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• W2010048393 abstract "The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) function in thylakoid biogenesis to target integral membrane proteins to thylakoids. Unlike cytosolic SRP receptors in eukaryotes, cpFtsY partitions between thylakoid membranes and the soluble stroma. Based on sequence alignments, a membrane-binding motif identified in Escherichia coli FtsY appears to be conserved in cpFtsY, yet whether the proposed motif is responsible for the membrane-binding function of cpFtsY has yet to be shown experimentally. Our studies show that a small N-terminal region in cpFtsY stabilizes a membrane interaction critical to cpFtsY function in cpSRP-dependent protein targeting. This membrane-binding motif is both necessary and sufficient to direct cpFtsY and fused passenger proteins to thylakoids. Our results demonstrate that the cpFtsY membrane-binding motif may be functionally replaced by the corresponding region from E. coli, confirming that the membrane-binding motif is conserved among organellar and prokaryotic homologs. Furthermore, the capacity of cpFtsY for lipid binding correlates with liposome-induced GTP hydrolysis stimulation. Mutations that debilitate the membrane-binding motif in cpFtsY result in higher rates of GTP hydrolysis, suggesting that negative regulation is provided by the intact membrane-binding region in the absence of a bilayer. Furthermore, NMR and CD structural studies of the N-terminal region and the analogous region in the E. coli SRP receptor revealed a conformational change in secondary structure that takes place upon lipid binding. These studies suggest that the cpFtsY membrane-binding motif plays a critical role in the intramolecular communication that regulates cpSRP receptor functions at the membrane. The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) function in thylakoid biogenesis to target integral membrane proteins to thylakoids. Unlike cytosolic SRP receptors in eukaryotes, cpFtsY partitions between thylakoid membranes and the soluble stroma. Based on sequence alignments, a membrane-binding motif identified in Escherichia coli FtsY appears to be conserved in cpFtsY, yet whether the proposed motif is responsible for the membrane-binding function of cpFtsY has yet to be shown experimentally. Our studies show that a small N-terminal region in cpFtsY stabilizes a membrane interaction critical to cpFtsY function in cpSRP-dependent protein targeting. This membrane-binding motif is both necessary and sufficient to direct cpFtsY and fused passenger proteins to thylakoids. Our results demonstrate that the cpFtsY membrane-binding motif may be functionally replaced by the corresponding region from E. coli, confirming that the membrane-binding motif is conserved among organellar and prokaryotic homologs. Furthermore, the capacity of cpFtsY for lipid binding correlates with liposome-induced GTP hydrolysis stimulation. Mutations that debilitate the membrane-binding motif in cpFtsY result in higher rates of GTP hydrolysis, suggesting that negative regulation is provided by the intact membrane-binding region in the absence of a bilayer. Furthermore, NMR and CD structural studies of the N-terminal region and the analogous region in the E. coli SRP receptor revealed a conformational change in secondary structure that takes place upon lipid binding. These studies suggest that the cpFtsY membrane-binding motif plays a critical role in the intramolecular communication that regulates cpSRP receptor functions at the membrane. Proper compartmentalization of proteins relies on the ability of protein localization pathways to transport proteins efficiently from their sites of synthesis to their sites of