Tic40 is important for reinsertion of proteins from the chloroplast stroma into the inner membrane

Abstract

Chloroplast inner-membrane proteins Tic40 and Tic110 are first imported from the cytosol into the chloroplast stroma, and subsequently reinserted from the stroma into the inner membrane. However, the mechanism of reinsertion remains unclear. Here we show that Tic40 itself is involved in this reinsertion process. When precursors of either Tic40 or a Tic110 C-terminal truncate, tpTic110-Tic110N, were imported into chloroplasts isolated from a tic40-null mutant, soluble Tic40 and Tic110N intermediates accumulated in the stroma of tic40-mutant chloroplasts, due to a slower rate of reinsertion. We further show that a larger quantity of soluble Tic21 intermediates also accumulated in the stroma of tic40-mutant chloroplasts. In contrast, inner-membrane insertion of the triose-phosphate/phosphate translocator was not affected by the tic40 mutation. Our data suggest that multiple pathways exist for the insertion of chloroplast inner-membrane proteins.

Figure 1

More in vitro imported iTic40 and Tic110N accumulated in the soluble fraction of tic40-mutant chloroplasts. (a, c) [³⁵S]-prCAB, prRBCS, prTic40 and tpTic110-Tic110N were imported into wild-type (WT) and tic40-null mutant chloroplasts for 20 min. Re-isolated intact chloroplasts (cpt) were separated into pellet (P) and supernatant (S) fractions by hypotonic lysis in 0.2 m NaCl (a) or by alkaline extraction (c). Samples were analyzed by SDS-PAGE and fluorography. TR, 2.5% of in-vitro translated precursor proteins added to the import reactions. pr, precursor form; i, intermediate; m, mature form. iTic40 and mature Tic40 are indicated with an asterisk and an arrowhead, respectively. (b, d) Ratio of soluble to membrane-inserted proteins in (a) and (c), respectively. In the gels shown in (a) and (c), an equal amount of proteins from the WT and tic40-mutnat pellets were loaded (lanes 3 and 5). Because tic40-mutant chloroplasts contain less protein (Chou et al., 2003), slightly more chloroplasts were loaded from the mutant. Each supernatant fraction then used three times the number of chloroplasts of its corresponding pellet fraction in order to have similar band intensities across the gel. The quantification in (b) and (d) has been corrected for the chloroplast numbers and represents the ratio within a chloroplast.

Figure 2

In vitro imported iTic40 and Tic110N accumulated in the stroma of tic40-mutant chloroplasts. [³⁵S]-prTic40 and [³⁵S]-tpTic110-Tic110N were imported into tic40-mutant chloroplasts for 20 min. After import chloroplasts were treated with various concentrations of trypsin. Re-isolated intact chloroplasts were separated into pellet (for analysis of Toc75) and supernatant [for analysis of Tic40, Tic110N and RuBP carboxylase small subunit (RBCS)] fractions by hypotonic lysis in 0.2 m NaCl (a, b) or by alkaline extraction (c, d). Samples were analyzed by SDS-PAGE. Proteins were detected by fluorography (Tic40 and Tic110N), Coomassie blue staining (RBCS) or immunoblots (Toc75).

Figure 3

Accumulation kinetics of membrane-inserted Tic40 and soluble iTic40 in wild-type (WT) and tic40-mutant chloroplasts. (a) [³⁵S]-prTic40 was pre-imported into WT and tic40-mutant chloroplasts for 2 min, re-isolated and then chased for the length of time indicated. Chloroplasts were treated with thermolysin and separated into membrane and supernatant fractions by alkaline extraction. Samples were analyzed by SDS-PAGE and fluorography. An equal amount of protein from all membrane-fraction samples was loaded, and each supernatant fraction then used three times the number of chloroplasts of its corresponding membrane fraction. TR, in vitro translated protein. i, intermediate; m, mature form. All WT samples were analyzed on one gel and all mutant samples on another. All gels were exposed for the same amount of time, except that the TR-lane image was from gels exposed for 1/6 the amount of time. (b) Quantification of the membrane-inserted mature Tic40 in (a). (c) Quantification of iTic40 in the supernatant fractions of (a). For both (b) and (c), the amount of membrane-inserted mature Tic40 in WT chloroplasts at 20 min was set as 100%.

