Title: atTic110 Functions as a Scaffold for Coordinating the Stromal Events of Protein Import into Chloroplasts
Abstract: The translocon of the inner envelope membrane of chloroplasts (Tic) mediates the late events in the translocation of nucleus-encoded preproteins into chloroplasts. Tic110 is a major integral membrane component of active Tic complexes and has been proposed to function as a docking site for translocation-associated stromal factors and as a component of the protein-conducting channel. To investigate the various proposed functions of Tic110, we have investigated the structure, topology, and activities of a 97.5-kDa fragment of Arabidopsis Tic110 (atTic110) lacking only the amino-terminal transmembrane segments. The protein was expressed both in Escherichia coli and Arabidopsis as a stable, soluble protein with a high α-helical content. Binding studies demonstrate that a region of the atTic110-soluble domain selectively associates with chloroplast preproteins at the late stages of membrane translocation. These data support the hypothesis that the bulk of Tic110 extends into the chloroplast stroma and suggest that the domain forms a docking site for preproteins as they emerge from the Tic translocon. The translocon of the inner envelope membrane of chloroplasts (Tic) mediates the late events in the translocation of nucleus-encoded preproteins into chloroplasts. Tic110 is a major integral membrane component of active Tic complexes and has been proposed to function as a docking site for translocation-associated stromal factors and as a component of the protein-conducting channel. To investigate the various proposed functions of Tic110, we have investigated the structure, topology, and activities of a 97.5-kDa fragment of Arabidopsis Tic110 (atTic110) lacking only the amino-terminal transmembrane segments. The protein was expressed both in Escherichia coli and Arabidopsis as a stable, soluble protein with a high α-helical content. Binding studies demonstrate that a region of the atTic110-soluble domain selectively associates with chloroplast preproteins at the late stages of membrane translocation. These data support the hypothesis that the bulk of Tic110 extends into the chloroplast stroma and suggest that the domain forms a docking site for preproteins as they emerge from the Tic translocon. Chloroplast biogenesis is dependent upon the import of ∼3000 different nucleus-encoded proteins (1Keegstra K. Cline K. Plant Cell. 1999; 11: 557-570Crossref PubMed Scopus (321) Google Scholar). The majority of these proteins are synthesized as preproteins carrying an amino-terminal transit peptide that serves as the essential signal for targeting to the organelle. The transit peptide is recognized by receptor components of the translocon at the outer envelope membrane of chloroplasts (Toc), and a GTP-regulated switch initiates translocation through the protein-conducting channel of the Toc complex. At this stage, the Toc complex associates with the translocon at the inner envelope membrane (Tic), and this Toc-Tic supercomplex mediates the direct transport of the preproteins from the cytoplasm into the chloroplast stroma (2Bauer J. Hiltbrunner A. Kessler F. Cell. Mol. Life Sci. 2001; 58: 420-433Crossref PubMed Scopus (54) Google Scholar). Although many mechanistic details remain to be defined, the activities of the Toc components have been extensively investigated. Two membrane-associated GTPases, Toc159 and Toc34/33, mediate transit peptide recognition and regulate the initiation of translocation (3Kessler F. Blobel G. Patel H.A. Schnell D.J. Science. 1994; 266: 1035-1039Crossref PubMed Scopus (245) Google Scholar, 4Bauer J. Chen K. Hiltbunner A. Wehrli E. Eugster M. Schnell D. Kessler F. Nature. 2000; 403: 203-207Crossref PubMed Scopus (289) Google Scholar, 5Hiltbrunner A. Bauer J. Vidi P.A. Infanger S. Weibel P. Hohwy M. Kessler F. J. Cell Biol. 2001; 154: 309-316Crossref PubMed Scopus (102) Google Scholar, 6Bauer 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 (73) Google Scholar, 7Smith M.D. Hiltbrunner A. Kessler F. Schnell D.J. J. Cell Biol. 2002; 159: 833-843Crossref PubMed Scopus (79) Google Scholar). The Toc GTPases form a complex with Toc75, an integral membrane protein that, along with Toc159, constitutes a major component of the protein-conducting channel (1Keegstra K. Cline K. Plant Cell. 1999; 11: 557-570Crossref PubMed Scopus (321) Google Scholar, 8Schnell D.J. Kessler F. Blobel G. Science. 