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• W2006551808 abstract "Tic40 is a component of the protein import apparatus of the inner envelope of chloroplasts, but its role in the import mechanism has not been clearly defined. The C terminus of Tic40 shares weak similarity with the C-terminal Sti1 domains of the mammalian Hsp70-interacting protein (Hip) and Hsp70/Hsp90-organizing protein (Hop) co-chaperones. Additionally, Tic40 may possess a tetratricopeptide repeat (TPR) protein-protein interaction domain, another characteristic feature of Hip/Hop co-chaperones. To investigate the functional importance of different parts of the Tic40 protein and to determine whether the homology between Tic40 and co-chaperones is functionally significant, different Tic40 deletion and Tic40:Hip fusion constructs were generated and assessed for complementation activity in the Arabidopsis Tic40 knock-out mutant, tic40. Interestingly, all Tic40 deletion constructs failed to complement tic40, indicating that each part removed is essential for Tic40 function; these included a construct lacking the Sti1-like domain (ΔSti1), a second lacking a central region, including the putative TPR domain (ΔTPR), and a third lacking the predicted transmembrane anchor region. Moreover, the ΔSti1 and ΔTPR constructs caused strong dominant-negative, albino phenotypes in tic40 transformants, indicating that the truncated Tic40 proteins interfere with the residual chloroplast protein import that occurs in tic40 plants. Remarkably, the Tic40:Hip fusion constructs showed that the Sti1 domain of human Hip is functionally equivalent to the Sti1-like region of Tic40, strongly suggesting a co-chaperone role for the Tic40 protein. Supporting this notion, yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated the in vivo interaction of Tic40 with Tic110, a protein believed to recruit stromal chaperones to protein import sites. Tic40 is a component of the protein import apparatus of the inner envelope of chloroplasts, but its role in the import mechanism has not been clearly defined. The C terminus of Tic40 shares weak similarity with the C-terminal Sti1 domains of the mammalian Hsp70-interacting protein (Hip) and Hsp70/Hsp90-organizing protein (Hop) co-chaperones. Additionally, Tic40 may possess a tetratricopeptide repeat (TPR) protein-protein interaction domain, another characteristic feature of Hip/Hop co-chaperones. To investigate the functional importance of different parts of the Tic40 protein and to determine whether the homology between Tic40 and co-chaperones is functionally significant, different Tic40 deletion and Tic40:Hip fusion constructs were generated and assessed for complementation activity in the Arabidopsis Tic40 knock-out mutant, tic40. Interestingly, all Tic40 deletion constructs failed to complement tic40, indicating that each part removed is essential for Tic40 function; these included a construct lacking the Sti1-like domain (ΔSti1), a second lacking a central region, including the putative TPR domain (ΔTPR), and a third lacking the predicted transmembrane anchor region. Moreover, the ΔSti1 and ΔTPR constructs caused strong dominant-negative, albino phenotypes in tic40 transformants, indicating that the truncated Tic40 proteins interfere with the residual chloroplast protein import that occurs in tic40 plants. Remarkably, the Tic40:Hip fusion constructs showed that the Sti1 domain of human Hip is functionally equivalent to the Sti1-like region of Tic40, strongly suggesting a co-chaperone role for the Tic40 protein. Supporting this notion, yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated the in vivo interaction of Tic40 with Tic110, a protein believed to recruit stromal chaperones to protein import sites. Most chloroplast proteins are nucleus-encoded, synthesized in precursor form (each one with a cleavable transit peptide), and post-translationally imported into the organelle (1Bédard J. Jarvis P. J. Exp. Bot. 2005; 56: 2287-2320Crossref PubMed Scopus (86) Google Scholar, 2Kessler F. Schnell D.J. Traffic. 