Chloroplast FtsY, Chloroplast Signal Recognition Particle, and GTP Are Required to Reconstitute the Soluble Phase of Light-harvesting Chlorophyll Protein Transport into Thylakoid Membranes
1999; Elsevier BV; Volume: 274; Issue: 38 Linguagem: Inglês
10.1074/jbc.274.38.27219
ISSN1083-351X
AutoresChao-Jung Tu, Danja Schuenemann, Norman E. Hoffman,
Tópico(s)Protist diversity and phylogeny
ResumoThe integration of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membrane proceeds in two steps. First, LHCP interacts with a chloroplast signal recognition particle (cpSRP) to form a soluble targeting intermediate called the transit complex. Second, LHCP integrates into the thylakoid membrane in the presence of GTP, at least one other soluble factor, and undefined membrane components. We previously determined that cpSRP is composed of 43- and 54-kDa polypeptides. We have examined the subunit stoichiometry of cpSRP and find that it is trimeric and composed of two subunits of cpSRP43/subunit of cpSRP54. A chloroplast homologue of FtsY, an Escherichia coli protein that is critical for the function of E. coli SRP, was found largely in the stroma unassociated with cpSRP. When chloroplast FtsY was combined with cpSRP and GTP, the three factors promoted efficient LHCP integration into thylakoid membranes in the absence of stroma, demonstrating that they are all required for reconstituting the soluble phase of LHCP transport. The integration of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membrane proceeds in two steps. First, LHCP interacts with a chloroplast signal recognition particle (cpSRP) to form a soluble targeting intermediate called the transit complex. Second, LHCP integrates into the thylakoid membrane in the presence of GTP, at least one other soluble factor, and undefined membrane components. We previously determined that cpSRP is composed of 43- and 54-kDa polypeptides. We have examined the subunit stoichiometry of cpSRP and find that it is trimeric and composed of two subunits of cpSRP43/subunit of cpSRP54. A chloroplast homologue of FtsY, an Escherichia coli protein that is critical for the function of E. coli SRP, was found largely in the stroma unassociated with cpSRP. When chloroplast FtsY was combined with cpSRP and GTP, the three factors promoted efficient LHCP integration into thylakoid membranes in the absence of stroma, demonstrating that they are all required for reconstituting the soluble phase of LHCP transport. signal recognition particle chloroplast SRP light-harvesting chlorophyll protein glutathione S-transferase polymerase chain reaction nitrilotriacetic acid polyacrylamide gel electrophoresis chloroplast FtsY chloroplast Sec SRP1 mediates the cotranslational targeting of endomembrane and secretory proteins to the endoplasmic reticulum in eukaryotes and of polytopic membrane proteins to the cytoplasmic membrane in prokaryotes (1Rapoport T.A. Jungnickel B. Kutay U. Annu. Rev. Biochem. 1996; 65: 271-303Crossref PubMed Scopus (492) Google Scholar, 2Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (713) Google Scholar, 3Ulbrandt N.D. Newitt J.A. Bernstein H.D. Cell. 1997; 88: 187-196Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Cytosolic forms of SRP are ubiquitous in eukaryotic and prokaryotic organisms. All contain, at a minimum, a 54-kDa GTPase subunit and an RNA (1Rapoport T.A. Jungnickel B. Kutay U. Annu. Rev. Biochem. 