function. The signal recognition particle (SRP) 2The abbreviations used are: SRP, signal recognition particle; cpSRP, chloroplast SRP; SR, SRP receptor; cpFtsY, chloroplast FtsY; LHCP, light-harvesting chlorophyll-binding protein; EcFtsY, E. coli FtsY; Tha4TM, Tha4 transmembrane domain; cp43, cpSRP43; RubSS, ribulose-bisphosphate carboxylase/oxygenase small subunit; ITC, isothermal titration calorimetry; GMP-PNP, guanosine 5′-(β,γ-iminotriphosphate); TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy.2The abbreviations used are: SRP, signal recognition particle; cpSRP, chloroplast SRP; SR, SRP receptor; cpFtsY, chloroplast FtsY; LHCP, light-harvesting chlorophyll-binding protein; EcFtsY, E. coli FtsY; Tha4TM, Tha4 transmembrane domain; cp43, cpSRP43; RubSS, ribulose-bisphosphate carboxylase/oxygenase small subunit; ITC, isothermal titration calorimetry; GMP-PNP, guanosine 5′-(β,γ-iminotriphosphate); TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy. and its receptor function in every kingdom of life to target proteins to the endoplasmic reticulum (eukaryotes), cytoplasmic membrane (prokaryotes), and thylakoid membrane (chloroplasts) (1Pool M. Mol. Membr. Biol. 2005; 22: 3-15Crossref PubMed Scopus (87) Google Scholar). The targeting function of SRP relies on a conserved 54-kDa SRP subunit (SRP54; Ffh in Escherichia coli and cpSRP54 in chloroplasts) as well as a conserved SRP receptor (SRα; FtsY in E. coli and cpFtsY in chloroplasts). For cytosolic SRPs (SRP54 and Ffh), interactions with a substrate signal sequence and an SRP RNA moiety are prerequisite for interaction with the SRP receptor (SRα and FtsY) (2Bradshaw N. Neher S.B. Booth D.S. Walter P. Science. 2009; 323: 127-130Crossref PubMed Scopus (68) Google Scholar). GTP binding and hydrolysis by both SRP54 and SRα coordinate substrate release from SRP to the translocon and release of SRP from SRα. In chloroplasts, cpFtsY functions along with a unique SRP (cpSRP) to post-translationally target nuclear encoded proteins to thylakoid membranes (3Henry R. Goforth R.L. Schunemann D. Tamanoi F. Dalbey R. Koehler C. The Enzymes: Molecular Machines Involved in Protein Transport across Cellular Membranes 25. Elsevier, St. Louis, MO2007: 493-521Google Scholar). Light-harvesting chlorophyll a/b-binding proteins (LHCPs) imported into the chloroplast stroma are bound by cpSRP to form a soluble targeting complex, which directs the LHCP substrate to the thylakoid membrane translocon Alb3 (Albino3) in a GTP- and cpFtsY-dependent manner (14Moore M. Goforth R.L. Mori H. Henry R. J. Cell Biol. 2003; 162: 1245-1254Crossref PubMed Scopus (77) Google Scholar, 36Asakura Y. Hirohashi T. Kikuchi S. Belcher S. Osborne E. Yano S. Terashima I. Barkan A. Nakai M. Plant Cell. 2004; 16: 201-214Crossref PubMed Scopus (59) Google Scholar). Although many general steps of SRP protein targeting seem largely conserved across evolutionary boundaries, the nature and dynamics of the receptor appear to have diverged.In eukaryotic systems, SRα is peripherally bound to the membrane through association with the integral membrane subunit SRβ. In contrast, no chloroplast or bacterial homolog of SRβ has been identified. cpFtsY and E. coli FtsY (EcFtsY) are found partitioned between the membrane and the stroma or cytosol, respectively. The membrane-binding capacity of EcFtsY serves to stimulate GTPase activity and appears critical in that only membrane-associated EcFtsY supports the release of nascent chains from SRP to the translocon (4de Leeuw E. te Kaat K. Moser C. Menestrina G. Demel R. de Kruijff B. Oudega B. Luirink J. Sinning I. EMBO J. 2000; 19: 531-541Crossref PubMed Scopus (122) Google Scholar, 5Valent Q.A. Scotti P.A. High S. De Gier 