Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein translocon

Abstract

The function of Tic40 during chloroplast protein import was investigated. Tic40 is an inner envelope membrane protein with a large hydrophilic domain located in the stroma. Arabidopsis null mutants of the atTic40 gene were very pale green and grew slowly but were not seedling lethal. Isolated mutant chloroplasts imported precursor proteins at a lower rate than wild-type chloroplasts. Mutant chloroplasts were normal in allowing binding of precursor proteins. However, during subsequent translocation across the inner membrane, fewer precursors were translocated and more precursors were released from the mutant chloroplasts. Cross-linking experiments demonstrated that Tic40 was part of the translocon complex and functioned at the same stage of import as Tic110 and Hsp93, a member of the Hsp100 family of molecular chaperones. Tertiary structure prediction and immunological studies indicated that the C-terminal portion of Tic40 contains a TPR domain followed by a domain with sequence similarity to co-chaperones Sti1p/Hop and Hip. We propose that Tic40 functions as a co-chaperone in the stromal chaperone complex that facilitates protein translocation across the inner membrane.

Fig. 1. The hydrophilic domain of Tic40 is located in the stroma. (A) Characterization of the atTic40 antibodies. Total chloroplast proteins from pea or Arabidopsis were analyzed by immunoblots with antibodies raised against the C-terminal portion of atTic40 or the preimmune serum. Intact chloroplasts containing 5 µg of chlorophyll or antigen containing 0.01 µg of protein were loaded. (B) Treatment of intact pea chloroplasts with various concentrations of trypsin. Digestion was performed at room temperature for 1 h and was terminated by adding a cocktail of protease inhibitors (Tu and Li, 2000). The protease inhibitors were present in all the solutions afterwards. Intact chloroplasts were repurified and analyzed by immunoblots with antibodies against various proteins as labeled on the left.

Fig. 2. Phenotypes of the tic40 mutants. (A) Positions of T-DNA insertion in the two tic40 mutant alleles: solid boxes, exons; open boxes, introns. (B) Both alleles are nulls. Chloroplast proteins from the mutants or the corresponding wild types were analyzed by immunoblotting with antibodies against atTic40: WS2, the Wassilewskija wild-type plant; Col, the Columbia wild-type plant. (C) The tic40-1 mutant and the corresponding WS2 wild-type plants at 20 days (20d). Plants were grown on MS media with 2% sucrose at 22°C under a 16 h day. The bar represents 0.4 cm. (D) The tic40-2 mutant and the corresponding Columbia wild-type plants at 10 days (10d), 4 weeks (4wk) and 8 weeks (8wk). Plants were grown on soil at 22°C under a 16 h day. The bar represents 1 cm.

Fig. 3. Electron micrographs of chloroplasts from tic40-1 mutant and WS2 wild-type plants. Samples were taken from cotyledons of 10-day-old seedlings. The bars in (A) and (C) represent 2 µm; those in (B) and (D) represent 200 nm.

Fig. 4. The levels of endogenous chloroplast proteins are reduced in the tic40 mutant. (A) Various amounts of total leaf proteins, as indicated at the top of the lanes, from the tic40-1 mutant and the wild type (WT) were analyzed by SDS–PAGE and stained with Coomassie Blue. Filled triangles indicate the three major chloroplast proteins, which were present in reduced amounts in the mutant. Open triangles indicate three proteins whose amounts are not reduced in the mutants. M, molecular mass markers. (B) Samples were the same as in (A) but were analyzed by immunoblotting with antibodies against OE33, POR, CAB and mitochondrial porin. In addition, in vitro translated OE33 and POR precursor proteins (prOE33 and prPOR) were analyzed alongside the leaf samples and detected using anti-OE33 and anti-POR antibodies.

Fig. 5. The tic40 mutant chloroplasts are defective in protein import. (A) Import of [³⁵S]prSS into chloroplasts isolated from Columbia wild-type and tic40-2 plants. Isolated chloroplasts were incubated with [³⁵S]prSS and 4 mM ATP in import buffer under natural light at 22°C. Import was stopped at different times by centrifuging the chloroplasts through 40% Percoll. Chloroplast proteins were subjected to SDS–PAGE. Data points represent the mean (± SE) of three independent experiments. The quantitity of mature SS imported into the wild type at 20 min was set at 100%. (B) Import of various other precursors into isolated chloroplasts. Import was performed at room temperature for 10 min: TP, 10% of the translated precursor proteins that were added to the import assay. The arrows mark the position of the imported mature proteins. The open triangle in the PC samples indicates the intermediate (iPC) in the stroma during PC import (Bauerle et al., 1991). (C) The tic40 mutant chloroplasts contained normal amounts of translocon components. Chloroplast proteins of the tic40-2 mutant and the wild-type plants were analyzed by immunoblotting with antibodies against translocon components as labeled on the left. The 86 kDa degradation product of atToc159 is also shown.