1996; 65: 271-303Crossref PubMed Scopus (492) Google Scholar, 2Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (713) Google Scholar). Membrane targeting is facilitated by an interaction between SRP and an SRP receptor (4Gilmore R. Blobel G. Walter P. J. Cell Biol. 1982; 95: 463-469Crossref PubMed Scopus (235) Google Scholar, 5Gilmore R. Walter P. Blobel G. J. Cell Biol. 1982; 95: 470-477Crossref PubMed Scopus (295) Google Scholar, 6Meyer D.I. Dobberstein B. J. Cell Biol. 1980; 87: 503-508Crossref PubMed Scopus (80) Google Scholar). In eukaryotes, the receptor consists of two GTPases, a peripheral protein (the SRP receptor α-subunit), and an integral membrane polypeptide (the SRP receptor β-subunit) (7Tajima S. Lauffer L. Rath V. Walter P. J. Cell Biol. 1986; 103: 1167-1178Crossref PubMed Scopus (117) Google Scholar, 8Miller J.D. Tajima S. Lauffer L. Walter P. J. Cell Biol. 1995; 128: 273-282Crossref PubMed Scopus (89) Google Scholar). The localization of the SRP receptor to the membrane may facilitate, but is not essential for, targeting (9Ogg S.C. Barz W.P. Walter P. J. Cell Biol. 1998; 142: 341-354Crossref PubMed Scopus (50) Google Scholar). A key feature of the SRP/SRP receptor interaction is the ability of the SRP receptor α-subunit and SRP54 to reciprocally stimulate their GTP hydrolysis activities upon mutual binding in the presence of SRP RNA and thereby to regulate the GTP hydrolysis cycle associated with SRP-dependent protein targeting (10Miller J.D. Bernstein H.D. Walter P. Nature. 1994; 367: 657-659Crossref PubMed Scopus (186) Google Scholar, 11Powers T. Walter P. Science. 1995; 269: 1422-1424Crossref PubMed Scopus (181) Google Scholar).Recently, a specialized SRP was found in the chloroplast (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar, 13Schuenemann D. Amin P. Hoffman N.E. Biochem. Biophys. Res. Commun. 1999; 254: 253-258Crossref PubMed Scopus (24) Google Scholar). cpSRP contains a homologue of SRP54 (14Franklin A.E. Hoffman N.E. J. Biol. Chem. 1993; 268: 22175-22180Abstract Full Text PDF PubMed Google Scholar), but differs from cytoplasmic forms, as it lacks an RNA, contains a novel 43-kDa subunit, and interacts with substrates post-translationally (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar, 15Klimyuk V.I. Persello-Cartieaux F. Havaux M. Contard P. Schuenemann D. Meiherhoff K. Gouet P. Jones J.D.G. Hoffman N.E. Nussaume L. Plant Cell. 1999; 11: 87-99Crossref PubMed Scopus (119) Google Scholar, 16Li X.X. Henry R. Yuan J.G. Cline K. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3789-3793Crossref PubMed Scopus (159) Google Scholar). Both genetic and biochemical evidence indicates that the 43-kDa subunit is essential for this post-translational interaction (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar, 15Klimyuk V.I. Persello-Cartieaux F. Havaux M. Contard P. Schuenemann D. Meiherhoff K. Gouet P. Jones J.D.G. Hoffman N.E. Nussaume L. Plant Cell. 1999; 11: 87-99Crossref PubMed Scopus (119) Google Scholar, 17Amin P. Sy D. Pilgrim M. Parry D. Nussaume L. Hoffman N.E. Plant Physiol. 1999; (Bethesda), in pressPubMed Google Scholar). The known substrates of cpSRP are the LHCPs, hydrophobic proteins that are synthesized in the cytoplasm and are post-translationally transported to the internal membranes of the chloroplast via the soluble phase (18Cline K. Fulsom D.R. Viitanen P.V. J. Biol. Chem. 1989; 264: 14225-14232Abstract Full Text PDF PubMed Google Scholar,19Reed J.E. Cline K. Stephens L.C. Bacot K.O. Viitanen P.V. Eur. J. Biochem. 1990; 194: 33-42Crossref PubMed Scopus (67) Google Scholar). The solubility of LHCP is maintained in the stroma by its binding to cpSRP to form the targeting intermediate termed the transit complex (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar, 16Li X.X. Henry R. Yuan J.G. Cline K. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3789-3793Crossref PubMed Scopus (159) Google Scholar, 20Payan L.A. Cline K. J. Cell Biol. 1991; 112: 603-613Crossref PubMed Scopus (82) Google Scholar).The transit complex can be reconstituted in vitro from purified cpSRP and LHCP, suggesting that it is composed of cpSRP54, cpSRP43, and LHCP. However, one unresolved issue is the subunit stoichiometry of cpSRP and the transit complex. The molecular weight estimate of the transit complex from nondenaturing gel analysis is 120,000 (20Payan L.A. Cline K. J. Cell Biol. 1991; 112: 603-613Crossref PubMed Scopus (82) Google Scholar), whereas the molecular weight of cpSRP from gel filtration is 200,000 (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar). Also