2006; 7: 248-257Crossref PubMed Scopus (106) Google Scholar, 3Reumann S. Inoue K. Keegstra K. Mol. Membr. Biol. 2005; 22: 73-86Crossref PubMed Scopus (109) Google Scholar, 4Soll J. Schleiff E. Nat. Rev. Mol. Cell Biol. 2004; 5: 198-208Crossref PubMed Scopus (334) Google Scholar). Import is mediated by translocon complexes in the outer and inner envelope membranes, termed TOC 2The abbreviations used are: TOC, translocon at the outer envelope membrane of chloroplasts; TIC, translocon at the inner envelope membrane of chloroplasts; BiFC, bimolecular fluorescence complementation; Hip, Hsp70-interacting protein; Hop, Hsp70/Hsp90-organizing protein; TPR, tetratricopeptide repeat; CaMV, cauliflower mosaic virus; GBD, Gal4 DNA-binding domain; GAD, Gal4 activation domain; YFP, yellow fluorescent protein; mtHsp70, matrix Hsp70; AtHip, A. thaliana Hip; hs, H. sapiens; ps, Pisum sativum; at, A. thaliana; rn, R. norvegicus. and TIC, respectively. Once translocation through the TOC is initiated, this complex associates with the TIC allowing preprotein transport across both membranes simultaneously. Several components of the TIC complex have been identified, including Tic40 and Tic110, but the functions of many of these proteins remain unclear (1Bédard J. Jarvis P. J. Exp. Bot. 2005; 56: 2287-2320Crossref PubMed Scopus (86) Google Scholar, 2Kessler F. Schnell D.J. Traffic. 2006; 7: 248-257Crossref PubMed Scopus (106) Google Scholar, 3Reumann S. Inoue K. Keegstra K. Mol. Membr. Biol. 2005; 22: 73-86Crossref PubMed Scopus (109) Google Scholar, 4Soll J. Schleiff E. Nat. Rev. Mol. Cell Biol. 2004; 5: 198-208Crossref PubMed Scopus (334) Google Scholar). The TIC complex also recruits stromal chaperones, which are thought to drive protein import and mediate the folding of newly imported proteins (5Kessler F. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7684-7689Crossref PubMed Scopus (169) Google Scholar, 6Akita M. Nielsen E. Keegstra K. J. Cell Biol. 1997; 136: 983-994Crossref PubMed Scopus (168) Google Scholar, 7Nielsen E. Akita M. Davila-Aponte J. Keegstra K. EMBO J. 1997; 16: 935-946Crossref PubMed Scopus (233) Google Scholar, 8Kouranov A. Chen X. Fuks B. Schnell D.J. J. Cell Biol. 1998; 143: 991-1002Crossref PubMed Scopus (213) Google Scholar). Tic40 was identified by its association with preproteins undergoing import (9Ko K. Budd D. Wu C. Seibert F. Kourtz L. Ko Z.W. J. Biol. Chem. 1995; 270: 28601-28608Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 10Wu C. Seibert F.S. Ko K. J. Biol. Chem. 1994; 269: 32264-32271Abstract Full Text PDF PubMed Google Scholar) and is located in the inner envelope membrane in close association with Tic110 (11Stahl T. Glockmann C. Soll J. Heins L. J. Biol. Chem. 1999; 274: 37467-37472Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Like Tic110, the bulk of Tic40 protrudes into the stroma where it may interact with stromal chaperones (12Chou M.L. Fitzpatrick L.M. Tu S.L. Budziszewski G. Potter-Lewis S. Akita M. Levin J.Z. Keegstra K. Li H.M. EMBO J. 2003; 22: 2970-2980Crossref PubMed Scopus (158) Google Scholar, 13Kovacheva S. Bédard J. Patel R. Dudley P. Twell D. Riíos G. Koncz C. Jarvis P. Plant J. 2005; 41: 412-428Crossref PubMed Scopus (158) Google Scholar). Interestingly, Tic40 shares homology with eukaryotic Hip and Hop co-chaperones (11Stahl T. Glockmann C. Soll J. Heins L. J. Biol. Chem. 1999; 274: 37467-37472Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 