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). However, the partitioning activity is not strictly required because EcFtsY tethered to the membrane is functional in vivo (37Zelazny A. Seluanov A. Cooper A. Bibi E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6025-6029Crossref PubMed Scopus (69) Google Scholar). Given the conserved nature of partitioning among bacterial and chloroplast SRP receptors, partitioning may play an, as of yet, unidentified role in protein targeting by SRP. Nevertheless, differences in lipid composition between bacterial and thylakoid membranes make it interesting to speculate that there are mechanistic differences in membrane partitioning.Like many prokaryotic FtsY homologs (e.g. Thermus aquaticus), cpFtsY lacks the N-terminal acidic domain (A domain) implicated in EcFtsY membrane binding (6Samuelsson T. Zwieb C. Nucleic Acids Res. 1999; 27: 169-170Crossref PubMed Scopus (13) Google Scholar). Although the highly conserved FtsY GTPase domain (NG domain) of EcFtsY (EcFtsYNG) fails to support protein targeting, the addition of the last A domain residue, Phe-196 of a conserved double-Phe motif (EcFtsYNG+1), restores protein targeting in vivo (7Eitan A. Bibi E. J. Bacteriol. 2004; 186: 2492-2494Crossref PubMed Scopus (48) Google Scholar). In vitro studies also show that EcFtsYNG+1 retains the capacity to bind membranes and support integration of SRP-dependent substrates, although at significantly reduced levels compared with full-length EcFtsY (8Angelini S. Boy D. Schiltz E. Koch H.-G. J. Cell Biol. 2006; 174: 715-724Crossref PubMed Scopus (55) Google Scholar). A resolved structure of EcFtsYNG+1 suggests that the amphipathic nature of the region containing Phe-196 plays a critical role in membrane association (9Parlitz R. Eitan A. Stjepanovic G. Bahari L. Bange G. Bibi E. Sinning I. J. Biol. Chem. 2007; 282: 32176-32184Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Furthermore, it has been demonstrated that liposomes stimulate GTP hydrolysis rates of SRP with EcFtsYNG+1, but not with EcFtsYNG, supporting the idea that the A domain in its entirety is not strictly required.For cpFtsY, the necessity and functional role(s) of partitioning between a thylakoid-bound and a soluble phase, as well as the role of N-terminal residues in these functions, remain unknown. In addition, both the conformational state of membrane-bound cpFtsY and EcFtsY and the mechanism responsible for controlling membrane partitioning and altered GTPase activity remain unclear. Because of the gain of function exhibited by EcFtsYNG+1 and the conserved nature of the surrounding motif (9Parlitz R. Eitan A. Stjepanovic G. Bahari L. Bange G. Bibi E. Sinning I. J. Biol. Chem. 2007; 282: 32176-32184Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), it seems likely that this conserved region is necessary to support membrane binding and corresponding functions not only in EcFtsY but also in FtsY homologs.To examine the functional role of the N-terminal region of cpFtsY, we have utilized deletion and point mutants in assays that reconstitute cpFtsY activities, including the cpSRP-dependent integration of LHCP. Together, our data indicate that the conserved lipid-binding motif identified in bacterial FtsY homologs is present in cpFtsY and is both necessary and sufficient for thylakoid binding and critical for LHCP targeting.EXPERIMENTAL PROCEDURESAll reagents and enzymes used were purchased commercially. All primers were from Integrated DNA Technologies. The plasmid used for in vitro transcription/translation of pLHCP (psAB80XD/4) has been described (10Cline K. Fulsom D.R. Viitanen P.V. J. Biol. Chem. 1989; 264: 14225-14232Abstract Full Text PDF PubMed Google Scholar). cpSRP43, cpFtsY, and cpSRP54 were prepared as described (38Yuan J. Kight A. Goforth R.L. Moore M. Peterson E.C. Sakon J. Henry R. J. Biol. Chem. 2002; 277: 32400-32404Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 39Goforth R.L. Peterson E.C. Yuan J. Moore M.J. Kight A.D. Lohse M.B. Sakon J. Henry R.L. J. Biol. Chem. 2004; 279: 43077-43084Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 40Jaru-Ampornpan P. Chandrasekar S. Shan S.