Fig. 6. The tic40 mutant chloroplasts are defective in translocation across the inner envelope membrane. (A) Chloroplasts were isolated from the tic40-1 mutant and kept on ice in the dark for 20 min to deplete internal ATP. Energy-depleted [³⁵S]prSS and the chloroplasts were incubated together under a dim green safelight at room temperature for 5 min in the presence (lanes 2 and 6) or absence (lanes 1 and 5) of 0.1 mM ATP. The chloroplasts were pelleted by centrifugation. Half of the chloroplasts that had been incubated with 0.1 mM ATP were incubated for a further 15 min in import buffer containing 5 mM ATP (0.1 + 5 mM ATP, lanes 3 and 7). The chloroplasts were again pelleted by centrifugation. The recovered chloroplasts were repurified through a 40% Percoll cushion and the contents were analyzed. The supernatant was dried down and dissolved in SDS–PAGE sample buffer. Samples containing equal amounts of protein were loaded in lanes 1–3 and 5–7. Samples containing an equal portion of the total sample were loaded in lanes 4 and 8. The positions of prSS and mature SS are indicated. (B) Quantification of the binding and translocation experiments shown in (A). The amount of prSS when no ATP was added [lanes 1 and 5 in (A)] is set at 100%. Data points represent the mean (± SE) of four independent experiments. The mean value is given at the top of each bar. (C) Quantification of the post-translocation supernatant shown in (A). The amount of SS in lanes 3, 4, 7 and 8 of (A) was compared with the amount of prSS within each sample. Data points represent the mean (± SE) of three independent experiments. The mean value is given at the top of each bar.

Fig. 7. Tic40 is a component of the translocation apparatus. (A) Intact pea chloroplasts were incubated with DSP for 15 min on ice. After the reaction was stopped, chloroplasts were re-isolated and lysed. Total membranes were isolated and solubilized. Proteins in the solubilized supernatant were immunoprecipitated with antibodies against Tic40. The cross-links in the immunoprecipitates were cleaved by the addition of gel loading buffer containing DTT and analyzed by immunoblotting with antibodies against various proteins as labeled on the left. A minor cross-reactivity of normal rabbit serum with Hsp93 was consistently observed with all the rabbits tested. (B) Reciprocal immunoprecipitation demonstrating association of Tic40 with Toc75. Experiments were the same as in (A), except that immunoprecipitation was performed using anti-Toc75 antibodies.

Fig. 8. Tic40 is associated with importing precursor proteins at the same stage as Tic110 and Hsp93. An import time course was performed with [³⁵S]prSS. Reactions were terminated at various time points by diluting with cold import buffer. Chloroplasts were re- isolated, cross-linked with 0.5 mM DSP and lysed hypotonically. Total membranes were isolated; and the proteins were solubilized with Triton X-100 and immunoprecipitated with antibodies against Toc75, Tic110, Tic40 or Hsp93. Immunoprecipitates were analyzed by SDS–PAGE under conditions that cleaved the cross-links. Autoradiographs of the gels showing prSS and SS associated with each translocon component are presented.

Fig. 9. Sequence analyses of Tic40. (A) Sequence alignment of Tic40 from various plant species. After isolation and sequencing a pea Tic40 cDNA clone, we found several discrepancies between our sequence and the published pea Tic40 cDNA sequence (DDBJ/EMBL/GenBank accession No. AJ243758) (Stahl et al., 1999). Our sequence is shown here and has been submitted to the DDBJ/EMBL/GenBank database under the accession No. AY157668. The partial maize sequence is from the EST clone of accession No. BG550516. The soybean sequence is compiled from EST sequences BF066367+BM522900+BI971146_revers+BE021537+BE211020, the barley sequence from EST sequences BI949343+BJ464357+BI959205+ BJ468493_revers and the partial tomato sequence from EST sequences AW398666+BI934934. The asterisks represent stop codons. The proposed processing site for the transit peptidase is marked by an arrow. The putative transmembrane domain and the seven α-helices of the TPR domain are underlined. The second, fifth and sixth helices in the tertiary structure prediction are composed of non-continuous sequences. The Sti1p/Hop/Hip domain is underlined by a gray box. (B) Sequence alignment of the Sti1p/Hop/Hip domain from yeast Sti1p (accession No. NP014670), human Hop (accession No. NM006819), human Hip (accession No. XP165401) and atTic40. (C) Tic40 was recognized by anti-TPR1 antibodies. In the upper panel, an E.coli overexpressed atTic40 hydrophilic domain was analyzed by SDS–PAGE and revealed by Coomassie Blue staining or immunoblotting with antibodies against Tic40, TPR1 or the preimmune serum of anti-TPR1. In the lanes labeled ‘chy’, the atTic40 proteins had been purposely degraded by chymotrypsin. In the lower panel, total chloroplast proteins from the wild type or the tic40-1 mutant were analyzed by SDS–PAGE and immunoblotting with antibodies against Tic40, TPR1 or the preimmune serum of anti-TPR1. (D) A proposed domain structure of Tic40 compared with that of Hop. The numbers shown are the amino acid residue numbers for human Hop (Scheufler et al., 2000) and atTic40. TM, transmembrane domain.