The Roles of Toc34 and Toc75 in Targeting the Toc159 Preprotein Receptor to Chloroplasts
2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês
10.1074/jbc.m307873200
ISSN1083-351X
AutoresTanya R. Wallas, Matthew D. Smith, Sobeida Sánchez‐Nieto, Danny J. Schnell,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe Toc complex at the outer envelope of chloroplasts initiates the import of nuclear-encoded preproteins from the cytosol into the organelle. The core of the Toc complex is composed of two receptor GTPases, Toc159 and Toc34, as well as Toc75, a β-barrel membrane channel. Toc159 is equally distributed between a soluble cytoplasmic form and a membrane-inserted form, suggesting that assembly of the Toc complex is dynamic. In the present study, we used the Arabidopsis thaliana orthologs of Toc159 and Toc34, atToc159 and atToc33, respectively, to investigate the requirements for assembly of the trimeric Toc complex. In addition to its intrinsic GTPase activity, we demonstrate that integration of atToc159 into the Toc complex requires atToc33 GTPase activity. Additionally, we show that the interaction of the two GTPase domains stimulates association of the membrane anchor of atToc159 with the translocon. Finally, we employ reconstituted proteoliposomes to demonstrate that proper insertion of the receptor requires both Toc75 and Toc34. Collectively these data suggest that Toc34 and Toc75 act sequentially to mediate docking and insertion of Toc159 resulting in assembly of the functional translocon. The Toc complex at the outer envelope of chloroplasts initiates the import of nuclear-encoded preproteins from the cytosol into the organelle. The core of the Toc complex is composed of two receptor GTPases, Toc159 and Toc34, as well as Toc75, a β-barrel membrane channel. Toc159 is equally distributed between a soluble cytoplasmic form and a membrane-inserted form, suggesting that assembly of the Toc complex is dynamic. In the present study, we used the Arabidopsis thaliana orthologs of Toc159 and Toc34, atToc159 and atToc33, respectively, to investigate the requirements for assembly of the trimeric Toc complex. In addition to its intrinsic GTPase activity, we demonstrate that integration of atToc159 into the Toc complex requires atToc33 GTPase activity. Additionally, we show that the interaction of the two GTPase domains stimulates association of the membrane anchor of atToc159 with the translocon. Finally, we employ reconstituted proteoliposomes to demonstrate that proper insertion of the receptor requires both Toc75 and Toc34. Collectively these data suggest that Toc34 and Toc75 act sequentially to mediate docking and insertion of Toc159 resulting in assembly of the functional translocon. The import of nucleus-encoded preproteins into chloroplasts is initiated by the translocon at the outer envelope membrane of chloroplasts (Toc) 1The abbreviations used are: Toc, translocon at the outer envelope membrane of chloroplasts; G-domain, GTPase domain; M-domain, membrane domain; Ni-NTA, nickel-nitrilotriacetic acid; atToc, Arabidopsis thaliana translocon at the outer envelope membrane of chloroplasts; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; GMP-P(NH)P, guanyl-5′-yl imidodiphosphate.1The abbreviations used are: Toc, translocon at the outer envelope membrane of chloroplasts; G-domain, GTPase domain; M-domain, membrane domain; Ni-NTA, nickel-nitrilotriacetic acid; atToc, Arabidopsis thaliana translocon at the outer envelope membrane of chloroplasts; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; GMP-P(NH)P, guanyl-5′-yl imidodiphosphate. (1Keegstra K. Cline K. Plant Cell. 1999; 11: 557-570Crossref PubMed Scopus (315) Google Scholar, 2Schleiff E. Soll J. Planta. 2000; 211: 449-456Crossref PubMed Scopus (74) Google Scholar, 3Bauer J. Hiltbrunner A. Kessler F. Cell. Mol. Life Sci. 2001; 58: 420-433Crossref PubMed Scopus (54) Google Scholar). The Toc complex is comprised of three core subunits, Toc159 (4Kessler F. Blobel G. Patel H.A. Schnell D.J. Science. 