unresolved is the fact that the soluble form of LHCP is incapable of inserting into the thylakoid membrane unless additional stroma is added (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar, 20Payan L.A. Cline K. J. Cell Biol. 1991; 112: 603-613Crossref PubMed Scopus (82) Google Scholar). The requirement for additional stroma has fueled the speculation that two stromal factors are involved in LHCP integration: one factor, cpSRP, binds LHCP to form the intermediate, and the second facilitates membrane insertion (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar, 20Payan L.A. Cline K. J. Cell Biol. 1991; 112: 603-613Crossref PubMed Scopus (82) Google Scholar). This idea is directly supported by the observation that LHCP integration does not occur when the stroma is immunodepleted of cpSRP, but does occur when the immunodepleted stroma is supplemented with cpSRP (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar).Whereas LHCP integration requires GTP hydrolysis (21Hoffman N.E. Franklin A.E. Plant Physiol. (Bethesda). 1994; 105: 295-304Crossref PubMed Scopus (69) Google Scholar), the formation of the transit complex is not GTP-dependent (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar, 20Payan L.A. Cline K. J. Cell Biol. 1991; 112: 603-613Crossref PubMed Scopus (82) Google Scholar). Therefore, it is likely that the second chloroplast protein participates in the regulation of the GTPase activity of cpSRP54; and hence, a likely candidate would be a homologue of the SRP receptor. An essential Escherichia coli protein, FtsY (22Gill D.R. Salmond G.P.C. Mol. Gen. Genet. 1987; 210: 504-508Crossref PubMed Scopus (35) Google Scholar), is homologous to the soluble α-subunit of the SRP receptor (23Bernstein H. Poritz M.A. Strub K. Hoben P. Brenner S. Walter P. Nature. 1989; 340: 482-486Crossref PubMed Scopus (376) Google Scholar). Recently, a putative ftsY gene was detected on chromosome II of Arabidopsis (Bacterial Artificial Clone number F4I18.25 and GenBankTM accession number ATAC004665). In the present work, we demonstrate that the FtsY homologue is a chloroplast protein and, together with cpSRP and GTP, is required for reconstituting the soluble phase of LHCP transport. Furthermore, using these functionally active proteins, we have measured the subunit stoichiometry of cpSRP.EXPERIMENTAL PROCEDURESDNA constructsGST43 Translation Vector (pSPUTKGSTchaos)The chaos cDNA encoding cpSRP43 was subcloned into the E. coli expression vector pGTK+ (generously provided by John Walker) by PCR amplification of the plasmid pBSSK+sschaos with the primers GGAATTCGCCGCCGTACAAAGAAAC, which introduces an Eco RI site just 5′ to the processing site, and GTAATACGACTCACTATAGGGC (T7 primer), which results in the introduction of a 3′-Xho I site from the polylinker. The resulting PCR product was digested with Eco RI and Xho I and subcloned into the same sites of pGTK+ to form plasmid pGTK+chaos. This plasmid encodes a GST43 fusion protein. To make the translation construct of GST43, the insert from pGTK+chaos was subcloned in two steps. First, pGTK+chaos was PCR-amplified with AGTATCCATGGCCCCTATACTAGG, which introduces an Nco I site at the initiation codon of GST, and CGGGGTACCTCATTCATTCATTGGTTGTTG, a reverse primer that hybridizes to the 3′-end of the chaos cDNA. The PCR product was digested with Nco I and Bam HI, and the 670-base pair fragment encoding GST was subcloned into the Nco I and Bam HI sites of pSPUTK to form pTU3. The remaining portion of the insert from pGTK+chaos was subcloned as a Bam HI-Cla I fragment into similarly digested pTU3 to form pSPUTKGSTchaos.GST54his Expression Vector (pGTK+ 54his)pNH4 (24Pilgrim M.L. van Wijk K.