1994; 266: 1007-1012Crossref PubMed Scopus (329) Google Scholar, 9Ma Y. Kouranov A. LaSala S. Schnell D.J. J. Cell Biol. 1996; 134: 315-327Crossref PubMed Scopus (140) Google Scholar, 10Hinnah S.C. Hill K. Wagner R. Schlicher T. Soll J. EMBO J. 1997; 16: 7351-7360Crossref PubMed Scopus (189) Google Scholar). In contrast to the Toc complex, the activities and functions of the Tic components are less well defined. The biochemical analysis of Tic function has been complicated by the fact that assembly of functional Tic complexes is dynamic and occurs in response to preprotein translocation (11Kouranov A. Chen X. Fuks B. Schnell D.J. J. Cell Biol. 1998; 143: 991-1002Crossref PubMed Scopus (211) Google Scholar). Therefore, the isolation of a stable Tic complex has thus far been elusive. Tic110 was the first Tic component identified and represents a major component of active Tic complexes (12Kessler F. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7684-7689Crossref PubMed Scopus (169) Google Scholar, 13Lubeck J. Soll J. Akita M. Nielsen E. Keegstra K. EMBO J. 1996; 15: 4230-4238Crossref PubMed Scopus (123) Google Scholar). It is an integral inner envelope membrane protein, and structural predictions suggest that it consists of two predicted transmembrane helices at its extreme amino terminus and a 97.5-kDa carboxyl-terminal region that is largely hydrophilic. Tic110 transiently associates with at least five other Tic proteins. These include Tic20, a polytopic membrane protein that is implicated in protein translocation at the inner membrane by covalent cross-linking and genetic studies (11Kouranov A. Chen X. Fuks B. Schnell D.J. J. Cell Biol. 1998; 143: 991-1002Crossref PubMed Scopus (211) Google Scholar, 14Chen X. Smith M.D. Fitzpatrick L. Schnell D.J. Plant Cell. 2002; 14: 641-654Crossref PubMed Scopus (103) Google Scholar); Tic22, a peripheral inner membrane protein that may regulate interactions between the Toc and Tic translocons by sensing preprotein emergence into the intermembrane space (11Kouranov A. Chen X. Fuks B. Schnell D.J. J. Cell Biol. 1998; 143: 991-1002Crossref PubMed Scopus (211) Google Scholar, 15Kouranov A. Wang H. Schnell D.J. J. Biol. Chem. 1999; 274: 25181-25186Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar); Tic40, an inner membrane protein that appears to interact with preproteins at late stage in inner membrane translocation (16Stahl T. Glockmann C. Soll J. Heins L. J. Biol. Chem. 1999; 274: 37467-37472Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar); and Tic55 and Tic62, two inner membrane redox components that are proposed to serve as regulators of the translocation reaction (17Caliebe A. Grimm R. Kaiser G. Lubeck J. Soll J. Heins L. EMBO J. 1997; 16: 7342-7350Crossref PubMed Scopus (142) Google Scholar, 18Kuchler M. Decker S. Hormann F. Soll J. Heins L. EMBO J. 2002; 21: 6136-6145Crossref PubMed Scopus (136) Google Scholar). Studies of Tic110 topology and molecular interactions have led to three models for its roles in protein import. In the first model, the carboxyl-terminal region of pea Tic110, psTic110, was predicted to extend into the intermembrane space between the outer and inner envelope membranes and thereby mediate the interactions between the Toc and Tic complexes during the translocation reaction (13Lubeck J. Soll J. Akita M. Nielsen E. Keegstra K. EMBO J. 1996; 15: 4230-4238Crossref PubMed Scopus (123) Google Scholar, 19Lubeck J. Heins L. Soll J. J. Cell Biol. 1997; 137: 1279-1286Crossref PubMed Scopus (66) Google Scholar). However, this model was discounted by detailed topology mapping studies (20Jackson D.T. Froehlich J.E. Keegstra K. J. Biol. Chem. 1998; 273: 16583-16588Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The results indicated that psTic110 exists in the opposite orientation with the bulk of the protein extending into the stroma and limited exposure to the intermembrane space. In the second model, the large hydrophilic domain of Tic110 is proposed to serve as a docking site for soluble stromal chaperones that assist in the translocation and folding of imported proteins (12Kessler F. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7684-7689Crossref PubMed Scopus (169) Google Scholar, 20Jackson D.T. Froehlich J.E. Keegstra K. J. Biol. Chem. 1998; 273: 16583-16588Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In this scenario, Tic110 would function in a manner analogous to Tim44, a protein that mediates mtHsp70 localization to the matrix face of the Tim23 translocon in mitochondria (21Schneider H.C. Berthold J. Bauer M.F. Dietmeier K. Guiard B. Brunner M. Neupert W. Nature. 1994; 371: 768-774Crossref PubMed Scopus (334) Google Scholar). This model is supported by the topology studies and by the observation that native psTic110 associates reversibly with the Hsp93 and Cpn60 chaperones of the chloroplast stroma (12Kessler F. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7684-7689Crossref PubMed Scopus (169) Google Scholar, 22Nielsen E. Akita M. Davila-Aponte J. Keegstra K. EMBO J. 1997; 16: 935-946Crossref PubMed Scopus (233) Google Scholar). Recently, this model has been challenged by a third hypothesis. This study proposed that Tic110 is a β-barrel membrane protein and functions as the protein-conducting channel of the Tic translocon (23Heins L. Mehrle A. Hemmler R. Wagner R. Kuchler M. Hormann F. Sveshnikov D. Soll J. EMBO J. 2002; 21: 2616-2625Crossref PubMed Scopus (111) Google Scholar). These conclusions were based on the observation that a urea-denatured fragment of psTic110 gave rise to ion channels when inserted into proteoliposomes. However, the relevance of the reconstitution data are unclear because native psTic110 does not form similar channels in reconstituted proteoliposomes, nor are similar channels observed in native chloroplast envelope membranes (23Heins L. Mehrle A. Hemmler R. Wagner R. Kuchler M. Hormann F. Sveshnikov D. Soll J. EMBO J. 2002; 21: 2616-2625Crossref PubMed Scopus (111) Google Scholar). To examine these three models, we have investigated the structure, topology, and function of Arabidopsis Tic110, atTic110. We demonstrate that the 97.5-kDa carboxyl-terminal region of atTic110 lacking only the two predicted transmembrane domains (atTic11093-996) exists as a soluble protein when expressed in Escherichia coli and when targeted to chloroplasts in vivo. Circular dichroism spectra indicate that this region is predominantly α-helical, consistent with secondary structure predictions. Furthermore, we demonstrate that native Tic110 purified from pea chloroplasts and recombinant atTic11093-996 bind selectively to chloroplast preproteins. The transit peptide-binding domain maps to a site on atTic11093-996 that is predicted to localize close to the stromal face of the Tic translocon. These data support the hypothesis that Tic110 acts as a scaffold to coordinate the stromal events of protein translocation into chloroplasts and are inconsistent with a role for Tic110 as a β-barrel channel. Construction of atTic110 Deletion Mutants and Arabidopsis Transformation—The pre-atTic110 cDNA was amplified from total Arabidopsis cDNA by reverse transcription-PCR using primers that introduced 5′ XhoI and BspHI sites spanning the start codon and a 3′ XbaI site following the stop codon. The cDNA was inserted into the XhoI and XbaI sites of pBluescript SK+ to generate pBS-pre-atTic110 and the NcoI and XbaI sites of pCAMBIA3300.1 to generate pCAMBIA-pre-atTic110. The mature atTic110 construct and the deletion construct atTic11093-966 were generated using PCR with primers that introduced XhoI and BspHI sites containing start codons immediately upstream from the amino-terminal codons of the constructs and XbaI sites following the stop codons of the open reading frames. A full-length atTic110 cDNA clone (pBS-pre-atTic110) or an identical clone encoding the addition of a carboxyl-terminal hexahistidine tag (pBS-atTic110-His) was used as the template. The PCR products were introduced into the XhoI and XbaI sites of pBlueScript SK+ to generate pBS-atTic110 and pBS-atTic11093-966. The pBluescript clones were digested with BspHI and NotI and subcloned into the NcoI and NotI sites of pET21d to generate