12Chou M.L. Fitzpatrick L.M. Tu S.L. Budziszewski G. Potter-Lewis S. Akita M. Levin J.Z. Keegstra K. Li H.M. EMBO J. 2003; 22: 2970-2980Crossref PubMed Scopus (158) Google Scholar). Weak sequence similarity between Tic40 and Hip/Hop is restricted to ∼60 C-terminal residues, which in the co-chaperones constitute a conserved region termed the Sti1 domain (Sti1 is the yeast homolog of Hop). However, in silico and immunological studies have revealed that the C-proximal region of Tic40 immediately upstream of the putative Sti1 domain likely contains a TPR domain, which is another characteristic feature of Hip/Hop co-chaperones (12Chou M.L. Fitzpatrick L.M. Tu S.L. Budziszewski G. Potter-Lewis S. Akita M. Levin J.Z. Keegstra K. Li H.M. EMBO J. 2003; 22: 2970-2980Crossref PubMed Scopus (158) Google Scholar). Hip interacts with Hsp70 and regulates its ATPase cycle by stabilizing the ADP-bound, high substrate affinity form of the chaperone (14Hoöhfeld J. Minami Y. Hartl F.U. Cell. 1995; 83: 589-598Abstract Full Text PDF PubMed Scopus (380) Google Scholar). In addition, Hip possesses intrinsic chaperone activity, binding specifically to unfolded proteins and preventing their aggregation (14Hoöhfeld J. Minami Y. Hartl F.U. Cell. 1995; 83: 589-598Abstract Full Text PDF PubMed Scopus (380) Google Scholar, 15Bruce B.D. Churchich J. Eur. J. Biochem. 1997; 245: 738-744Crossref PubMed Scopus (13) Google Scholar). Hop interacts with both Hsp70 and Hsp90 to mediate their association (16Chen S. Smith D.F. J. Biol. Chem. 1998; 273: 35194-35200Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 17Johnson B.D. Schumacher R.J. Ross E.D. Toft D.O. J. Biol. Chem. 1998; 273: 3679-3686Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar) but, unlike Hip, does not seem to function as a chaperone itself (18Bose S. Weikl T. Bugl H. Buchner J. Science. 1996; 274: 1715-1717Crossref PubMed Scopus (319) Google Scholar, 19Freeman B.C. Toft D.O. Morimoto R.I. Science. 1996; 274: 1718-1720Crossref PubMed Scopus (289) Google Scholar). Together, Hip and Hop facilitate the transfer of some Hsp70-bound protein substrates (e.g. steroid hormone receptors) to Hsp90, in order for them to undergo further folding and reach their final conformation (20Frydman J. Hoöhfeld J. Trends Biochem. Sci. 1997; 22: 87-92Abstract Full Text PDF PubMed Scopus (257) Google Scholar). In addition to Tic40, the Arabidopsis genome encodes two Hip homologs, AtHip-1 and AtHip-2 (21Webb M.A. Cavaletto J.M. Klanrit P. Thompson G.A. Cell Stress Chaperones. 2001; 6: 247-255Crossref PubMed Google Scholar). The former is structurally similar to mammalian Hip along its entire length, and so it is likely to function as a canonical Hip co-chaperone. The latter possesses a truncated Hip-like region and a thioredoxin domain, suggesting an involvement in redox regulation. Neither are closely related to Tic40. The homology between Tic40 and Hip/Hop co-chaperones suggests that Tic40 may play similar (co)chaperone roles during chloroplast import. In mitochondrial protein import (which is functionally similar to chloroplast import), matrix Hsp70 (mtHsp70) is the key component of the presequence translocase-associated motor, the complex that drives preprotein translocation (22Wiedemann N. Frazier A.E. Pfanner N. J. Biol. Chem. 2004; 279: 14473-14476Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 23Neupert W. Brunner M. Nat. Rev. Mol. Cell Biol. 2002; 3: 555-565Crossref PubMed Scopus (301) Google Scholar). In addition to mtHsp70, the presequence translocase-associated motor contains Tim44, which recruits mtHsp70 to the import site, and a range of co-chaperones that regulate mtHsp70 activity (22Wiedemann N. Frazier A.E. Pfanner N. J. Biol. Chem. 2004; 279: 