-J. Parry D.H. Sy D.A.C. Hoffman N.E. Plant J. 1998; 13: 177-186Crossref PubMed Scopus (53) Google Scholar) was digested with Hin dIII and partially digested with Eco RI. The 1500-base pair fragment was subcloned into the same sites in pGTK+ to form pGTK+54his.GST54his Translation Vector (pSPUTK54+his)pGTK+54his was digested with Bam HI and Hin dIII and cloned into the 3.6-kilobase vector fragment from pSPUTKGSTchaos digested with Bam HI and partially digested with Hin dIII to form pSPUTK+54his.ftsY RNA was extracted (RNeasy kit, QIAGEN Inc.) from Arabidopsis leaf tissue and used to amplify the ftsY cDNA by reverse transcription-PCR using a kit from Life Technologies, Inc. To clone the FtsY precursor into a translation vector, the forward and reverse primers CTCTAGCACAACTGCCATGGCAACTTCT and GGTTCTAAAGCTTAAGAGAATATAGCATTCAC, respectively, were used to introduce Nco I and Hin dIII sites at the initiation methionine and stop codons of the open reading frame, and the resulting PCR product was cloned into the same sites of pSS6.5NcoI (14Franklin A.E. Hoffman N.E. J. Biol. Chem. 1993; 268: 22175-22180Abstract Full Text PDF PubMed Google Scholar) to form pTU1. For overexpressing FtsY in E. coli, the forward primer GGGGATCCGCCGGACCGAGCGGATTCTTC, which introduces a Bam HI site at the predicted processing site, and the above reverse primer were used to PCR amplify cDNA that was cloned into the Bam HI and Hin dIII sites of pQE30 to form pTU2. The GST43 expression vector (pGEX4Tchaos(m)), the cpSRP54 translation vector (pAF1), and the Lhcb1 translation vector (pAB80) have been previously described (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar, 13Schuenemann D. Amin P. Hoffman N.E. Biochem. Biophys. Res. Commun. 1999; 254: 253-258Crossref PubMed Scopus (24) Google Scholar, 18Cline K. Fulsom D.R. Viitanen P.V. J. Biol. Chem. 1989; 264: 14225-14232Abstract Full Text PDF PubMed Google Scholar)Antibodies and Immunoblot AnalysisRecombinant FtsY was expressed from pTU2 in the E. coli strain XL1-Blue. Cells were grown in LB medium containing 100 μg/ml ampicillin and 25 μg/ml kanamycin to an absorbance of 0.6–1.0, and expression was induced by the addition of 0. 1 mm isopropyl-β-d-thiogalactopyranoside for 3 h. Cells were harvested and frozen at −80 °C until use. Overexpressed protein was purified on Ni2+-NTA-agarose (QIAGEN Inc.) as suggested by the manufacturer. Recombinant protein was further purified by SDS-PAGE, eluted from gel slices, and injected into rabbits to raise antibodies (Cocalico Biologicals, Inc., Reamstown, PA). The IgG fraction was prepared from crude serum and affinity-purified on antigen cross-linked to Affi-Gel 10 (25Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Immunoblot analysis was done as described (24Pilgrim M.L. van Wijk K.-J. Parry D.H. Sy D.A.C. Hoffman N.E. Plant J. 1998; 13: 177-186Crossref PubMed Scopus (53) Google Scholar). Antibodies against LHCP (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar), cpSRP43 (15Klimyuk V.I. Persello-Cartieaux F. Havaux M. Contard P. Schuenemann D. Meiherhoff K. Gouet P. Jones J.D.G. Hoffman N.E. Nussaume L. Plant Cell. 1999; 11: 87-99Crossref PubMed Scopus (119) Google Scholar), and cpSRP54 (17Amin P. Sy D. Pilgrim M. Parry D. Nussaume L. Hoffman N.E. Plant Physiol. 1999; (Bethesda), in pressPubMed Google Scholar) were previously described.Cross-linking20 ng of recombinant cpSRP43 or GST-cpSRP43 were incubated with 1 mm disuccinimidyl tartarate in 20 mmHEPES-KOH, pH 8.0, and 150 mm NaCl for 2 h on ice in a final volume of 20 μl. The cross-linking reaction was quenched by the addition of 1.5 μl of 1 m Tris-HCl, pH 8.0, and incubation for 15 min at room temperature. Samples were separated on 8% SDS-polyacrylamide gels and detected by immunoblotting as described (24Pilgrim M.L. van Wijk K.