Article Figures & Data
Figures
- Fig. 1.
Toc86 is a proteolytic fragment of a larger polypeptide. Chloroplasts were isolated from pea seedlings in the absence (−) or presence (+) of a protease inhibitor cocktail. The chloroplasts were lysed and fractionated to yield an envelope membrane fraction. Envelope membranes (25 μg of protein) were resolved by SDS-PAGE and immunoblotted with anti-Toc86 (anti-Toc86), anti-Toc75 (anti-Toc75), or anti-Toc34 (anti-Toc34) IgG. The molecular masses of known proteins are shown to the left of the figure.
- Fig. 2.
Deduced amino acid sequence of the pea Toc159 cDNA. A, The deduced amino acid sequence of the complete pea Toc159 cDNA. Repetitive sequences are aligned vertically and highlighted by shaded boxes. The amino acid sequences of peptides obtained from the Toc159 polypeptide are underlined. An arrowhead indicates the position of the N-terminal amino acid of the previously proposed Toc86 precursor. B, Northern-blot analysis of pea seedling poly(A+) RNA hybridized to a Toc159 cDNA 3′ fragment that corresponds to the original cDNA for Toc86. The sizes of standard RNA markers are indicated in kilobases to the right of the figure. C, Proposed tripartite domain structure of Toc159. The positions of the acidic, repetitive domain (A-domain), the GTPase domain (G-domain), and the membrane-anchor domain (M-domain) are indicated by the numbers of the corresponding amino acids in the deduced sequence in A.
- Fig. 3.
Comparison of the amino acid sequences of pea and Arabidopsis Toc159. The complete deduced amino acid sequences of the pea Toc159 (psToc159) cDNA and a predicted gene encoding a related protein in Arabidopsis (atToc159) (GenBank accession no. AF069298) are aligned to maximize the occurrence of identical residues. Identical amino acids are highlighted by shaded boxes.
- Fig. 4.
Effects of Toc159 proteolysis on preprotein binding and import. Intact chloroplasts or chloroplasts treated with 10 μg/mL thermolysin were incubated with in vitro-synthesized [35S]preSSU in a standard import reaction containing 25 μm ATP or 1 mm ATP as indicated. The chloroplasts were re-isolated and analyzed by SDS-PAGE. A, Immunoblots of envelope membrane fractions from thermolysin-treated (+) or control (−) chloroplasts. Membrane proteins (25 μg of chlorophyll) were resolved by SDS-PAGE and immunoblotted with affinity-purified antibodies to Toc86, Toc75, and Toc34. The positions of the known Toc components and the 52-kD membrane-protected fragment of Toc159 (52 kD) are indicated to the left. Tr, 10% of the [35S]preSSU translation product added to each reaction. B, Phosphor imager analysis of [35S]preSSU import. C, Quantitative analysis of the data presented in B. The numbers of the lanes in B that were used for quantitation are indicated at the bottom of the graph in C.
- Fig. 5.
Label-transfer cross-linking of pre-SSU to intact and protease-treated chloroplasts. Intact chloroplasts (−) or chloroplasts treated with 10 μg/mL thermolysin (+) were incubated with 200 nm 125I-pS-1 in a standard import reaction containing apyrase or 25 μm ATP as indicated. The chloroplasts were irradiated with UV light, re-isolated, and fractionated to yield a total envelope membrane fraction. The envelope membrane proteins were analyzed by SDS-PAGE and phosphor imager analysis. A, 125I-pS-1 cross-linking to intact chloroplasts isolated in the presence of protease inhibitors. The protein samples were resolved on 8% (w/v) polyacrylamide gels. B,125I-pS-1 cross-linking to chloroplasts isolated in the absence of protease inhibitors. The protein samples were resolved on 12% (w/v) polyacrylamide gels. The positions of known Toc and Tic components and 125I-pS-1 (pS-1) are indicated to the left of A and the right of B.
- Fig. 6.
Effect of a GTP analog on the binding and import of preSSU in intact or protease-treated chloroplasts. Intact chloroplasts or chloroplasts treated with 10 μg/mL thermolysin were incubated with in vitro synthesized [35S]preSSU in a standard import reaction containing 25 μm ATP, or 1 mm ATP as indicated. GTPγS was included in the binding or import reactions at the concentrations indicated. The chloroplasts were re-isolated and analyzed by SDS-PAGE. A, Phosphor imager analysis of SDS-PAGE-resolved chloroplast proteins. Tr, 10% of the [35S]preSSU translation product added to each reaction. B, Quantitative analysis of the data presented in A. The numbers of the lanes in A that were used for quantitation are indicated at the bottom of the graph in B.
- Fig. 7.
Effect of a GTP analog on the import of a preSSU early import intermediate in intact or protease-treated chloroplasts. Intact chloroplasts or chloroplasts treated with 10 μg/mL thermolysin were incubated with in vitro-synthesized [35S]preSSU in a standard import reaction containing apyrase or 25 μm ATP as indicated (Import inter.). The chloroplasts were re-isolated and resuspended in import buffer containing 1 mm ATP and 1 mm GTPγS as indicated and incubated at 26°C for 10 min (Chase). The chloroplasts were re-isolated and analyzed by SDS-PAGE and phosphor imager analysis.