14473-14476Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). In chloroplasts, Hsp70 is not associated functionally with the TIC (6Akita M. Nielsen E. Keegstra K. J. Cell Biol. 1997; 136: 983-994Crossref PubMed Scopus (168) Google Scholar, 7Nielsen E. Akita M. Davila-Aponte J. Keegstra K. EMBO J. 1997; 16: 935-946Crossref PubMed Scopus (233) Google Scholar, 8Kouranov A. Chen X. Fuks B. Schnell D.J. J. Cell Biol. 1998; 143: 991-1002Crossref PubMed Scopus (213) Google Scholar). Instead, the Hsp100 homolog, Hsp93, associates with the translocon in an ATP-dependent fashion and is thought to be functionally analogous to mtHsp70 (6Akita M. Nielsen E. Keegstra K. J. Cell Biol. 1997; 136: 983-994Crossref PubMed Scopus (168) Google Scholar, 7Nielsen E. Akita M. Davila-Aponte J. Keegstra K. EMBO J. 1997; 16: 935-946Crossref PubMed Scopus (233) Google Scholar, 8Kouranov A. Chen X. Fuks B. Schnell D.J. J. Cell Biol. 1998; 143: 991-1002Crossref PubMed Scopus (213) Google Scholar). Tic110 has been proposed to recruit Hsp93 to the stromal face of the TIC (cf. Tim44 in mitochondria) and to provide an initial binding site for preproteins as they emerge into the stroma (6Akita M. Nielsen E. Keegstra K. J. Cell Biol. 1997; 136: 983-994Crossref PubMed Scopus (168) Google Scholar, 7Nielsen E. Akita M. Davila-Aponte J. Keegstra K. EMBO J. 1997; 16: 935-946Crossref PubMed Scopus (233) Google Scholar, 8Kouranov A. Chen X. Fuks B. Schnell D.J. J. Cell Biol. 1998; 143: 991-1002Crossref PubMed Scopus (213) Google Scholar, 24Inaba T. Li M. Alvarez-Huerta M. Kessler F. Schnell D.J. J. Biol. Chem. 2003; 278: 38617-38627Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). More recently, Tic40 was reported to mediate the regulation of Hsp93 activity and the transferral of translocating preproteins between other components of the TIC complex (25Chou M.L. Chu C.C. Chen L.J. Akita M. Li H.M. J. Cell Biol. 2006; 175: 893-900Crossref PubMed Scopus (92) Google Scholar). The former role is supported by the fact that yeast Hop (Sti1) is able to interact with Hsp104, another Hsp100 chaperone (26Abbas-Terki T. Donzé O. Briand P.A. Picard D. Mol. Cell. Biol. 2001; 21: 7569-7575Crossref PubMed Scopus (81) Google Scholar). Interestingly, Tic110, Hsp93, and Tic40 were found to associate with a translocating preprotein at a similar late stage of import (12Chou M.L. Fitzpatrick L.M. Tu S.L. Budziszewski G. Potter-Lewis S. Akita M. Levin J.Z. Keegstra K. Li H.M. EMBO J. 2003; 22: 2970-2980Crossref PubMed Scopus (158) Google Scholar). Furthermore, the Arabidopsis knock-out mutations, tic110, hsp93, and tic40, interact genetically, supporting the notion that the proteins cooperate functionally in vivo (13Kovacheva S. Bédard J. Patel R. Dudley P. Twell D. Riíos G. Koncz C. Jarvis P. Plant J. 2005; 41: 412-428Crossref PubMed Scopus (158) Google Scholar). To further investigate the hypothesis that Arabidopsis Tic40 (atTic40) plays (co)chaperone roles during import, we assessed its functional similarity with human Hip; domain-swap constructs were generated and tested for their ability to complement the tic40 knock-out mutation in Arabidopsis plants. Additionally, to determine the importance of different domains of Tic40, a series of atTIC40 deletion constructs were used in similar tic40 complementation studies. Plant Materials and Growth—All Arabidopsis thaliana plants were of the Columbia-0 ecotype. The tic40-4 mutant used has been described previously (13Kovacheva S. Bédard J. Patel R. Dudley P. Twell D. Riíos G. Koncz C. Jarvis P. Plant J. 2005; 41: 412-428Crossref PubMed Scopus (158) Google Scholar). Plants were grown on Murashige-Skoog medium using published procedures (13Kovacheva S. Bédard J. Patel R. Dudley P. Twell D. Riíos G. Koncz C. Jarvis P. Plant