Stimulation of transit-peptide release and ATP hydrolysis by a cochaperone during protein import into chloroplasts

Abstract

Three components of the chloroplast protein translocon, Tic110, Hsp93 (ClpC), and Tic40, have been shown to be important for protein translocation across the inner envelope membrane into the stroma. We show the molecular interactions among these three components that facilitate processing and translocation of precursor proteins. Transit-peptide binding by Tic110 recruits Tic40 binding to Tic110, which in turn causes the release of transit peptides from Tic110, freeing the transit peptides for processing. The Tic40 C-terminal domain, which is homologous to the C terminus of cochaperones Sti1p/Hop and Hip but with no known function, stimulates adenosine triphosphate hydrolysis by Hsp93. Hsp93 dissociates from Tic40 in the presence of adenosine diphosphate, suggesting that Tic40 functions as an adenosine triphosphatase activation protein for Hsp93. Our data suggest that chloroplasts have evolved the Tic40 cochaperone to increase the efficiency of precursor processing and translocation.

Figure 1.

Constructs of Tic40, Tic110, and Hsp93 used in this study. Numbers below each construct specify the amino acid residue numbers, with the first amino acid of the mature protein as 1 and the residues for transit peptides negative. Proteins from A. thaliana are specified with “at.” Others are from pea. Numbers in parentheses specify the corresponding residue numbers of the A. thaliana proteins. TP, transit peptide; TM, transmembrane region; pr, precursor form; GAL4-BD and -AD, GAL4 DNA binding and activation domains, respectively, for yeast two-hybrid assays.

Figure 2.

Tic40 TPR domain binds to Tic110. (A) Yeast two-hybrid analyses of interacting domains between Tic40 and Tic110. (top) Yeast cells containing each of the activation domain constructs were transformed with each of the binding domain constructs and plated on the synthetic dropout (SD) medium containing 5 mM 3-amino-1,2,4-triazole (3-AT) without tryptophan, leucine, and histidine. (bottom) Filter replicates from the SD plates were then assayed for β-galactosidase activity. (B) GST pull-down assay analyzing direct interaction between Tic40 and Tic110. GST fused to the entire Tic40 stromal soluble domain (GST-atTic40S), the TPR subdomain (GST-atTic40TPR), or the Hip/Hop subdomain (GST-atTic40Hip/Hop), or GST itself was incubated with atTic110S-His₆ at 4°C for 1 h. GST fusion proteins were recovered by binding to glutathione resin and eluted with glutathione.

Figure 3.

Tic40 does not bind import substrates directly. 10 μl of [³⁵S]prRBCS or an equal molar amount of [³⁵S]RBCS was incubated with 48 pmol of OE33-His₆, GST-atTic40S, or atTic110S-His₆ at 4°C for 2 h. TALON or glutathione resin was then added, and incubation continued for another 30 min. Proteins eluted from resin were analyzed by SDS-PAGE (A) and quantified by a PhosphorImager (B).

Figure 4.

Precursors increase the affinity of Tic110 to Tic40. (A) 83 pmol of atTic110S-His₆ was mixed with buffer (lane 1), 83 pmol of recombinant RBCS (lane 2), or prRBCS (lane 3). 83 pmol of GST-atTic40S was then added, and the mixture was incubated at 4°C for 2 h. Proteins associated with GST-atTic40S were recovered by glutathione resin, eluted by glutathione, and analyzed by immunoblotting using antibodies against Tic110 and Tic40. Approximately 0.5% of atTic110S-His₆ added was coeluted with GST-atTic40S. (B) The experimental procedure was the same as in A, except RBCS and prRBCS were replaced with 83 pmol of synthetic prFD transit peptides (lane 2) or SynB2 control peptides (lane 3).

Figure 5.