1994; 266: 1035-1039Crossref PubMed Scopus (244) Google Scholar, 5Kouranov A. Schnell D.J. J. Cell Biol. 1997; 139: 1677-1685Crossref PubMed Scopus (165) Google Scholar), Toc33/34 (6Gutensohn M. Schulz B. Nicolay P. Flügge U.L. Plant J. 2000; 23: 771-783Crossref PubMed Google Scholar, 7Sveshnikova N. Soll J. Schleiff E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4973-4978Crossref PubMed Scopus (116) Google Scholar), and Toc75 (8Schnell D.J. Kessler F. Blobel G. Science. 1994; 266: 1007-1012Crossref PubMed Scopus (328) Google Scholar, 9Ma Y. Kouranov A. LaSala S. Schnell D.J. J. Cell Biol. 1996; 134: 1-13Crossref PubMed Scopus (139) Google Scholar, 10Hinnah S.C. Hill K. Wagner R. Schlicher T. Soll J. EMBO J. 1997; 16: 7351-7360Crossref PubMed Scopus (187) Google Scholar). Toc159 and Toc34 are homologous GTPases that recognize the transit peptides of preproteins (4Kessler F. Blobel G. Patel H.A. Schnell D.J. Science. 1994; 266: 1035-1039Crossref PubMed Scopus (244) Google Scholar, 5Kouranov A. Schnell D.J. J. Cell Biol. 1997; 139: 1677-1685Crossref PubMed Scopus (165) Google Scholar, 6Gutensohn M. Schulz B. Nicolay P. Flügge U.L. Plant J. 2000; 23: 771-783Crossref PubMed Google Scholar, 7Sveshnikova N. Soll J. Schleiff E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4973-4978Crossref PubMed Scopus (116) Google Scholar, 11Jarvis P. Chen L.-J. Li H. Peto C.A. Fankhauser C. Chory J. Science. 1998; 282: 100-103Crossref PubMed Scopus (271) Google Scholar, 12Schleiff E. Soll J. Sveshnikova N. Tien R. Wright S. Dabney-Smith C. Subramanian C. Bruce B.D. Biochemistry. 2002; 41: 1934-1946Crossref PubMed Scopus (70) Google Scholar, 13Perry S.E. Keegstra K. Plant Cell. 1994; 6: 93-105Crossref PubMed Scopus (204) Google Scholar) and regulate membrane translocation via a GTPase cycle (5Kouranov A. Schnell D.J. J. Cell Biol. 1997; 139: 1677-1685Crossref PubMed Scopus (165) Google Scholar, 14Chen K. Chen X. Schnell D.J. Plant Physiol. 2000; 122: 813-822Crossref PubMed Scopus (101) Google Scholar, 15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar). These two components associate with Toc75 to form a translocation pore across the outer membrane (8Schnell D.J. Kessler F. Blobel G. Science. 1994; 266: 1007-1012Crossref PubMed Scopus (328) Google Scholar, 9Ma Y. Kouranov A. LaSala S. Schnell D.J. J. Cell Biol. 1996; 134: 1-13Crossref PubMed Scopus (139) Google Scholar, 10Hinnah S.C. Hill K. Wagner R. Schlicher T. Soll J. EMBO J. 1997; 16: 7351-7360Crossref PubMed Scopus (187) Google Scholar). Although the mechanism of preprotein import into chloroplasts has garnered considerable attention, less information is available on the assembly of the translocon itself. Toc34 and Toc75 are exclusively localized to the outer envelope membrane in approximately a 1:1 stoichiometry (16Schleiff E. Soll J. Kuchler M. Kuhlbrandt W. Harrer R. J. Cell Biol. 2003; 160: 541-551Crossref PubMed Scopus (174) Google Scholar). In contrast, Toc159 is found both in a soluble cytoplasmic form and integrated into the Toc complex in substoichiometric amounts relative to the other Toc components (16Schleiff E. Soll J. Kuchler M. Kuhlbrandt W. Harrer R. J. Cell Biol. 2003; 160: 541-551Crossref PubMed Scopus (174) Google Scholar, 17Hiltbrunner A. Bauer J. Vidi P.A. Infanger S. Weibel P. Hohwy M. Kessler F. J. Cell Biol. 2001; 154: 309-316Crossref PubMed Scopus (100) Google Scholar). These observations suggest that Toc complex assembly is dynamic and have led to the proposal that Toc159 may act as a cycling receptor that targets preproteins from the cytoplasm to the Toc translocon (17Hiltbrunner A. Bauer J. Vidi P.A. Infanger S. Weibel P. Hohwy M. Kessler F. J. Cell Biol. 2001; 154: 309-316Crossref PubMed Scopus (100) Google Scholar). Previous studies have examined the structural features of Toc159 that are required for assembly into the Toc complex (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar, 17Hiltbrunner A. Bauer J. Vidi P.A. Infanger S. Weibel P. Hohwy M. Kessler F. J. Cell Biol. 2001; 154: 309-316Crossref PubMed Scopus (100) Google Scholar). We recently demonstrated that the GTPase domain (G-domain) of