-o. Mol. Biol. Cell. 2007; 18: 2636-2645Crossref PubMed Scopus (37) Google Scholar).Construction of cpFtsY Clones for Transcription and Translation—cpFtsY clones for transcription/translation were designed to match the mature coding sequence of Arabidopsis thaliana cpFtsY starting with the predicted mature amino acid sequence CSAGPSGF. PCR amplification was used to create deletion and substitution mutants with incorporation of a Kozak sequence (Met-Ala) and restriction sites for insertion into pGEM-4Z. Expression clones were created by subcloning into pET-32b. The cpSRP43 transcription/translation clone was designed to match the mature coding sequence of A. thaliana cpSRP43 starting with Ala-Ala-Val-Gln-Arg-Asn and incorporating a Kozak sequence and restriction sites for insertion into pGEM-7Z. Chimeric sequence transcription/translation clones for Tha4TM-cpFtsY, Tha4TM-F48A, and Tha4TM-F48A/F49A are exact fusions constructed by overlap extension (11Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2628) Google Scholar) of Pisum sativum Tha4 beginning with Ala-Phe-Phe-Gly-Leu-Gly and ending with Val-Phe-Gly-Pro-Lys-Lys and A. thaliana cpFtsY beginning with Met-Ala-Cys-Ser-Ala-Gly-Pro-Ser. Likewise, cpFtsY39–56-cp43, EcFtsY186–204-cp43, and cpFtsY39–56-RubSS constructs are exact fusions of mature A. thaliana cpFtsY construct residues 39–56 (Met-Ala-Cys-Ser-Ala-Gly-Pro-Ser-Gly-Phe-Phe-Thr-Arg-Leu-Gly-Arg-Leu-Ile) or E. coli FtsY residues 186–204 (Glu-Gln-Glu-Lys-Pro-Thr-Lys-Glu-Gly-Phe-Phe-Ala-Arg-Leu-Lys-Arg-Ser-Leu-Leu), a linker (Val-Phe-Gly-Pro-Lys-Lys), and either the predicted mature sequence of A. thaliana cpSRP43 or the P. sativum RubSS beginning Gln-Val-Trp-Pro-Pro-Ile-Gly-Lys. The RubSS transcription/translation clone includes the linker described above and the predicted mature sequence of the P. sativum RubSS. Kozak sequences and restriction sites for insertion into pGEM-4Z were included in the primer for all above-mentioned transcription/translation clones. All cloned sequences were verified by DNA sequencing (Molecular Resource Laboratory, University of Arkansas for Medical Sciences).Preparation of Chloroplasts and Radiolabeled Precursors—Intact chloroplasts were isolated from 10–12-day-old pea seedlings (P. sativum cv. Laxton's Progress) and used to prepare thylakoids and stroma as described (12Cline K. Henry R. Li C. Yuan J. EMBO J. 1993; 12: 4105-4114Crossref PubMed Scopus (172) Google Scholar). Chlorophyll content was determined as described previously (13Arnon D.I. Plant Physiol. 1949; 24: 1-15Crossref PubMed Google Scholar). Thylakoids were isolated from lysed chloroplasts by centrifugation and salt-washed two times with 1 m potassium acetate in import buffer (50 mm HEPES-KOH (pH 8.0) and 0.33 m sorbitol) and two times with import buffer with 10 mm MgCl2 (IBM buffer) prior to use. For protease treatment, salt-washed thylakoids were diluted to 0.5 mg/ml chlorophyll in import buffer with 0.2 mg/ml thermolysin and 1 mm CaCl2, incubated for 40–60 min, combined with EDTA in import buffer to 20 mm EDTA, and applied to a 7.5% (v/v) Percoll gradient in import buffer containing 10 mm EDTA. Pellets were washed once with import buffer containing 10 mm EDTA and twice with IBM buffer. Protease-treated thylakoids were resuspended at 1 mg/ml chlorophyll in IBM buffer.In vitro-transcribed