-J. Parry D.H. Sy D.A.C. Hoffman N.E. Plant J. 1998; 13: 177-186Crossref PubMed Scopus (53) Google Scholar).Subunit StoichiometrycpSRP was assembled by incubating 3.4 μCi of cpSRP54his and 2.7 μCi of GST43 translation products with incubation buffer (20 mm HEPES-KOH, pH 8.0, 50 mm KOAc, and 10 mm MgCl2) for 15 min at 25 °C. The reaction was mixed end-over-end with glutathione-Sepharose beads (Amersham Pharmacia Biotech) for 1 h at 4 °C. The beads were washed three times with 1.5 ml of washing buffer (20 mm HEPES-KOH, pH 8.0, 0.3 m KCl, 10 mm MgCl2 and 1% Tween 20). The beads were transferred to Wizard minicolumns (Promega), and the protein was eluted in 50 μl of 10 mm glutathione in incubation buffer. The eluted sample was diluted to 120 μl with incubation buffer and incubated with Ni2+-NTA-agarose beads for 1 h at 4 °C. The beads were transferred to a second Wizard column, washed three times with 1.5 ml of washing buffer, and eluted in 45 μl of 200 mm imidazole in 20 mm HEPES-KOH, pH 8.0. The sample was analyzed by SDS-PAGE on 13% acrylamide gels, and radioactivity was quantitated by radioimaging on a PhosphorImager (Molecular Dynamics, Inc.). Normalized pixel values were calculated by dividing the total pixels in each band by the number of methionines within the protein, and the ratio of the normalized values was used to calculate the molar ratio of the two subunits. To investigate cpSRP43-mediated dimerization of cpSRP54, 0.5 μg of GST54 was incubated with 3.4 μCi of cpSRP54his, 0.5 μg of cpSRP43, or both under standard conditions; purified on glutathione-Sepharose; and analyzed by SDS-PAGE as described above.Isolation of Stroma, Salt Washing of Thylakoids, and Gel FiltrationThe stroma was collected from chloroplasts lysed in 20 mm HEPES-KOH, pH 8.0, 5 mm MgCl2, 1 mm dithiothreitol, and 1 mmphenylmethylsulfonyl fluoride (lysis buffer) at 2 mg of chlorophyll/ml and centrifuged for 10 min in a microcentrifuge. The thylakoid pellet was resuspended in lysis buffer, centrifuged, and resuspended in lysis buffer containing the indicated salt solution. Samples were rotated end-over-end for 10 min at 4 °C and centrifuged, and the pellet was washed a second time as described before and resuspended in lysis buffer at a concentration of 0.5 mg of chlorophyll/ml.Arabidopsis stroma, pea stroma, or purified recombinant protein was fractionated on a Superose 6HR gel filtration column (Amersham Pharmacia Biotech) in 20 mm HEPES-KOH, pH 7.0, 5 mm MgCl2, and 180 mm NaCl at 0.5 ml/min and analyzed as described (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar).ReconstitutionPea thylakoids were washed one time in 20 mmHEPES-KOH, pH 8.0, and 2 m KOAc and two times in 50 mm HEPES-KOH, pH 8.0, and 330 mm sorbitol. Thylakoids containing 25 μg of chlorophyll were resuspended in a total of 100 μl of the indicated reaction mixture. Each reaction mixture contained 10 mm MgCl2, 10 mm methionine, 0.15 mm GTP, 82 mmsorbitol, and 13 mm HEPES-KOH, pH 8.0. cpSRP54, cpFtsY, and LHCP were translated either in a wheat germ translation extract or a commercial rabbit reticulocyte lysate. The amounts added for wheat germ and rabbit reticulocyte lysate translations were as follows: cpSRP54, 20 and 20 μl, respectively; cpFtsY, 40 and 30 μl, respectively; and LHCP, 5 and 40 μl, respectively. Recombinant cpSRP43 (150 ng), pea stroma (equivalent to 90 μg of chlorophyll), and apyrase (1 unit) were added as noted. Reactions were incubated for 30 min at 25 °C, followed by trypsin treatment and analysis by SDS-PAGE and fluorography.GeneralPreviously described conditions were used for import of FtsY into chloroplasts (26Adam Z. Hoffman N.E. Plant Physiol. (Bethesda). 1993; 102: 35-43Crossref PubMed Scopus (34) Google Scholar), in vitro transcription/translation (26Adam Z. Hoffman N.E. Plant Physiol. (Bethesda). 1993; 102: 35-43Crossref PubMed Scopus (34) Google Scholar), and expression and purification of GST fusion proteins (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar). Mature cpSRP43 was generated by thrombin cleavage of GST43 using biotinylated thrombin and purified from GST and thrombin using streptavidin-agarose (Novagen) as suggested by the manufacturer.DISCUSSIONThis work establishes four new and important points. First, we show that cpSRP43 is a dimer. Second, we demonstrate that cpSRP is a trimer consisting of two cpSRP43 subunits and one cpSRP54 subunit (Fig.6). Third, we show that cpFtsY is a