the Arabidopsis Toc159 orthologue, atToc159, mediates targeting of the receptor to the chloroplast surface via a direct interaction with the GTPase domain of atToc33, the Arabidopsis Toc34 orthologue (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar, 18Bauer J. Hiltbrunner A. Weibel P. Vidi P.A. Alvarez-Huerta M. Smith M.D. Schnell D.J. Kessler F. J. Cell Biol. 2002; 159: 845-854Crossref PubMed Scopus (71) Google Scholar). Tight association of the Toc GTPases is triggered by conversion of atToc159 to its GDP-bound form via intrinsic GTP hydrolysis. Hydrolysis is required for insertion of the membrane anchor (M-domain) of the receptor into the outer membrane and results in the assembly of atToc159 into the Toc complex (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar). These studies highlighted the role for a GTP-driven switch in regulating assembly of the Toc translocon. In this report, we have investigated the roles of Toc34 and Toc75 in targeting Toc159 to the outer membrane. We demonstrate that the GTPase activity of atToc33 participates in atToc159 targeting, indicating that the coordinate activities of the two GTPases are required for Toc assembly. To define the minimal requirements of Toc159 targeting, we reconstituted Toc complex assembly in proteoliposomes containing Toc34 and/or Toc75. These studies confirm the role of Toc34 in docking of the Toc159 receptor at the membrane surface and demonstrate that Toc75 mediates the insertion of the M-domain of Toc159 into the membrane. Toc33/34 Mutations and AtToc159 Deletion Constructs—Point mutations were introduced into the conserved G1 motif of the GTP binding domain (amino acids 1–265) of atToc33 (atToc33G) (17Hiltbrunner A. Bauer J. Vidi P.A. Infanger S. Weibel P. Hohwy M. Kessler F. J. Cell Biol. 2001; 154: 309-316Crossref PubMed Scopus (100) Google Scholar) using the overlap extension technique (19Ling M.M. Robinson B.H. Anal. Biochem. 1997; 254: 157-178Crossref PubMed Scopus (180) Google Scholar) and pET21d-atToc33GHis (17Hiltbrunner A. Bauer J. Vidi P.A. Infanger S. Weibel P. Hohwy M. Kessler F. J. Cell Biol. 2001; 154: 309-316Crossref PubMed Scopus (100) Google Scholar) as a template. PCR primers were used that resulted in the following point mutations: codon GGC was changed to CGT resulting in a Gly to Arg mutation at amino acid 45; codon AAA was changed to AAT resulting in a Lys to Asn mutation at amino acid 49; and codon TCA was changed to CGA resulting in a Ser to Arg mutation at amino acid 50. The mutated PCR fragment was cloned into the BamHI and XbaI sites of pET21d, resulting in an in-frame fusion with a COOH-terminal hexahistidine tag, which gave rise to clone pET21d-atToc33GHis-G45R/K49N/S50R. Construction of pET21d-atToc159, pET21d-atToc159-A864R, pET21d-atToc159-K868R, pET21d-atToc159GHis, pET21d-atToc159GHis-K868R, and pET21a-atToc159MHis has been described previously (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar). Constructs encoding atToc159G (pET21d-atToc159G) and atToc159M (pET21a-atToc159M) lacking hexahistidine tags were generated by modifying pET21d-atToc159GHis and pET21a-atToc159MHis, respectively, by PCR to replace the coding sequence of the tag with stop codons. In Vitro Translation and Expression in Escherichia coli—[35S]Methionine-labeled translation products of atToc159, atToc159-A864R, and atToc159-K868R were obtained using a coupled transcription-translation system containing reticulocyte lysate according to the manufacturer's recommendations (Promega). mRNA for atToc159 deletion mutants atToc159G and atToc159M was generated using the RiboMAX in vitro transcription system (Promega). [35S]Methionine-labeled translation products were subsequently obtained using an in vitro translation system containing reticulocyte lysate as recommended by the manufacturer (Promega). For bacterial overexpression, pET21d-atToc33GHis (17Hiltbrunner A. Bauer J. Vidi P.A. Infanger S. Weibel P. Hohwy M. Kessler F. J. Cell Biol. 2001; 154: 309-316Crossref PubMed Scopus (100) Google Scholar), pET21d-atToc33GHis-G45R/K49N/S50R, pET21d-atToc159GHis (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar), pET21d-atToc159GHis-K868R (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar), pET21a-atToc159MHis (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar), pET21d-IAP34 (psToc34) (20Chen D. Schnell D.J. J. Biol. Chem. 1997; 272: 6614-6620Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), pET21d-IAP34-G48R/K52N/S53R (psToc34-G48R/K52N/S53R) (20Chen D. Schnell D.J. J. Biol. Chem. 1997; 272: 6614-6620Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), and pET21b-IAP75 (psToc75) (9Ma Y. Kouranov A. LaSala S. Schnell D.J. J. Cell Biol. 1996; 134: 1-13Crossref PubMed Scopus (139) Google Scholar) were transformed into E. coli BL-21(DE3). Expression of atToc33GHis, atToc33GHis-G45R/K49N/S50R, atToc159GHis, atToc159GHis-K868R, and atToc159MHis was achieved using 2 g/liter of INDUCER (Molecular Research Labs, Inc.) for 14–16 h at 22 °C. The hexahistidine-tagged proteins were then purified from the soluble fraction of cleared E. coli lysates under nondenaturing conditions using Ni-NTA chromatography (Novagen, Inc.) and eluted into HMK buffer (50 mm Hepes-KOH, pH 7.5, 40 mm KOAc, 2 mm MgCl2) containing 250 mm imidazole. Purified atToc33GHis, atToc33GHis-G45R/K49N/S50R, atToc159GHis, and atToc159GHis-K868R as well as all [35S]methionine-labeled in vitro translation products were depleted of free nucleotides by gel filtration prior to use as previously described (20Chen D. Schnell D.J. J. Biol. Chem. 1997; 272: 6614-6620Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Expression of psToc75, psToc34, and psToc34-G48R/K52N/S53R was achieved by induction with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 3 or 4 h at 37 °C, and purification from inclusion bodies using Ni-NTA chromatography under denaturing conditions was performed as previously described (9Ma Y. Kouranov A. LaSala S. Schnell D.J. J. Cell Biol. 1996; 134: 1-13Crossref PubMed Scopus (139) Google Scholar, 20Chen D. Schnell D.J. J. Biol. Chem. 1997; 272: 6614-6620Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Guanine Nucleotide Binding and Hydrolysis Assays—GTP binding activities of purified atToc33GHis and atToc33GHis-G45R/K49N/S50R were measured using a filter binding assay as previously described (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar, 20Chen D. Schnell D.J. J. Biol. Chem. 1997; 272: 6614-6620Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) with minor modifications. Protein was diluted to 2 μm and incubated with 1.0 μm [α-32P]GTP (150 mCi/μmol) and 10 mm MgCl2 for 30 min at 25 °C. The radioactivity retained on the membrane with the protein was quantitated using scintillation counting. This concentration of [α-32P]GTP was chosen because it minimized the level of nonspecific binding observed in the assay. Nucleotide binding at this concentration gives <10% saturation but yields the highest signal to noise ratio. Nucleotide binding to hexahistidine-tagged cellular retinoic acid-binding protein (21Clark P.L. Weston B.F. Gierasch L.M. Fold Des. 1998; 3: 401-412Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) purified from E. coli using Ni-NTA chromatography was used as background reference. GTP hydrolysis was measured in a soluble phase assay as described (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar). Here, 1.6 μm purified atToc33GHis or atToc33GHis-G45R/K49N/ S50R was incubated with 2 μm [α-32P]GTP (150 mCi/μmol) and samples were resolved using TLC. Plates were dried and quantitated using a Storm 840 PhosphorImager and ImageQuant version 5.2 software. Hydrolysis of GTP by overexpressed hexahistidine-tagged preferredoxin-protein A (5Kouranov A. Schnell D.J. J. Cell Biol. 1997; 139: 1677-1685Crossref PubMed Scopus (165) Google Scholar) purified from E. coli was used as a background reference. GTP hydrolysis activity of proteoliposomes containing psToc34 or psToc34-G48R/K52N/S53R was measured as described for purified atToc33GHis proteins, with the exception that the proteoliposomes were diluted to give a final protein concentration of 1.2 