pET21d-atTic110 and pET21d-atTic11093-966. The fragments of atTic110185-966 and atTic110370-966 were generated by PCR with primers that introduced XhoI sites and BspHI sites containing start codons immediately upstream from the amino-terminal codons of the constructs and NotI site following the stop codons of the open reading frames. The PCR products were directly digested with BspHI and NotI and subcloned into the NcoI and NotI sites of pET21d to generate pET21d-atTic110185-966 and pET21d-atTic110370-966. The pBS-atTic1101-602 construct encoding atTic1101-602 was generated by digestion of pBS-atTic110 with EcoRV and PstI, treatment with Pfu DNA polymerase to generate blunt ends, and self-ligation. The pBS-atTic1101-602 was then digested with BspHI and NotI and subcloned into the NcoI and NotI sites of pET21d to generate pET21d-atTic1101-602. The pCAMBIA-pre-atTic11093-966 construct encoding pre-atTic11093-966 was generated by digesting pCAMBIA-pre-atTic110 with NcoI and XbaI and replacing the atTic110 3′ fragment with the BspHI-XbaI fragment of pBS-atTic11093-966. All of the construts were confirmed by DNA sequencing. The pCAMBIA-pre-atTic11093-966 and pCAMBIA-pre-atTic110 constructs were introduced into Arabidopsis thaliana (ecotype Wassilewskija) via Agrobacterium tumefaciens-mediated transformation by the floral dip method (14Chen X. Smith M.D. Fitzpatrick L. Schnell D.J. Plant Cell. 2002; 14: 641-654Crossref PubMed Scopus (103) Google Scholar). Transformed plants were selected on BASTA, and the presence of the transgene was confirmed by PCR of genomic DNA using transgene-specific primers (data not shown). In Vitro Translation and Expression in E. coli—All of the 35S-labeled in vitro translation products were generated in a coupled transcription-translation system containing reticulocyte lysate according to the manufacturer's recommendations (Promega) using the pET21d constructs encoding the atTic110 constructs. For bacterial expression, pET21d-atTic11093-966-His, pET21d-atTic110185-966-His, and pET21d-atTic110370-966-His were transformed into E. coli BL21(DE3). Expression was induced with 0.2 mm isopropyl-1-thio-β-d-galactopyranoside overnight at 20 °C. The proteins were purified from soluble E. coli extracts of under nondenaturing conditions using nickel-NTA 1The abbreviations used are: NTA, nitrilotriacetic acid agarose; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DHFR, dihydrofolate reductase; PIRAC, protein import related anion channel; CRABP, cellular retinoic acid-binding protein; rubisco, ribulosebisphosphate carboxylase/oxygenase; APDP, N-[4-(p-azidosalicylamido)butyl]-3′(2-pyridyldithio)propionamide; SSU, small subunit of rubisco. resin (Novagen, Inc.). The proteins were stored in 40 mm Tris-HCl, pH 8.0, 1 m NaCl, 250 mm imidazole, and 0.02% NaN3 at +4 °C or -80 °C. Structural Analysis—CD spectra of proteins were measured in 50 mm sodium phosphate buffer, pH 8.0, at a concentration of 5 μm proteins on an AVIV 62DS CD spectrophotometer (Aviv Associates, Inc., Lakewood, NJ). The α-helical content was estimated from the mean residue ellipticity at 222 nm according to Ref. 24Pelton J.T. McLean L.R. Anal. Biochem. 2000; 277: 167-176Crossref PubMed Scopus (966) Google Scholar. The secondary structure was also predicted by the PSIPRED secondary structure prediction method (25Jones D.T. J. Mol. Biol. 1999; 292: 195-202Crossref PubMed Scopus (4467) Google Scholar). Arabidopsis Chloroplast Isolation and Immunoblotting—Wild type and transgenic Arabidopsis were grown on soil under 18-h day conditions. Chloroplasts were isolated from 3-week-old seedlings as described previously (26Smith M.D. Fitzpatrick L.M. Keegstra K. Schnell D.J. Bonifacino J.S. Harford J.B. Lippincott-Schwartz J. Yamada K.M. Current Protocols in Cell Biology. John Wiley & Sons, Inc., New York2002: 11.16.11-11.16.21Google Scholar) with the omission of bovine serum albumin from all buffers. Isolated intact chloroplasts were separated into membrane and soluble fractions by lysis in 0.1 m Na2CO3, pH 11.5, followed by centrifugation at 200,000 × g for 20 min. The pellets (membrane fraction) were directly dissolved in 350 mm Tris base, pH 11.0, 5% sodium dodecyl sulfate, 10% glycerol, and 80 mm dithiothreitol (SDS-PAGE sample buffer). The soluble proteins were recovered by precipitation with trichloroacetic acid and dissolved into SDS-PAGE sample buffer. Intact chloroplasts were treated with 20 μg/ml trypsin as described previously (11Kouranov A. Chen X. Fuks B. Schnell D.J. J. Cell Biol. 1998; 143: 991-1002Crossref PubMed Scopus (211) Google Scholar). The reisolated chloroplasts were analyzed by SDS-PAGE and immunoblotting. Total protein extracts from Arabidopsis were obtained by directly homogenizing leaves in SDS-PAGE sample buffer. For fractionation of total membrane and soluble proteins, Arabidopsis leaves were homogenized in 50 mm Hepes-KOH, pH 7.5, 0.45 m sucrose, 2 mm dithiothreitol, and 2 mm EDTA. The homogenate was filtered through two layers of Miracloth and centrifuged at 500 × g for 2 min. The supernatant was diluted to either 2 mm ice-cold EDTA or 0.1 m Na2CO3, pH 11.5, and separated into membrane and soluble fractions as described above and analyzed by SDS-PAGE and immunoblotting. All of the samples were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-atTic110, anti-atToc33, and anti-atToc75 serums as described previously (14Chen X. Smith M.D. Fitzpatrick L. Schnell D.J. Plant Cell. 2002; 14: 641-654Crossref PubMed Scopus (103) Google Scholar). Pea Chloroplast Isolation and Preprotein Cross-linking—Intact chloroplasts were isolated from 10-14-day-old pea seedlings (Pisum sativum var. Green Arrow) as previously described (11Kouranov A. Chen X. Fuks B. Schnell D.J. J. Cell Biol. 1998; 143: 991-1002Crossref PubMed Scopus (211) Google Scholar). The modification of pFd-protA with [125I]APDP and covalent cross-linking reactions with intact chloroplasts were performed as previously described (9Ma Y. Kouranov A. LaSala S. Schnell D.J. J. Cell Biol. 1996; 134: 315-327Crossref PubMed Scopus (140) Google Scholar, 27Kouranov A. Schnell D.J. J. Cell Biol. 1997; 139: 1677-1685Crossref PubMed Scopus (166) Google Scholar). The cross-linked products were analyzed on 7.5-15% SDS-PAGE gradient gels to resolve proteins in the 60-150-kDa range. Thermolysin treatment of intact chloroplasts was performed as described previously (8Schnell D.J. Kessler F. Blobel G. Science. 1994; 266: 1007-1012Crossref PubMed Scopus (329) Google Scholar) using 0.2 mg protease/ml for 30 min on ice. To prepare chloroplast envelope membranes, intact chloroplasts were lysed under hypertonic conditions and separated into soluble and membrane fractions by differential centrifugation as described by Keegstra and Yousif (28Keegstra K. Yousif A.E. Methods Enzymol. 1986; 118: 316-325Crossref Scopus (140) Google Scholar). The total membrane fraction was separated into envelope and thylakoid membrane fractions by flotation into linear sucrose gradients as previously described (8Schnell D.J. Kessler F. Blobel G. Science. 1994; 266: 1007-1012Crossref PubMed Scopus (329) Google Scholar). The envelope fractions were analyzed by SDS-PAGE. The radioactive signals in dried gels were captured and quantitated using a PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA) with the IPLab Gel Scientific Image Processing version 1.5c program (Signal Analytics, Vienna, VA). Immunoaffinity Chromatography and Immunoprecipitation Reactions—Envelope membranes corresponding to a mixed outer/inner membrane population were used for all immunoaffinity chromatography reactions. The envelope membranes were purified by the method described previously (11Kouranov A. Chen X. Fuks B. Schnell D.J. J. Cell Biol. 1998; 143: 991-1002Crossref PubMed Scopus (211) Google Scholar). For immunoaffinity chromatography under native conditions, the membranes (100 μg of protein) were solubilized in 50 mm Tricine-KOH, 2 mm EDTA, and 150 mm NaCl (TES buffer) with 1% (w/v) Triton X-100 for 10 min on ice. The extract was clarified by centrifugation at 100,000 × g for 30 min to remove insoluble aggregates. The supernatant was diluted in half and incubated in the presence of 2 mm MgCl2 and 1 mm ATP at 26 °C for 5 min. The sample was applied sequentially to IgG-Sepharose of anti-psToc34 IgG-Sepharose, anti-psToc86 IgG-Sepharose, and anti-psToc110 IgG-Sepharose (1 ml of packed matrix containing 5 mg of bound IgG). The Sepharose was washed with 10 