capped RNA was translated in the presence of [35S]methionine using a wheat germ system to produce radiolabeled proteins (12Cline K. Henry R. Li C. Yuan J. EMBO J. 1993; 12: 4105-4114Crossref PubMed Scopus (172) Google Scholar). Constructs were labeled with ratios of labeled and unlabeled Met such that equal 35S signal represented equimolar protein. For example, to prepare equimolar amounts of two proteins with six and seven Met residues, respectively, the first construct would be prepared with 100% [35S]Met, whereas the second construct would be prepared with 86% [35S]Met and 14% unlabeled Met. Constructs were quantified by comparing the 35S signal from a given protein band as analyzed by SDS-PAGE and phosphorimaging. Equimolar amounts of proteins were added to each experiment (supplemental Fig. S2). Precursor LHCP translation products were diluted 2-fold with 30 mm unlabeled Met in import buffer prior to use.Protein Integration Assays Using Isolated Salt-washed Thylakoids—Integration assays included salt-washed thylakoids (equal to 50 μg of chlorophyll) in IBM buffer, 5 mm ATP, 1 mm GTP, 12.5 μl of radiolabeled pLHCP translation products, and stromal extract (equivalent to 50 μg of chlorophyll) or 25 μl of radiolabeled cpFtsY translation products and recombinant cpSRP43 and cpSRP54. Stromal extract, containing cpSRP and cpFtsY, was used as a positive control. Import buffer was used to bring the final volume to 150 μl. The mixtures were incubated at 25 °C for 30 min with light. Membranes were collected by centrifugation at 3200 × g for 6 min at 4 °C and protease-treated with thermolysin. Protease-treated membranes were solubilized in SDS buffer and heated. Amounts equivalent to 10 μg of chlorophyll/assay were analyzed by SDS-PAGE and phosphorimaging.Assays for Determining Membrane Binding and Partitioning—Partitioning assays included thylakoids (equal to 75 μg of chlorophyll) in IBM buffer and radiolabeled translation products. Reactions were incubated for 30 min in light at 25 °C. Thylakoids were centrifuged at 3200 × g for 6 min at 4 °C, washed in 1 ml of IBM buffer, and transferred to clean tubes. Thylakoids were then pelleted, solubilized in SDS buffer, and heated. Amounts equivalent to 7.5 μg of chlorophyll/sample were analyzed by SDS-PAGE and phosphorimaging.cpFtsY Membrane Binding Saturation Curves—Salt-washed or protease-treated thylakoids (equal to 50 μg of chlorophyll) were incubated with 0, 1, 2, 4, 8, 16, 32, or 64 μg of cpFtsY in a final volume of 100 μl of IBM buffer. Thylakoids were re-isolated, washed, and resuspended to a final volume of 50 μl, and 5 μl of each sample was analyzed by SDS-PAGE. Separated samples were transferred to BioTrace™ polyvinylidene difluoride membrane (Pall Life Sciences) and incubated with rabbit anti-A. thaliana cpFtsY polyclonal antibodies (14Moore M. Goforth R.L. Mori H. Henry R. J. Cell Biol. 2003; 162: 1245-1254Crossref PubMed Scopus (77) Google Scholar). Horseradish peroxidase-labeled mouse IgG (SouthernBiotech) was used as a secondary antibody. Proteins reacting with antibodies were revealed by incubation with SuperSignal® West Pico chemiluminescent substrate (Pierce).Imaging Acquisition—SDS-polyacrylamide gels were imaged using a Typhoon 8600 (GE Healthcare) and analyzed with IQ Solutions software (Molecular Dynamics). Western blots were imaged using a FluorChem™ 8900 (Alpha Innotech) and analyzed with the corresponding AlphaEase® FC StandAlone software.Isothermal Titration Calorimetry (ITC)—The binding of GMP-PNP/GDP to cpFtsY or F48A was analyzed by measuring heat change during titration of nucleotide into a protein solution using a VP-ITC titration microcalorimeter (MicroCal Inc.). All solutions were degassed under vacuum