soluble chloroplast protein that has a weak affinity for cpSRP. Fourth, we conclusively demonstrate that cpFtsY is the second soluble factor that is required to reconstitute the soluble phase of LHCP transport.cpSRP is distinctive in its ability to interact with members of the LHCP protein family post-translationally. It is likely that this specialized role is mediated directly or indirectly through cpSRP43. From an analysis of Arabidopsis mutants that lack cpSRP43, it appears that only members of the LHCP protein family are adversely affected (15Klimyuk V.I. Persello-Cartieaux F. Havaux M. Contard P. Schuenemann D. Meiherhoff K. Gouet P. Jones J.D.G. Hoffman N.E. Nussaume L. Plant Cell. 1999; 11: 87-99Crossref PubMed Scopus (119) Google Scholar, 17Amin P. Sy D. Pilgrim M. Parry D. Nussaume L. Hoffman N.E. Plant Physiol. 1999; (Bethesda), in pressPubMed Google Scholar). Thus, it appears that cpSRP43 functions primarily, if not exclusively, in LHCP biogenesis. Mutant plants lacking cpSRP54 show wider effects; chloroplast encoded proteins whose targeting is cotranslational are affected in addition to LHCP (17Amin P. Sy D. Pilgrim M. Parry D. Nussaume L. Hoffman N.E. Plant Physiol. 1999; (Bethesda), in pressPubMed Google Scholar, 24Pilgrim M.L. van Wijk K.-J. Parry D.H. Sy D.A.C. Hoffman N.E. Plant J. 1998; 13: 177-186Crossref PubMed Scopus (53) Google Scholar). A substantial pool of cpSRP54 is dissociated from cpSRP43 and associated with 70 S ribosomes (14Franklin A.E. Hoffman N.E. J. Biol. Chem. 1993; 268: 22175-22180Abstract Full Text PDF PubMed Google Scholar). Furthermore, cpSRP54 has been shown to directly interact with the nascent chain of a chloroplast protein synthesized in a chloroplast translation extract (30Nilsson R. Brunner J. Hoffman N.E. van Wijk K.-J. EMBO J. 1999; 18: 733-742Crossref PubMed Scopus (100) Google Scholar). Therefore, we think it likely that cpSRP54 free of cpSRP43 mediates cotranslational targeting, whereas the presence of cpSRP43 enables the post-translational interaction between cpSRP and LHCP. Cross-linking data clearly demonstrate that cpSRP54 directly interacts with LHCP (16Li X.X. Henry R. Yuan J.G. Cline K. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3789-3793Crossref PubMed Scopus (159) Google Scholar). It remains to be shown whether cpSRP43 is also able to interact directly with LHCP or whether it simply modifies the conformation of cpSRP54 to facilitate the post-translational interaction. Previously, we entertained the possibility that cpSRP43 effectively dimerized cpSRP54 and thereby created a novel interaction between the SRP54 homologue and its substrate (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar). The results from the present study clearly indicate that cpSRP, like cytoplasmic SRP, contains a single SRP54 subunit that presumably binds a single substrate molecule. Thus, the predicted molecular mass of cpSRP is 123 kDa. The large deviation of the mass estimate by gel filtration from the predicted value suggests that cpSRP is not a globular protein.Previous work provided strong evidence that LHCP integration required multiple soluble factors (12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (161) Google Scholar, 20Payan L.A. Cline K. J. Cell Biol. 1991; 112: 603-613Crossref PubMed Scopus (82) Google Scholar), a hypothesis that has now been validated. The present data also provide strong evidence that cpSRP, cpFtsY, and GTP are sufficient for reconstituting the soluble phase of LHCP transport. Purifying an active form of cpSRP54 is an obstacle that must be overcome to prove this point conclusively. For reconstitution experiments, recombinant and highly purified cpSRP43 was used, whereas cpSRP54, cpFtsY, and LHCP were synthesized by translation in either wheat germ extracts or rabbit reticulocyte lysates (data not shown). The fact that LHCP integration can be reconstituted using translation products synthesized in rabbit reticulocyte lysates indicates that no other chloroplast factors are required for the reaction.In E. coli, SRP-dependent proteins are inserted into the membrane via the Sec translocon (31Valent Q.A. Scotti P.A. High S. deGier J.W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar), which minimally consists of SecA/E/Y (32Schatz P.J. Beckwith J. Annu. Rev. Genet. 