J. 2005; 41: 412-428Crossref PubMed Scopus (158) Google Scholar, 27Aronsson H. Jarvis P. FEBS Lett. 2002; 529: 215-220Crossref PubMed Scopus (163) Google Scholar). Constructs were stably introduced into tic40-4 plants using the floral dip method (28Clough S.J. Bent A.F. Plant J. 1998; 16: 735-743Crossref PubMed Google Scholar). Complementation Construct Generation—cDNA fragments for construct assembly were amplified using Proof Start polymerase (Qiagen, Crawley, UK). The atTic40 template for amplification was cDNA 144K24T7 (accession T76608), whereas the hsHip template included the full coding sequence between the NcoI and KpnI sites of pSPUTK (Stratagene, La Jolla, CA). Constructs were assembled from PCR fragments and cDNA restriction fragments downstream of the atTIC40 promoter in pSP72 (Promega, Madison, WI). Promoter-construct fusions were transferred to a pBluescript II KS (Stratagene) derivative containing the cauliflower mosaic virus (CaMV) 35S terminator. These cassettes were transferred to the pBin19 T-DNA vector for Agrobacterium and plant transformation. Because the atTIC40 promoter used in these constructs failed to drive robust expression, it was replaced by the CaMV 35S promoter. A detailed account of these cloning procedures is provided as Supplemental Material. Characterization of Transgenic Lines—Chlorophyll quantification, immunoblotting, reverse transcription-PCR, electron microscopy, chloroplast isolation, and protein import assays were all conducted using standard procedures, as described previously (13Kovacheva S. Bédard J. Patel R. Dudley P. Twell D. Riíos G. Koncz C. Jarvis P. Plant J. 2005; 41: 412-428Crossref PubMed Scopus (158) Google Scholar, 27Aronsson H. Jarvis P. FEBS Lett. 2002; 529: 215-220Crossref PubMed Scopus (163) Google Scholar). Yeast Two-hybrid Analysis—Assays were done using the Matchmaker GAL4 Two-hybrid System 3 (Clontech). Gal4 DNA-binding domain (GBD) fusions were made in the pGBKT7 vector, and Gal4 activation domain (GAD) fusions were made in the pGADT7 vector. Sequence encoding the soluble domain of atTic40 (Tic40ΔN; residues 129-447 of the precursor) was cloned from cDNA 144K24T7 as a DraI-BamHI fragment into SmaI-BamHI-cut pGBKT7 or pGADT7; note that the pGBKT7-Tic40 clone was unusable for interaction analysis, because it caused strong auto-activation of the HIS3 histidine reporter. Sequence encoding the soluble domain of atTic110 (Tic110ΔN; residues 144-1016 of the precursor) was amplified from cDNA RAFL09-95-B13 (accession AY099850) using the following primers: Tic110 SmaI forward, 5′-acc cgg gtg tac cgg agg tag ctg-3′; Tic110 XhoI reverse, 5′-act cga gga ttt aaa aga cga aat tgc c-3′. After sequencing, the SmaI-XhoI-cut Tic110 fragment was cloned into SmaI-XhoI-cut pGADT7 and into SmaI-SalI-cut pGBKT7. The constructs were co-transformed into yeast strain AH109 for the plate growth assays. The control clones, pGBKT7-p53 (pGBKT7-53) and pGADT7-SV40T (pGADT7-T), were supplied by the manufacturer (Clontech). Quantitative growth assays were conducted using yeast strain HF7c, essentially as described previously (29Maple J. Aldridge C. Møller S.G. Plant J. 2005; 43: 811-823Crossref PubMed Scopus (114) Google Scholar) except that cultures were grown to an A600 of 0.5 prior to spotting. The difference in growth on synthetic dropout (SD) medium lacking histidine, tryptophan, and leucine (SD-HTL) versus SD medium lacking tryptophan and leucine (SD-TL) was measured after 1 day only. Fluorescence Microscopy—Full-length coding sequences for atTic40 and atTic110, as well as the atTic40 ΔTM deletion mutant (all three lacking stop codons), were amplified by PCR using cDNA templates described above and the ΔTM binary construct with the following primers: Tic40 XhoI