μm in a 50-μl reaction volume. Solid Phase Binding Assays—Direct interaction between atToc159 or atToc159G and atToc33GHis or atToc33GHis-G45R/K49N/S50R was measured using solid phase binding assays adapted from Smith et al. (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar). Briefly, 20 μg of purified, nucleotide-depleted atToc33GHis or atToc33GHis-G45R/K49N/S50R were bound to 8 μl of packed Ni-NTA resin in the presence of 0.1 mm GTP or GMP-P(NH)P, and incubated with 1–5 μl of in vitro translated [35S]atToc159 or [35S]atToc159G. To measure atToc159M binding to atToc33GHis, 6 μl of packed Ni-NTA resin was first saturated with 80 μg of atToc33GHis, and then incubated with 5–10 μl of in vitro translated [35S]atToc159M in the absence or presence of 20 μm purified atToc159MHis, or increasing concentrations of atToc159GHis. All eluted proteins from solid phase binding assays were resolved by SDS-PAGE and stained with Coomassie Blue to detect the resin-immobilized proteins (data not shown). [35S]Methionine-labeled proteins were detected in dried gels using a Storm 840 PhosphorImager and ImageQuant version 5.2 software. Chloroplast Isolation and Toc159M Targeting Assays—Intact chloroplasts were isolated from ∼2-week-old Arabidopsis thaliana (ecotype Wassilewskija) seedlings grown on 0.8% (w/v) phytagar plates containing Murashige and Skoog growth medium and 1% (w/v) sucrose, as described (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar, 22Smith M.D. Fitzpatrick L.M. Keegstra K. Schnell D.J. Bonifacino J.S. Lippincott-Schwartz J. Harford J.B. Yamada K.M. Current Protocols in Cell Biology. 14th Ed. John Wiley and Sons, Inc., New York2002: 11.16.11-11.16.21Google Scholar, 23Fitzpatrick L.M. Keegstra K. Plant J. 2001; 27: 59-65Crossref PubMed Scopus (86) Google Scholar). Targeting of atToc159M to chloroplasts was carried out using intact chloroplasts equivalent to 50 μg of chlorophyll in 100 μl of HS buffer (50 mm Hepes-KOH, pH 8.0, 330 mm sorbitol) containing 50 mm KOAc and 4 mm MgOAc (import buffer). Targeting reactions were initiated with the addition of [35S]atToc159M in the absence or presence of 5 μm purified atToc159GHis, atToc159GHis-K868R, or atToc33GHis. Following the standard import reactions chloroplasts were incubated in the presence or absence of 100 μg/ml thermolysin as described (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar, 22Smith M.D. Fitzpatrick L.M. Keegstra K. Schnell D.J. Bonifacino J.S. Lippincott-Schwartz J. Harford J.B. Yamada K.M. Current Protocols in Cell Biology. 14th Ed. John Wiley and Sons, Inc., New York2002: 11.16.11-11.16.21Google Scholar). Reisolated chloroplasts were hypotonically lysed in 10 mm Tris-HCl, pH 8.0, 1 mm EDTA, and the total chloroplast membrane fraction was recovered by centrifugation at 100,000 × g for 20 min. The membrane fractions were resolved by SDS-PAGE, and [35S]atToc159M was detected and quantitated in dried gels using a Storm 840 PhosphorImager and ImageQuant version 5.2 software. Reconstitution of Proteoliposomes—Liposomes were generated using azolectin (type IV-S, Sigma) and a method adapted from Hinnah et al. (10Hinnah S.C. Hill K. Wagner R. Schlicher T. Soll J. EMBO J. 1997; 16: 7351-7360Crossref PubMed Scopus (187) Google Scholar). Azolectin was resuspended in 10 mm MOPS-KOH, pH 6.9, to give a final concentration of 50 mg/ml and pulse sonicated for 30 s to facilitate even dispersion. The liposomes were frozen at –80 °C for 1 h. Purified, urea-soluble psToc75, psToc34, or psToc34-G48R/K52N/S53R was diluted with an equal volume of 24 mm CHAPS, and incubated for 1 h on ice. The liposomes were thawed and added to the proteins at a ratio of 200 μl of liposomes/90 μg of protein. The mixture was pulse sonicated for 10 s, incubated for 1 h on ice, and dialyzed overnight into 50 mm Hepes-KOH, pH 7.5. Proteoliposomes were recovered by centrifugation at 90,000 × g for 40 min, and resuspended in HS buffer, containing 1 mm dithiothreitol. Carbonate extraction of proteoliposomes was achieved by resuspending pelleted proteoliposomes containing psToc34 and psToc75 