volumes of TES buffer containing 0.2% (w/v) Triton X-100 and eluted with 0.2 m glycine, pH 2.2, containing 0.2% (w/v) decyl maltoside. The elutes and unbound fractions were analyzed by SDS-PAGE and immunoblotting. The binding of [35S]pre-SSU and [35S]SSU to immunoadsorbed psTic110 was performed with anti-psTic110 IgG-Sepharose (0.5 ml of packed matrix containing 2.5 mg of bound IgG) containing bound psTic110 from envelope membranes (50 μg of protein) that had been subjected to the sequential immunoprecipitation as given above. The [35S]pre-SSU and [35S]SSU translation products were diluted 40-fold with 50 mm Hepes-KOH, pH 7.5, 50 mm KOAc, 4 mm MgCl2, 0.1% Triton X-100 (wash buffer) and incubated with the anti-psTic110 IgG-Sepharose containing bound psTic110 for 30 min at 4 °C. The resin was washed five times with an excess of wash buffer, and bound protein was eluted with 0.2 m glycine, pH 2.2, 0.1% Triton X-100. The elutes were analyzed by SDS-PAGE and phosphorimaging. Immunoprecipitation of psTic110 from chloroplast envelope membranes under denaturing conditions following covalent cross-linking was performed by the method of Anderson and Blobel (29Anderson D.J. Blobel G. Methods Enzymol. 1983; 96: 111-120Crossref PubMed Scopus (208) Google Scholar). Before immunoprecipitation, the envelope membranes (100 μg of protein) were reduced with β-mercaptoethanol to cleave the cross-linker as described previously (9Ma Y. Kouranov A. LaSala S. Schnell D.J. J. Cell Biol. 1996; 134: 315-327Crossref PubMed Scopus (140) Google Scholar). atTic110 Binding Assays—pFd-protA, Fd-protA, pSSU-DHFR, and DHFR were expressed in E. coli and purified on nickel-NTA resin as described previously (27Kouranov A. Schnell D.J. J. Cell Biol. 1997; 139: 1677-1685Crossref PubMed Scopus (166) Google Scholar). Purified pFd-protA and Fd-protA (30 pmol each), or pSSU-DHFR and DHFR (250 pmol each) were diluted in 20 mm Hepes-KOH, pH 7.5, 200 mm NaCl, and 0.1% Triton X-100 (binding buffer) to give a final concentration of 40 mm imidazole. The samples were bound to 8 μl of packed nickel-NTA resin at room temperature for 30 min under constant mixing. The resin was washed once with 400 μl of binding buffer containing 40 mm imidazole. The resin was incubated with in vitro translated [35S]atTic110 or the [35S]atTic110 deletion mutants in binding buffer containing 40 mm imidazole for 1 h at room temperature under constant mixing. The resin was washed three times with 400 μl of ice-cold binding buffer containing 40 mm imidazole. The bound proteins were eluted from the resin with SDS-PAGE sample buffer containing 1 m imidazole and resolved by SDS-PAGE. For the competition assays, purified pFd-protA (30pmol) was diluted in 20 mm Tris-HCl, pH 7.5, 150 mm NaCl (TBS buffer), and the samples were bound to 8 μl of packed IgG-Sepharose at room temperature for 1 h. The IgG-Sepharose was washed once with 400 μl of TBS buffer. The IgG-Sepharose was incubated with purified competitor (atTic11093-966, atTic110185-966, atTic110370-966, or CRABP) for 30 min at room temperature under constant mixing. After preincubation with competitor, in vitro translated [35S]atTic110 or [35S]atTic1101-602 was added and incubated for 1 h at room temperature under constant mixing. The resin was washed three times with 400 μl of ice-cold TBS buffer. The proteins were eluted from IgG-Sepharose with incubation in 0.2 m glycine, pH 2.2, for 15 min at room temperature under constant mixing. The eluates were resolved directly by SDS-PAGE. All of the samples were analyzed on a Storm 840 PhosphorImager using ImageQuant 1.2 software (Molecular Dynamics). The quantitative binding data are presented as percentages of maximal binding observed with each translation product. Structural and Topological Analyses of Recombinant atTic110 —As a first step in our analysis of atTic110 function, we examined the structural features of the native protein. The primary structure of atTic110 contains two distinguishing features, a short amino-terminal region (residues 1-93) containing two α-helical transmembrane segments and a 97.5-kDa carboxyl terminus that is largely hydrophilic in nature (Fig. 1). We generated a series of truncated atTic110 constructs lacking only the two