forward, 5′-act cga gat atg gag aac ctt acc cta g-3′; Tic40 KpnI reverse, 5′-agg tac ccg tca ttc ctg gga aga gct g-3′; Tic110 XhoI forward, 5′-tct cga gac cat gaa tcc ctc act c-3′; and Tic110 KpnI reverse, 5′-tgg tac caa aga cga aat tgc cct c-3′. To generate full-length YFP fusions, all three sequences were cloned as XhoI-KpnI fragments into pWEN18 (30Kost B. Spielhofer P. Chua N.H. Plant J. 1998; 16: 393-401Crossref PubMed Google Scholar). To generate half-YFP fusions for the bimolecular fluorescence complementation (BiFC) assays, the two full-length sequences were cloned the same way into pWEN-NY and pWEN-CY (29Maple J. Aldridge C. Møller S.G. Plant J. 2005; 43: 811-823Crossref PubMed Scopus (114) Google Scholar). Protoplasts were prepared from 14-day-old, wild-type Arabidopsis seedlings essentially as described previously (31Jin J.B. Kim Y.A. Kim S.J. Lee S.H. Kim D.H. Cheong G.W. Hwang I. Plant Cell. 2001; 13: 1511-1526Crossref PubMed Scopus (304) Google Scholar). Seedlings were chopped with a razor blade in solution containing 2% cellulose and 0.08% macerozyme and incubated for 3.5 h. For transfection, 5 μg of plasmid DNA (per clone) was used with 100 μl of protoplast suspension (2 × 106 per ml) and 110 μl of 40% PEG4000 solution. Fluorescence analysis was performed using a Nikon Eclipse TE-2000E inverted fluorescence microscope equipped with filters for analyzing YFP (exciter HQ500/20x, emitter HQ535/30m) and chlorophyll autofluorescence (exciter D480/30x, emitter D660/50m) (Chroma Technologies, Rockingham, VT). For BiFC analysis, samples were analyzed 16-24 h after (co)transfection. The pWEN-NY-Tic110 with pWEN-CY-Tic40 combination was repeated three times and with relevant controls; the empty vectors (pWEN-NY and pWEN-CY) and the pWEN-NY-Tic110 with pWEN-CY-Tic40 clones did not produce detectable fluorescence when transformed alone (data not shown; supplemental Fig. S5). atTic40 Contains Several Conserved Domains—To identify regions of Tic40 that may be important for function, Tic40 sequences from different plant species were aligned (supplemental Fig. S1). The degree of conservation varied considerably along the length of the alignment. Distinct, highly conserved regions corresponding to the transit peptide cleavage site and the predicted transmembrane domain (11Stahl T. Glockmann C. Soll J. Heins L. J. Biol. Chem. 1999; 274: 37467-37472Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) were detected. In addition, three conserved motifs were found in the central part, upstream of a highly conserved C-terminal region. Very high conservation in the C-terminal ∼150 residues suggested that this region may contain one or more important functional domains. Interestingly, the C terminus of atTic40 was found to display weak similarity to the C termini of eukaryotic Hip and Hop co-chaperones, as reported previously (11Stahl T. Glockmann C. Soll J. Heins L. J. Biol. Chem. 1999; 274: 37467-37472Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 12Chou M.L. Fitzpatrick L.M. Tu S.L. Budziszewski G. Potter-Lewis S. Akita M. Levin J.Z. Keegstra K. Li H.M. EMBO J. 2003; 22: 2970-2980Crossref PubMed Scopus (158) Google Scholar). However, only an ∼60 residue C-terminal region of atTic40 shows clear homology to the Hip/Hop proteins (Fig. 1A); previous comparisons of Hip and Hop led to the detection of this conserved domain, termed the Sti1 domain (14Hoöhfeld J. Minami Y. Hartl F.U. Cell. 1995; 83: 589-598Abstract Full Text PDF PubMed Scopus (380) Google Scholar). The human Hip (hsHip) Sti1 domain shares low but significant similarity with the human Hop (hsHop) Sti1 domain, equivalent to ∼25% identity across 60 residues. As shown in Fig. 1B, the atTic40 C terminus is more similar to the Sti1 domains of