in 40 μlof 0.1 m Na2CO3, pH 11.5, and incubation for 20 min on ice. Proteoliposomes were recovered by centrifugation for 30 min at 18,000 × g, and the pellet and supernatant fractions were analyzed by SDS-PAGE and Coomassie Blue staining. Immunoprecipitation of Proteoliposome Proteins—Proteoliposomes were dissolved in 1% (v/v) Triton X-100 and applied to a column containing anti-psToc34 IgG coupled to Sepharose as previously described (9Ma Y. Kouranov A. LaSala S. Schnell D.J. J. Cell Biol. 1996; 134: 1-13Crossref PubMed Scopus (139) Google Scholar). Bound proteins were eluted, resolved by SDS-PAGE, and immunoblotted with anti-psToc34 and anti-psToc75 (9Ma Y. Kouranov A. LaSala S. Schnell D.J. J. Cell Biol. 1996; 134: 1-13Crossref PubMed Scopus (139) Google Scholar). AtToc159 Proteoliposome Targeting Assays—Proteoliposome targeting assays were performed using 1–5 μl of freshly prepared proteoliposomes (corresponding to approximately 5 μg of protein) in import buffer containing 0.1 mg/ml bovine serum albumin, 2 mm ATP, and 2 mm GTP. Targeting reactions were initiated with the addition of [35S]methionine-labeled in vitro translation products and were incubated for 20 min at 26 °C in the dark. The reactions were stopped with the addition of ice-cold HS buffer and recovered by centrifugation for 30 min at 14,000 × g. The proteoliposomes were resuspended in 50 μl of HS buffer and incubated with or without thermolysin (10 μg/ml) for 30 min on ice. Protease treatments were terminated by adding EDTA to a final concentration of 10 mm in 1 ml of ice-cold HS buffer, and the liposomes were collected again by centrifugation. Finally, the proteoliposomes were resolved using SDS-PAGE and radioactive signals in dried gels were detected and quantitated using a Strom 840 PhosphorImager and ImageQuant version 5.2 software. Counts from proteolytic fragments of atToc159 (i.e. atToc159M) in thermolysin-treated proteoliposomes were normalized to reflect the number of methionine residues lost because of proteolysis. The GTPase Activity of AtToc33 Is Required for AtToc159 Binding—Our previous studies demonstrated that chloroplast binding and membrane insertion of atToc159 were promoted by its intrinsic GTPase activity and involved a direct interaction with atToc33 (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar, 18Bauer J. Hiltbrunner A. Weibel P. Vidi P.A. Alvarez-Huerta M. Smith M.D. Schnell D.J. Kessler F. J. Cell Biol. 2002; 159: 845-854Crossref PubMed Scopus (71) Google Scholar). To investigate whether GTP binding and the GTPase activity of atToc33 are also involved in atToc159 targeting, we studied the binding of atToc159 to a mutant of atToc33 with altered nucleotide binding and hydrolysis activities. The mutant, atToc33-G45R/K49N/S50R, contains three point mutations in the consensus G1 motif (P-loop) of the conserved GTP-binding site (Fig. 1A). A comparable mutant of pea Toc34, psToc34-G48R/K52N/S53R, previously was shown to inhibit GTP binding and hydrolysis (20Chen D. Schnell D.J. J. Biol. Chem. 1997; 272: 6614-6620Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). To confirm the effects of the mutations, 29-kDa amino-terminal fragments of wild type atToc33 (atToc33GHis) and atToc33-G45R/K49N/S50R (atToc33GHis-G45R/K49N/S50R) containing hexahistidine tags and lacking the carboxyl-terminal transmembrane segment were expressed in E. coli, purified by Ni-NTA chromatography (Fig. 1B), and assayed for nucleotide binding and hydrolysis. As predicted, the GTP hydrolytic activity (Fig. 1C) and GTP binding capacity (Fig. 1D) of atToc33GHis-G45R/K49N/S50R were reduced to 19% of wild type atToc33GHis levels. To examine the effects of the atToc33 mutations on atToc159 binding, we incubated in vitro translated full-length atToc159 or a deletion mutant corresponding to the atToc159 GTPase domain, atToc159G, with immobilized atToc33GHis or atToc33GHis-G45R/K49N/S50R in a solid phase binding assay. The assays were performed either in the presence of GTP or the nonhydrolyzable GTP analog, GMP-P(NH)P. As previously shown, atToc33GHis binds