Tic40 causes the release of bound transit peptides from Tic110. (A) 48 pmol of atTic110S-His₆ was incubated with 10-fold excess of ³H-labeled prFD transit peptides at 4°C for 2 h, TALON resin was added, and incubation continued for another 30 min. The resin was washed, and ∼3% of the added ³H-prFD transit peptides were bound to Tic110-His₆ resin. Washed resin was then incubated with the same molar amount (48 pmol) or 10- or 20-fold excess of GST or GST-atTic40S at 4°C for 2 h. The resin was pelleted, and ³H counts in the supernatant were measured. Approximately 14% of bound ³H-prFD was released at 20-fold GST-atTic40S. (B) Membrane-bound processed mature proteins are delayed in appearance, and their association with Tic110 is reduced in the tic40 mutant chloroplasts. Isolated wild-type and tic40 mutant chloroplasts were used to perform an import time course experiment with [³⁵S]prRBCS. Total membranes were isolated from chloroplasts of each time point and either directly analyzed by SDS-PAGE (top) or solubilized and immunoprecipitated by the anti-Tic110 antibody. (C) Quantification of the 30-min samples (B, lanes 5 and 10) of the gels shown in B. The amount of prRBCS in wild type at 30 min (prRBCS in lane 5) of each gel was taken as 1. The counts for RBCS have been corrected for the number of methionine residues compared with prRBCS.

Figure 6.

Tic40 Hip/Hop domain activates Hsp93 ATPase activity. (A) His₆-Hsp93 was incubated with ATP or ATP plus various other proteins, as indicated at the bottom of the graph. The amount of Pi hydrolyzed by Hsp93 incubated with other proteins was divided by the amount of Pi hydrolyzed by Hsp93 alone. Values represent mean ± SEM; n = 3. (B) The three mutant forms of GST-atTic40Hip/Hop have the same protease sensitivity as the wild-type GST-atTic40Hip/Hop. GST-atTic40Hip/Hop and its three mutants, N320A, N329A, and N342A, were incubated with trypsin at a concentration of 4 U/g protein at 37°C for an increasing amount of time (0,10, 20, 30, and 40 min). The samples were analyzed by SDS-PAGE and silver staining. (C) His₆-Hsp93 and ATP were incubated with the wild-type (WT) GST-atTic40Hip/Hop or one of the mutants, as indicated at the bottom of the graph. Stimulation of ATP hydrolysis was calculated as in A.

Figure 7.

ATP preloading can partially rescue the tic40 mutant import defect. (A) Isolated chloroplasts from wild-type and tic40 mutant plants were preincubated with 3 mM ATP (closed bar) or import buffer (open bar) for 5 min at room temperature in the dark, reisolated, and used to perform import experiments with [³⁵S]prRBCS. The amount of mature RBCS imported at 30 min was quantified, and the ratio of imported RBCS in tic40 chloroplasts to that in wild-type chloroplasts was plotted. (B) Hsp93 has a higher ATPase activity in the ATP concentration after chloroplasts have been preloaded with ATP. The ATP concentration within chloroplasts was calculated to be ∼0.07 mM before and 2.1 mM after preloading with 3 mM ATP (see Materials and methods). ATPase activity of Hsp93 was assayed under these two ATP concentrations. The ATPase activity at 0.07 mM ATP was taken as 1. (C) Association of Tic40 with Hsp93 decreases in the presence of ADP. 138 nM Hsp93-His₆ was incubated with 690 nM ATP, ADP, or AMP-PNP at room temperature for 10 min. An equal amount of atTic110S-His₆, prFD transit peptide, and GST-atTic40S was added with a 1-h incubation at 4°C after each addition. Tic40-containing complexes were recovered by glutathione resin, washed in PBS containing the nucleotides, eluted by glutathione, and analyzed by immunoblotting with antibodies against Hsp93 and Tic40.

Figure 8.

A working model for sequential steps of protein translocation into the chloroplast stroma. The lines moving across the two membrane channels represent a precursor protein, with the zigzag line representing the transit peptide. SPP, stromal processing peptidase; Hsp93T and Hsp93D, Hsp93 bound with ATP or ADP, respectively. The names of other translocon components are labeled only in A, for simplicity.