1990; 24: 215-248Crossref PubMed Scopus (270) Google Scholar, 33Akimaru J. Matsuyama S.I. Tokuda H. Mizushima S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6545-6549Crossref PubMed Scopus (170) Google Scholar). Homologues of all three proteins are found in the chloroplast and are required for the translocation of the luminal33-kDa oxygen-evolving protein OE33 (34Yuan J.G. Henry R. Mccaffery M. Cline K. Science. 1994; 266: 796-798Crossref PubMed Scopus (143) Google Scholar, 35Nakai M. Goto A. Nohara T. Sugita D. Endo T. J. Biol. Chem. 1994; 269: 31338-31341Abstract Full Text PDF PubMed Google Scholar, 36Laidler V. Chaddock A.M. Knott T.G. Walker D. Robinson C. J. Biol. Chem. 1995; 270: 17664-17667Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 37Schuenemann D. Amin P. Hartmann E. Hoffman N.E. J. Biol. Chem. 1999; 274: 12177-12182Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). One unresolved question is whether the Sec translocon is required for LHCP integration. The results presented here are inconsistent with the involvement of cpSecA in LHCP integration. cpSecA is a soluble protein that needs to be added as stroma or purified protein to reconstitute efficient translocation of OE33 across the thylakoid membranes (34Yuan J.G. Henry R. Mccaffery M. Cline K. Science. 1994; 266: 796-798Crossref PubMed Scopus (143) Google Scholar, 38Hulford A. Hazell L. Mould R.M. Robinson C. J. Biol. Chem. 1994; 269: 3251-3256Abstract Full Text PDF PubMed Google Scholar, 39Yuan J.G. Cline K. J. Biol. Chem. 1994; 269: 18463-18467Abstract Full Text PDF PubMed Google Scholar). In the present work, efficient integration of LHCP occurred without specifically adding cpSecA. Together with the observations that azide (an inhibitor of cpSecA) does not inhibit LHCP integration (34Yuan J.G. Henry R. Mccaffery M. Cline K. Science. 1994; 266: 796-798Crossref PubMed Scopus (143) Google Scholar), that OE33 is not a competitor of LHCP integration (40Cline K. Henry R. Li C.J. Yuang J.G. EMBO J. 1993; 12: 4105-4114Crossref PubMed Scopus (172) Google Scholar), and that maize SecA mutants have normal levels of LHCP (41Voelker R. Barkan A. EMBO J. 1995; 14: 3905-3914Crossref PubMed Scopus (130) Google Scholar), the data imply either that the cpSRP and cpSec pathways do not converge at the Sec translocon or, alternatively, that cpSecY/E is active in the absence of cpSecA. Either case represents a fundamental departure from translocation events in E. coli.We have now shown that cpFtsY is required for the activity of the specialized cpSRP. It remains to be determined whether cpFtsY also functions with cpSRP54 in the biogenesis of chloroplast encoded proteins. In either case, FtsY may regulate the GTPase activity of cpSRP54 and play a role in piloting SRP-dependent substrates to the thylakoid membrane. The fact that LHCP transport can now be reconstituted from defined components will allow detailed mechanistic studies to be conducted on this pathway. SRP1 mediates the cotranslational targeting of endomembrane and secretory proteins to the endoplasmic reticulum in eukaryotes and of polytopic membrane proteins to the cytoplasmic membrane in prokaryotes (1Rapoport T.A. Jungnickel B. Kutay U. Annu. Rev. Biochem. 1996; 65: 271-303Crossref PubMed Scopus (492) Google Scholar, 2Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (713) Google Scholar, 3Ulbrandt N.D. Newitt J.A. Bernstein H.D. Cell. 1997; 88: 187-196Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Cytosolic forms of SRP are ubiquitous in eukaryotic and prokaryotic organisms. All contain, at a minimum, a 54-kDa GTPase subun
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