and equilibrated at 25 °C prior to titration. The sample cell (1.4 ml) contained 0.1 mm protein in 10 mm Tris buffer (pH 7.0) and 50 mm KCl. The reference cell contained MilliQ water. Upon equilibration, 5 mm GMP-PNP/GDP was injected in 20 × 6-μl aliquots using the default injection rate. Titration curves were corrected for protein-free buffer and analyzed using Origin ITC software (MicroCal Inc.).Liposome Preparation and Fluorescence Quenching Experiments—Soybean total extract (Avanti Polar Lipids) lipids were dissolved at 100 mg/ml in chloroform, dried under nitrogen, and vacuum-desiccated overnight. Lipid pellets were resuspended to 10 mg/ml (13 mm) in 10 mm HEPES-KOH (pH 7), 100 mm KCl, and 1 mm EDTA. The lipid solution was subjected to 15-s sonication/15-s rest cycles for 2 min. Liposomes were clarified by centrifugation at 11,700 × g for 10 min at 4 °C and stored at 4 °C for up to 1 month. Liposomes were sized (Avanti mini-extruder) by passing through polycarbonate filters seven times. Brominated lipids were obtained by bromine addition to the unsaturated carbons of the soybean phosphatidylcholine fatty acyl chain as described (15Carney J. East J.M. Mall S. Marius P. Powl A.M. Wright J.N. Lee A.G. Curr. Protocols Protein Sci. 2006; PubMed Google Scholar). The brominated lipid mixture was extruded through 80-nm polycarbonate membranes and homogenized via freeze/thaw cycles.Fluorescence quenching was measured using a SpectraMax Gemini XS spectrofluorometer (Molecular Devices) set for maximum sensitivity and 282-nm excitation/330-nm emission wavelengths. 10 μg of protein in 50 μl of 10 mm HEPES-KOH (pH 8) and 10 mm MgCl2 and 0–50 μl of liposomes were mixed and equilibrated for 20 min at 25 °C, and the fluorescence was measured. For each concentration, six measurements of five separate samples were acquired. Fluorescence quenching was estimated as the normalized value of (F0 – F)/F0, where F0 is the average fluorescence of the samples without liposomes, and F is the average fluorescence for each concentration.GTPase Assays—GTPase activity assays were conducted at 22 °C and contained 100 nm cpFtsY or F48A, 0.5 μm [α-32P]GTP (400 Ci/mmol), and liposomes in a final volume of 5 μl of buffer (50 mm HEPES (pH 8.0), 150 mm potassium acetate, 10 mm potassium chloride, 2 mm magnesium acetate, 0.01% (v/v) C12E8 (octaethylene glycol mono-N-dodecyl ether), and 2 mm dithiothreitol). Aliquots were removed at frequent time points and spotted onto polyethyleneimine-cellulose thin layer plates as described (16Connolly T. Gilmore R. J. Cell Biol. 1993; 123: 799-807Crossref PubMed Scopus (49) Google Scholar).NMR Structural Studies—All NMR spectra were acquired at 25 °C on a Bruker AVANCE DMX-500 MHz spectrometer equipped with a 5-mm triple resonance cryoprobe. NMR samples (∼1 mm concentration) were prepared both in 90% (v/v) H2O + 10% (v/v) D2O (pH 7.0) containing 100 mm NaCl and in Me2SO-d6. Two-dimensional 1H TOCSY and NOESY (17Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., Hoboken, NJ1986Crossref Google Scholar) data were acquired with 2048 data points in the f2 dimension and 512 increments in the f1 dimension over a spectral width corresponding to 12 ppm. Two-dimensional 1H TOCSY data were acquired with mixing times of 60 and 75 ms. NOE-based distance restraints were derived from two-dimensional 1H NOESY data obtained with various mixing times (200, 250, 300, and 350 ms). All NMR spectra were processed using XWIN-NMR and Sparky software (18Goddard T.D. Kneller D.G. Sparky3. University of California, San Francisco1997Google Scholar). The backbone dihedral angle restraints derived from 3JNHαH coupling constants and the χ1 dihedral angles derived from the TOCSY data were used as additional constraints for the structure calculation (19Wang Y. Nip A.M. Wishart D.S. J. Biomol. NMR. 1997; 10: 373-382Crossref PubMed Scopus (41) Google Scholar).Distance restraints were derived from the NOESY spectrum of the peptides. NOE cross-peak intensities were measured and converted into distance. Structure calculation was performed using ARIA-CNS (Version 1.2) (20Linge J.P. O'Donoghue S.I. Nilges M. Methods Enzymol. 