Figure 4

Accumulation kinetics of Tic110N in the membrane and supernatant fractions in wild-type (WT) and tic40-mutant chloroplasts. (a) [³⁵S]-tpTic110-Tic110N was pre-imported into WT and tic40-mutant chloroplasts for 2 min, re-isolated and then chased for the length of time indicated. Chloroplasts were treated with thermolysin and separated into pellet and supernatant fractions by alkaline extraction. Samples were analyzed by SDS-PAGE and fluorography. An equal amount of protein from all membrane-fraction samples was loaded, and each supernatant fraction then used three times the number of chloroplasts of its corresponding membrane fraction. TR, in-vitro translated protein. m, mature form. All wild-type samples were analyzed on one gel and all mutant samples on another. All gels were exposed for the same amount of time. (b) Quantification of the membrane-inserted mature Tic110N in (a). (c) Quantification of Tic110N in the supernatant fractions of (a). For both (b) and (c), the amount of membrane-inserted matureTic110N in the wild type at 20 min was set as 100%.

Figure 5

Tic40 is important for the membrane insertion of Tic21 but not phosphate translocator (PHT). (a) [³⁵S]-prTic21 and [³⁵S]-prPHT were imported into wild-type (WT) and tic40-mutant chloroplasts for 20 min. Re-isolated intact chloroplasts (cpt) were separated into pellet (P) and supernatant (S) fractions by hypotonic lysis in 0.2 m NaCl. Samples were analyzed by SDS-PAGE and fluorography. TR, 2.5% of in vitro translated precursor proteins added to the import reactions. pr, precursor form; i, intermediate; m, mature form. For the prTic21 panel, the TR and cpt portion was a shorter exposure from the same gel of the pellet (P) and supernatant (S) portion. (b) Ratio of supernatant to membrane-inserted iTic21 and mature Tic21 as shown in (a). In the gels shown in (a), an equal amount of protein from the WT and tic40-mutant pellets was loaded (lanes 3 and 5). Each supernatant fraction then used three times the number of chloroplasts of its corresponding pellet fraction in order to have similar band intensities across the gel. The quantification in (b) has been corrected for the chloroplast numbers and represents the ratio within a chloroplast. (c) In vitro imported iTic21 and mature Tic21 accumulated in the stroma of tic40-mutant chloroplasts. [³⁵S]-prTic21 was imported into tic40-mutant chloroplasts for 20 min. After import chloroplasts were treated with various concentrations of trypsin. Re-isolated intact chloroplasts were separated into pellet (for analyzing Toc75) and supernatant [for analyzing Tic21and RuBP carboxylase small subunit (RBCS)] fractions by hypotonic lysis in 0.2 m NaCl. Samples were analyzed by SDS-PAGE. Proteins were detected by fluorography (Tic21), Coomassie blue staining (RBCS) or immunoblots (Toc75).

Figure 6

Accumulation kinetics of membrane-inserted Tic21 and soluble iTic21 in wild-type (WT) and tic40-mutant chloroplasts. (a) [³⁵S]-prTic21 was pre-imported into WT and tic40-mutant chloroplasts for 2 min, re-isolated and then chased for the length of time indicated. Chloroplasts were treated with thermolysin and separated into membrane and supernatant fractions by alkaline extraction. Samples were analyzed by SDS-PAGE and fluorography. An equal amount of protein from all membrane-fraction samples was loaded, and each supernatant fraction then used three times the number of chloroplasts of its corresponding membrane fraction. TR, in vitro translated protein. i, intermediate; m, mature form. All WT samples were analyzed on one gel and all mutant samples on another. All gels were exposed for the same amount of time. (b) Quantification of the membrane-inserted mature Tic21 in (a). (c) Quantification of iTic21 in the supernatant fractions of (a). For both (b) and (c), the amount of membrane-inserted mature Tic21 in WT chloroplasts at 20 min was set as 100%.

Figure 7

Conserved proline residues are present in Tic40, Tic110 and iTic21 but not in phosphate translocator (PHT) and accumulation and replication of chloroplasts 6 (ARC6). Sequence alignment of the N-terminal portion of prTic21 and mature Tic40, Tic110 and PHT, and the C-terminal portion of ARC6. Multiple sequence alignments were created using the Wisconsin Package (version 10.3). The transit-peptide processing site of Arabidopsis prTic21 is indicated with an upward arrow. First transmembrane domains (TM1) are underlined. Proline residues are highlighted in white letters on a black background. Residues are numbered with the initiation methionine as residue 1.