transmembrane domains (atTic11093-966) or the transmembrane domains and additional portions of the amino terminus (atTic110185-966 and atTic110370-966) (Fig. 1). All of the constructs were successfully expressed and purified as soluble proteins in E. coli (Fig. 2A) with only a minor fraction of each (∼10-15%) found in insoluble inclusion bodies (data not shown). The yields ranged from 5 to 10 mg/liter of E. coli culture. The purified fractions were stable to freeze-thaw cycles at concentration up to 5 mg/ml. These data indicate that the proteins are expressed in their native conformations and establish that the 97.5-kDa carboxyl-terminal region folds into a stable, soluble domain.Fig. 2Expression of atTic110 deletion mutants in E. coli and their circular dichroism spectra. A, SDS-PAGE profile of the indicated recombinant atTic110 deletion mutants purified from soluble E. coli extracts by nickel-NTA chromatography. The molecular sizes of standard proteins (lane 1) are indicated to the left of the figure. The polypeptides were visualized by staining with Coomassie blue. B-D, circular dichroism spectra of atTic11093-966 (B), atTic110185-966 (C), and atTic110370-966 (D). Note that the negative bands at 208 and 222 nm are typically observed in proteins with substantial α-helical structure.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A variety of secondary structure predictions of both atTic110 and psTic110 suggest that the carboxyl-terminal region contains predominantly α-helical content. We examined the secondary structure of recombinant atTic11093-966 by CD spectroscopy. The recombinant protein exhibited two strong negative bands at 208 and 222 nm that are typically observed in proteins with substantial α-helical content (Fig. 2B). The protein is predicted to contain at least 60-80% α-helices based on the θ222 value of -29,088 (24Pelton J.T. McLean L.R. Anal. Biochem. 2000; 277: 167-176Crossref PubMed Scopus (966) Google Scholar). The CD spectra of atTic110185-966 and atTic110370-966 give slightly less negative θ222 values, yet both are predicted to contain at least 50% α-helical content (Fig. 2, C and D). These results indicate that the bulk of native atTic110 consists of α-helical structure. To further examine whether atTic11093-966 was soluble or membrane-integrated, we generated transgenic Arabidopsis expressing either pre-atTic110, the authentic precursor to atTic110, or pre-atTic11093-966, a construct corresponding to atTic11093-966 fused to the atTic110 transit peptide (Fig. 1). The expression of both proteins was confirmed by immunoblotting of total tissue extracts using anti-atTic110 antibodies. The results from representative transformants for each construct are shown in Fig. 3A. As predicted, the pre-atTic110 transformant expressed elevated levels of atTic110 compared with wild type (Fig. 3A, compare lanes 1 and 2), whereas the pre-atTic11093-966 transformant contained an additional 99.6-kDa band not observed in wild type plants (Fig. 3A, lane 3, arrow). The 99.6-kDa polypeptide corresponds to the hexahistidine-tagged atTic11093-966 after processing to remove the transit peptide by the stromal processing peptidase. Separation of the soluble and membrane fractions by lysis in buffer at neutral or alkaline pH demonstrated that authentic atTic110 was exclusively membrane-associated (Fig. 3B, compare lanes 1 and 2 and lanes 3 and 4). In contrast, atTic11093-966 was observed exclusively in the soluble fraction under either lysis condition (Fig. 3B, lanes 6 and 8, arrow). To confirm that pre-atTic11093-966 was targeted to chloroplasts, we isolated chloroplasts from wild type and atTic11093-966 transgenic plants. The relative proportion of authentic atTic110 and atTic11093-966 was similar to those observed in the total plant extracts (Fig. 3, A and C), indicating that pre-atTic11093-966 was imported into chloroplasts. Separation of chloroplasts into soluble and membrane fractions indicated that atTic110 was almost exclusively found in the membrane fraction (Fig. 3C, compare lanes 2 and 3). However, atTic11093-966 appeared only in the soluble fraction (Fig. 3C, compare lanes 5 and 6), indicating that the protein localized to the chloroplast stroma. The stromal lo