both full-length [35S]atToc159 (17Hiltbrunner A. Bauer J. Vidi P.A. Infanger S. Weibel P. Hohwy M. Kessler F. J. Cell Biol. 2001; 154: 309-316Crossref PubMed Scopus (100) Google Scholar) and [35S]atToc159G (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar) in the presence of GTP (Fig. 2, A, lane 2, and B, GTP). Binding is inhibited by GMP-P(NH)P (Fig. 2, A, compare lanes 2 and 5, and B) consistent with the proposal that GTP hydrolysis is required for high affinity binding (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar). The binding of atToc159 or atToc159G to atToc33GHis-G45R/K49N/S50R was decreased by 50 and 65%, respectively, relative to binding to wild type atToc33G (Fig. 2, A, compare lanes 2 and 3, and B, GTP). Binding to the mutant in the presence of GMP-P(NH)P was similar to the levels of binding in the presence of GTP (Fig. 2A, compare lanes 3 and 6, and B) and to the levels of binding to wild type proteins observed in the presence of GMP-P(NH)P (Fig. 2, A, compare lanes 5 and 6, and B, GMP-PNP). These results provide additional evidence that the binding of the Toc GTPases is nucleotide regulated and confirm a role for the GTPase activity of atToc33 in the binding of atToc159 to the Toc complex. The GTPase Domains of AtToc159 and AtToc33 Mediate Targeting of the Membrane Anchor of AtToc159 —The results presented in Fig. 2 and those of previous studies (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar, 18Bauer J. Hiltbrunner A. Weibel P. Vidi P.A. Alvarez-Huerta M. Smith M.D. Schnell D.J. Kessler F. J. Cell Biol. 2002; 159: 845-854Crossref PubMed Scopus (71) Google Scholar) provide convincing evidence that atToc159 targeting to the outer envelope membrane is mediated by a GTP-regulated cognate interaction between the atToc159 and atToc33 G-domains. We previously proposed that the binding of the G-domains and subsequent GTP hydrolysis results in the integration of the atToc159 M-domain into the outer envelope and assembly of functional Toc complexes (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar). This proposal predicts that the activities of the G-domains are required for specific targeting and integration of the atToc159 M-domain at the chloroplast surface. The complementary roles of the M-domain and G-domains in receptor binding are supported by the observation that full-length atToc159 binds with greater efficiency to both isolated chloroplasts (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar) and atToc33GHis (Fig. 2B) than either atToc159G or atToc159M alone. To test this hypothesis, we examined the role of the G-domains in M-domain targeting. As a first step, we studied the influence of atToc159G on the binding of in vitro translated M-domain ([35S]atToc159M) to isolated chloroplasts in trans. Isolated chloroplasts were incubated with [35S]atToc159M in the presence or absence of E. coli-expressed atToc159GHis, atToc159GHis-K868R, a mutant with reduced GTP binding and hydrolytic activity (15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar), or atToc33GHis. The amount of bound [35S]atToc159M was determined after reisolation of the chloroplasts through a Percoll cushion. Integration of [35S]atToc159M into the outer membrane was assessed by treating equivalent samples of chloroplasts in the presence or absence of thermolysin. The ∼52-kDa M-domain is protected from proteolysis upon proper integration of atToc159 (14Chen K. Chen X. Schnell D.J. Plant Physiol. 2000; 122: 813-822Crossref PubMed Scopus (101) Google Scholar, 15Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (77) Google Scholar, 24Hirsch S. Muckel E. Heemeyer F. von Heijne G. Soll J. Science. 1994; 266: 1989-1992Crossref PubMed Scopus (190) Google Scholar). In the absence of any additions, atToc159M binds with low efficiency to isolated chloroplasts and remains protease sensitive (Fig. 3, A, lanes 2 and 3, and B). Previous studies have shown that this low level of binding represents a nonspecific interaction with the chloroplast surface (15Smith M.D. H
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