transit complex formation, LHCP insertion into the thylakoid membrane requires both CD2 and a previously unexplored chromodomain, CD1. Loss of LHCP integration would be expected by using a CD2 deletion of cpSRP43; CD2 is required for transit complex formation, which is a prerequisite for LHCP integration. However, LHCP integration is nearly lost in the absence of CD1, whereas transit complex formation appears unhindered. This demonstrates for the first time that cpSRP43 functions downstream from the formation of a cpSRP⅐LHCP transit complex and supports a much expanded role for cpSRP43 that extends beyond its im- portance in allowing cpSRP to bind full-length substrates. The absence of CD1 interaction with other components in transit complex, along with a requirement for CD1 to promote efficient integration, suggests the possibility that CD1 may be interacting with an integral membrane protein necessary for integration of LHCPs. However, the recently defined functional interactions between cpSRP, FtsY, and the ALB3 translocase demonstrate that cpSRP43 is not required for functional association of these components (11). Therefore, it is not surprising that deletion of CD1 has no impact on the ability to form this complex at the membrane. 2 The fact that removal of CD1 from cpSRP43 increases by 4-fold the rate at which GTP is hydrolyzed by cpSRP/cpFtsY alone (Fig. 7) suggests an alternative explanation. It now seems likely that cpSRP43 coordinates activities at the membrane by regulating the GTPase cycle of cpSRP/cpFtsY. Specifically, CD1 appears to act as a negative regulator because its removal leads to increased rates of GTP hydrolysis. By this same view, a region of cpSRP43 outside of CD1 appears to act as a positive regulator of GTP hydrolysis because removal of CD1 leads to elevated GTPase activity of cpSRP/cpFtsY beyond the level of stimulation seen when fulllength cpSRP43 is added. It is noteworthy that the amount of GTP hydrolysis is low in all cases relative to the amount of recombinant protein present in the assays. This may indicate that additional factors are required for high (catalytic) rates of GTP hydrolysis. In bacteria, lipid interactions stimulate the GTPase activity of FtsY (39), and interaction of available translocase components with targeting machinery initiates GTP hydrolysis in targeting to the ER (40). In that context, the low rate of GTP hydrolysis by cpSRP54/cpFtsY in solution is likely of biological significance because high rates of GTP hydrolysis in solution would lead to a potentially deleterious futile cycle of GTP hydrolysis in stroma.
The ability of the Ank1-CD2 portion of cpSRP43 to stimulate GTP hydrolysis, whereas CD2 alone does not, indicates that the ankyrin repeat region of cpSRP43 is part of the mechanism by which cpSRP43 is able to regulate the GTP hydrolysis cycle. Recent reports (41,42) on the crystal structure of the GTPase core of the SRP targeting complex have shown that FtsY and Ffh, stabilized by extensive surface interactions, form a composite active site at their interface and suggest a unique mechanism by which Ffh-FtsY complex formation and disassembly are driven by nucleotide binding and hydrolysis. Our studies suggest that for post-translational targeting by cpSRP/cpFtsY, reciprocal activation of GTP binding/hydrolysis by these proteins is influenced by cpSRP43. A detailed understanding of how cpSRP43 influences the timing and course of LHCP integration remains to be investigated. Yet, in light of a recent report (17) illustrating the regulatory role of the ribosome in SRP54/SR␣ GTPase activity, it is attractive to speculate that cpSRP43 may have evolved to replace the regulatory function(s) of the ribosome. In this context, cpSRP43 may bind both cpSRP54 and cpFtsY at the membrane, which is supported by the fact that the addition of cross-linking reagents to a cpSRP-cpFtsY complex at the membrane results in a cross-linking adduct composed minimally of cpSRP54, cpSRP43, and cpFtsY along with the translocase component, ALB3 (11). A detailed understanding of cpSRP43 interactions at the membrane is likely to uncover interactions mediated by the Ank repeat and CD1, which we hypothesize are used to regulate the timing of GTP binding/hydrolysis by cpSRP/cpFtsY and LHCP release from cpSRP to ALB3.