2001; 339: 71-90Crossref PubMed Scopus (329) Google Scholar). Several cycles of ARIA were performed using standard protocols by varying the chemical shift tolerance between 0.04 and 0.01 ppm. Assignments and violations were analyzed after each cycle. An ensemble of 12 structures was chosen (from a pool of 50 structures) on the basis of lowest energy terms associated with violation of experimentally derived constraints. The ensemble of the best overlapping structures (with least root mean square deviation) of peptides was viewed using MOLMOL (21Koradi R. Billeter M. Wuthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6468) Google Scholar).Circular Dichroism—CD spectra were recorded on a Jasco J-715 spectropolarimeter using a sandwich quartz cell of 0.1-mm path length. Spectra were acquired at 0.1-nm intervals and at scan speeds of 10 nm/min. All measurements were made after incubation of peptides (50 μm final concentration) in appropriate concentrations of liposomes for 5 min at room temperature. The results are expressed as mean residue ellipticity ([θ]), defined as [θ] = θobs/nlc, where θobs is the observed ellipticity in degrees, n is the number of amino acids in the peptide, c is peptide concentration, and l is the light path (centimeters). Spectra were averaged over 10 scans and corrected for background absorption.Sequence Alignments—Sequence alignments of A. thaliana chloroplast, E. coli, Thermotoga maritima, and T. aquaticus FtsY homologs were performed using ClustalW (22Chenna R. Sugawara H. Koike T. Lopez R. Gibson T.J. Higgins D.G. Thompson J.D. Nucleic Acids Res. 2003; 31: 3497-3500Crossref PubMed Scopus (4013) Google Scholar). Sequences were input in FASTA format, and ClustalW was run using default settings. Alignment files were viewed using Jalview Version 2.0 (23Clamp M. Cuff J. Searle S.M. Barton G.J. Bioinformatics. 2004; 20: 426-427Crossref PubMed Scopus (1208) Google Scholar).Organeller cpFtsY sequences were obtained by searching for short, nearly exact matches using protein-protein BLAST (24Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59110) Google Scholar). Residues 41–366 of A. thaliana cpFtsY were blasted against Eukaryota with a word size of two and otherwise default settings. A non-redundant set of six cpFtsY sequences was obtained and aligned for a consensus sequence using ClustalW as described. A prokaryotic FtsY consensus sequence was obtained by blasting the same cpFtsY sequence against bacteria with 500 descriptions. Sequences were shortened to contain only the NG domain plus 25 N-terminal residues. Resulting sequences were reduced to a non-redundant set of 375 and aligned using ClustalW. The percentage of each residue represented in an alignment column represents the total number of appearances of an amino acid divided by the total number of residues in that column.RESULTSThe N-terminal Region of Mature cpFtsY Is Necessary for LHCP Integration and Thylakoid Membrane Binding—To understand whether the cpFtsY N terminus is functionally important in targeting of LHCP by cpSRP, cpFtsY was replaced with N-terminal deletion mutants (Fig. 1) in assays that reconstitute LHCP integration into isolated thylakoids. Proper integration of LHCP results in a protease-resistant degradation product, as shown in Fig. 2A. Deletion of cpFtsY residues 41–46 