mammalian Hip proteins (35% identity) than to those of mammalian Hop proteins (19% identity). This homology with the Hip/Hop Sti1 domains suggests that this region of atTic40 may constitute a conserved Sti1 domain. Moreover, the fact that it resides at the extreme C terminus suggests that it may perform a similar function. Deletion Mutants of the atTic40 Protein—To investigate the functional importance of different parts of the Tic40 protein, three deletion constructs were made from a full-length atTIC40 cDNA (Fig. 1C): ΔTM lacked codons 95-136, including the predicted transmembrane domain; ΔSti1 lacked codons 391-447 and contained a premature in-frame stop codon (introduction of these mutations also generated an I390L missense mutation); and ΔTPR lacked the entire region between the predicted transmembrane and Sti1 domains (codons 139-389), including the C-proximal region proposed to constitute a TPR domain (an in-frame fusion between residues 138 and 390 was generated, as well as an A137V missense mutation). The deletion constructs (and the full-length atTIC40 cDNA, termed WT40) were inserted into a T-DNA vector carrying the CaMV 35S promoter and terminator sequences and used to transform tic40 knock-out plants. Multiple (≥19) transformants (T1 plants) were identified for each of the four constructs. Single insertion lines (segregating three resistant T2 plants for every sensitive T2 plant) were further propagated to identify homozygous lines. These were subjected to PCR genotyping, to confirm homozygosity of the tic40 mutation and the presence of the appropriate atTic40 transgene (supplemental Fig. S2), before further analysis. As expected, the positive control construct, WT40, complemented tic40 fully, restoring wild-type-like appearance and growth in most transformants (Fig. 2A). By contrast, none of the atTic40 deletion constructs gave clear tic40 complementation (Fig. 2A). A total of 24, 34, and 38 transformants were identified for ΔTM, ΔSti1, and ΔTPR, respectively. All ΔTM transformants displayed a tic40-like phenotype (Fig. 2A). Interestingly, identification of ΔTPR transformants (and, to a lesser extent, ΔSti1 transformants) was more difficult, because many plants displayed a more severe phenotype than the tic40 mutant: i.e. seedlings with very small, albino cotyledons. Most ΔTPR transformants (23 of 38) produced T2 progenies containing seedlings with albino cotyledons, whereas only three of the ΔSti1 transformants expressed the albino phenotype (Fig. 2A). The effects of the various deletion constructs were quantified by measuring chlorophyll concentrations in several representative lines (Fig. 2B). As expected, chlorophyll levels in WT40 plants were comparable with those in the wild type, and those in tic40-like ΔTM and ΔSti1 transformants were comparable with those in tic40. By contrast, albino ΔTPR seedlings contained drastically reduced levels of chlorophyll (∼11% of the amount in tic40). atTic40 Deletion Mutants Are Expressed and Targeted to Chloroplasts—Failure of the deletion constructs to complement tic40 suggested that the deleted proteins were nonfunctional or that they were not expressed and/or targeted to chloroplasts properly. To eliminate the latter possibilities, we assessed accumulation of the proteins by immunoblotting, using an antibody raised against the pea Tic40 (psTic40) C terminus (residues 128-436) (11Stahl T. Glockmann C. Soll J. Heins L. J. Biol. Chem. 1999; 274: 37467-37472Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). As expected, WT40 control plants overexpressed a Tic40 protein of the same size (∼44 kDa) as that in wild type (Fig. 3A); that the apparent size of mature atTic40 is larger than predicted (41 kDa) is consistent with previous observations and