had little effect on LHCP integration, whereas further deletions (Δ41–49, Δ41–52, and Δ41–56) decreased integration by ∼90% relative to cpFtsY.FIGURE 2cpFtsY residues 47–49 (GFF) are required for LHCP integration and efficient thylakoid partitioning. A, integration of LHCP was reconstituted with salt-washed thylakoids using stromal extract or recombinant proteins and equimolar amounts of in vitro-translated cpFtsY construct as indicated. See “Experimental Procedures” and supplemental Fig. S1 for more information regarding the production of equimolar amounts of in vitro-translated constructs. Correctly integrated LHCP migrated as a protease-resistant (thermolysin) degradation product (DP). A lane of pLHCP (translation product (TP)) is shown for comparison. LHCP integration was calculated from a minimum of three separate experiments and is shown relative to the level of integration observed for the stroma. B, the membrane binding of radiolabeled cpFtsY constructs as indicated was examined by incubation with salt-washed (SW) thylakoids. Thylakoids were re-isolated, washed, and analyzed by SDS-PAGE and phosphorimaging. The level of each membrane-bound cpFtsY construct was calculated from three separate experiments and is shown relative to bound cpFtsY.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To address membrane-partitioning competency, salt-washed thylakoids were incubated with radiolabeled cpFtsY N-terminal deletion constructs and repurified to remove unbound protein. Deletion of the first six residues (Δ41–43 and Δ41–46) reduced membrane binding to 40–50% of that observed for cpFtsY (Fig. 2B). Further N-terminal deletions (Δ41–49, Δ41–52, and Δ41–56) reduced membrane binding to only 13% of that seen for cpFtsY, correlating with the precipitous drop in LHCP integration observed for the same cpFtsY deletions (Fig. 2, A and B).Phe-48 and Phe-49 Are Required for Efficient Thylakoid Membrane Binding and LHCP Integration—cpFtsYNG+1 and cpFtsYNG+2, consisting of the cpFtsY NG domain (residues 50–366) and Phe-49 (+1) or Phe-48 and Phe-49 (+2), respectively (Fig. 1), were examined for their ability to support LHCP integration and bind thylakoids. Although cpFtsYNG+2 bound membranes with ∼50% lower efficiency than cpFtsY, this construct supported efficient (∼90% relative to cpFtsY) LHCP integration in vitro (Fig. 3, A and B). cpFtsYNG+1 associated with thylakoids with 25% the efficiency of cpFtsY and exhibited integration efficiency comparable with that found in assays conducted without added cpFtsY. These data imply that the cpFtsY N terminus plays an active role in thylakoid binding and that membrane binding retained by cpFtsYNG+1 is not productive in terms of supporting the role of cpFtsY in LHCP localization.FIGURE 3cpFtsY double-Phe motif (Phe-48 and Phe-49) plays a critical role in LHCP integration and thylakoid partitioning. A, integration of radiolabeled LHCP was reconstituted as described in the legend to Fig. 2A. Integration efficiency was calculated from three separate experiments and is presented relative to integration observed in the presence of the stroma. TP, translation product; DP, degradation product" @default.
• W2010048393 created "2016-06-24" @default.
• W2010048393 creator A5005109729 @default.
• W2010048393 creator A5043294726 @default.
• W2010048393 creator A5059576120 @default.
• W2010048393 creator A5059710385 @default.
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• W2010048393 date "2009-05-01" @default.
• W2010048393 modified "2023-10-13" @default.
• W2010048393 title "The Membrane-binding Motif of the Chloroplast Signal Recognition Particle Receptor (cpFtsY) Regulates GTPase Activity" @default.
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