may be due to its high proline content (12Chou M.L. Fitzpatrick L.M. Tu S.L. Budziszewski G. Potter-Lewis S. Akita M. Levin J.Z. Keegstra K. Li H.M. EMBO J. 2003; 22: 2970-2980Crossref PubMed Scopus (158) Google Scholar). This protein could also be detected in isolated chloroplasts, where it was resistant to exogenously applied thermolysin protease, demonstrating proper targeting to the organelle (Fig. 3B). Expression products were also detected in ΔTM, ΔSti1, and ΔTPR plants (Fig. 3A). In the ΔTM lines, two faint bands were detected: one at ∼43 kDa and another more diffuse band at ∼36 kDa (Fig. 3A). It was recently reported that the atTic40 precursor is subjected to two distinct proteolytic processing events during its targeting to the inner envelope membrane (32Li M. Schnell D.J. J. Cell Biol. 2006; 175: 249-259Crossref PubMed Scopus (70) Google Scholar). The first processing event is carried out by the stromal processing peptidase, which cleaves after residue 42 to yield an intermediate form of the protein in the stroma; the second event occurs concomitantly with inner membrane insertion and is mediated by a thus far unidentified, envelope-associated peptidase that cleaves after residue 76 (supplemental Fig. S1). Because the transmembrane domain of atTic40 is essential for membrane insertion (32Li M. Schnell D.J. J. Cell Biol. 2006; 175: 249-259Crossref PubMed Scopus (70) Google Scholar), our ΔTM protein is predicted to accumulate as an intermediate in the stroma. Thus, the ∼43-kDa band in Fig. 3A most likely corresponds to the intermediate form of the ΔTM protein, which has a calculated size of 40 kDa but is expected to have a larger apparent size (12Chou M.L. Fitzpatrick L.M. Tu S.L. Budziszewski G. Potter-Lewis S. Akita M. Levin J.Z. Keegstra K. Li H.M. EMBO J. 2003; 22: 2970-2980Crossref PubMed Scopus (158) Google Scholar). Consistent with this notion, a ΔTM:YFP fusion protein yielded distinctly stromal fluorescence in transfected Arabidopsis protoplasts, which was in marked contrast with the envelope-associated fluorescence provided by a full-length atTic40:YFP fusion (supplemental Fig. S3). The low abundance of the ΔTM intermediate (Fig. 3A) probably reflects the instability of the protein when mis-localized to the stroma. We therefore suggest that the additional, diffuse band at ∼36 kDa includes stromal proteolytic fragments of the ΔTM protein (Fig. 3A). Consistent with this interpretation, this band was resolved into at least two distinct fragments in Fig. 3B. As expected, all detected ΔTM forms (∼43- and ∼36-kDa fragments) were completely protected from protease in isolated chloroplasts, indicating that all bands contained fully imported protein (Fig. 3B). In the ΔSti1 lines, a single and abundant protein of ∼35 kDa was detected. This is most likely the mature, processed ΔSti1 protein (calculated size, 34 kDa), because it was also detected in isolated, protease-treated chloroplasts (Fig. 3B). Finally, a protein of the expected size (13 kDa) was detected in ΔTPR seedlings (Fig. 3A). It was not possible to isolate chloroplasts from these albino seedlings, but the strong effect of the ΔTPR protein on chloroplast biogenesis (Fig. 2, A and B) strongly suggests that it too is properly targeted to the organelle. The fact that the ΔTM, ΔSti1, and ΔTPR proteins are expressed (and, in the case of ΔTM and ΔSti1, properly targeted to chloroplasts) means that their failure to complement tic40 can be attributed to effects o" @default.
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• W2006551808 title "Functional Similarity between the Chloroplast Translocon Component, Tic40, and the Human Co-chaperone, Hsp70-interacting Protein (Hip)" @default.
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