Characterization of the translocon of the outer envelope of chloroplasts - PubMed (original) (raw)

Characterization of the translocon of the outer envelope of chloroplasts

Enrico Schleiff et al. J Cell Biol. 2003.

Abstract

The protein translocon of the outer envelope of chloroplasts (Toc) consists of the core subunits Toc159, Toc75, and Toc34. To investigate the molecular structure, the core complex was purified. This core complex has an apparent molecular mass of approximately 500 kD and a molecular stoichiometry of 1:4:4-5 between Toc159, Toc75, and Toc34. The isolated translocon recognizes both transit sequences and precursor proteins in a GTP-dependent manner, suggesting its functional integrity. The complex is embedded by the lipids phosphatidylcholine and digalactosyldiacylglyceride. Two-dimensional structural analysis by EM revealed roughly circular particles consistent with the formation of a stable core complex. The particles show a diameter of approximately 130 A with a solid ring and a less dense interior structure. A three-dimensional map obtained by random conical tilt reconstruction of electron micrographs suggests that a "finger"-like central region separates four curved translocation channels within one complex.

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Figures

Figure 1.

Figure 1.

The purified core complex of the chloroplast outer envelope translocon. The outer envelope fraction was solubilized as described in Materials and methods, separated on a 25–70% sucrose density gradient (A) followed by separation of the indicated fractions (gray underlay) on a sucrose step gradient (B). A silver-stained gel of the fractions is shown. Proteins are assigned according to results of immunoblot analysis where Toc159* indicates the 86-kD fragment of Toc159. Numbers indicate the fractions of the gradient (on top), the molecular weight (on the side), and the sucrose concentration (wt/vol; on the bottom). (C) The protein pattern of outer envelopes (lane 1), the combined fractions (gray underlay in A) of the first gradient (lane 2), or the isolated core complex in fractions 17–20 of the step gradient (lane 3) were probed by immunodetection using antibodies against outer and inner envelope proteins as indicated. (D) The purified core complex (fraction 17–20) was subjected to an S500-sephacryl size exclusion chromatography. The fractions indicated on top were subjected to SDS-PAGE followed by immunodecoration using Toc159, Toc75, or Toc34 antisera. The retention time of standard proteins is indicated by their molecular mass (numbers on the bottom). (E) The purified core complex was subjected to a superose 6 column, and peak fractions were used for EM (inset, left). The micrograph shows negatively stained particles (oval) at a magnification of 60,000 using the deep stain technique. The arrow points to tobacco mosaic virus, which was added for calibration purpose and as a standard to visualize the quality of the stain. The inset shows the profile of the size exclusion chromatography (triangles show the positions of thyroglobin and ferritin, respectively; the fraction at 0.9 ml represented the void volume and the fraction at 1.8 ml the salt peak). Bar, 100 nm.

Figure 2.

Figure 2.

Stoichiometry of the Toc components in the isolated core complex. (A) Different amounts of calibration proteins (st.; lane 2, 4, 5, and 7) and outer envelope (OE; lane 1, 3, 6, and 8) were separated on SDS-PAGE followed by Coomassie brilliant blue staining. Toc34, Toc75, and the 86-kD fragment of Toc159 are indicated. (B) The molar quantity of the proteins was compared and normalized to the amount of Toc159 according to the staining in A. (C) Different amounts of outer envelope (lanes 1–5) and of the isolated Toc core complex (lanes 6 and 7) were separated on SDS-PAGE and immunodecorated using Toc159, Toc75, and Toc34 antibodies. (D) The molar amount of the proteins was compared and normalized to Toc159 (as in C). (E) The Toc core complex was treated with 3H-NEM. Incorporated radioactivity is counted and normalized to the number of cysteines in each protein. Histograms in B, D, and E show the average of at least three independent experiments. A representative figure is shown in A and C, respectively. (F) A Western blot analysis of the used purified Toc complex using antisera against Toc34 (lane 1), Toc75 (lane 2), and Toc159 (lane 3) is shown.

Figure 3.

Figure 3.

Lipid analysis of the Toc core complex. Lipids of the outer envelope (OE) and the core complex (Toc) after (A) and before (B) lipase treatment were separated using a mixture of acetone/benzene/water (A) or chloroform/methanol/water/acetic acid/acetone as solvent system (B). PC, PG, phosphatidylinositol (PI), MGDG, and DGDG are shown as standards. The inset shows the same result as in B with less contrast. x shows the position of PG and * shows the position of DGDG.

Figure 4.

Figure 4.

Functional analysis of the isolated complex. (A) Radioactive-labeled preSSU was subjected on top of a step gradient before (top) and after incubation with the isolated Toc complex in the presence of 0.1 mM EDTA (middle) or 1.0 mM MgCl2 (bottom) and 0.5 mM GTP and centrifuged. Fraction numbers are indicated on top, and the percentage of the sucrose is given below the figure panel. (B) Fraction 9 of the gradient containing EDTA (left) or MgCl2 (right) was probed using antisera against Toc159, Toc75, and Toc34 as indicated. (C) Heterologously expressed preSSU-His6 was coupled to an affinity matrix and incubated with the isolated Toc complex in the absence (lane 1–3) or presence of 0.5 mM GTP and 1 mM MgCl2 (lanes 4–6). Proteins in flow through (FT), wash step (WS), and elution with 0.25 M imidazol (ES) were precipitated, resolved in Laemmli sample buffer, separated by SDS-PAGE, and immunoblotted using αToc159, αToc75, and αToc34 antibodies or silver stained to visualize preSSU. (D) The experiment was performed as in C, but only eluted fractions are shown. Lane 1 shows the loaded complex (20%) and lanes 2 to 7 show the eluted fractions after binding in the presence of 0.25 mM GTP/0.25 mM ATP (lane 2), 0.25 mM GTP (lane 3), 0.25 mM ATP (lane 4), 0.25 mM GTP/0.25 mM GDP (lane 5), 0.25 mM GDP (lane 6), and without nucleotides (lane 7). (E) Purified Toc complex was added to preSSU (lanes 2–5 and 14–19), mSSU (lanes 10–13), or synthetic B1 peptide (lanes 6–9) coupled to Toyopearl matrix in the absence (lanes 2, 3, 6, 7, 10, and 11) or presence of GMP-PNP (lanes 4, 5, 8, 9, and 12–19) after preincubation with 5 μM (lanes 16 and 17) and 50 μM (lanes 18 and 19) B1 peptide. 50% of the used Toc fraction (lane 1), the flow through (F, even lanes), and the bound fractions (B, uneven lanes) were separated on SDS-PAGE followed by immunodecoration using antisera against Toc159, Toc75, and Toc34. (F) Transmission EM of Toc complex treated with the peptide covalently linked to nanogold particles in the absence (right) or presence of 0.5 mM GTP and 1 mM MgCl2 (left). In the presence of 0.5 mM GTP, 90% of the observed gold particles (arrowheads) were shown to be associated to Toc complex clusters, whereas under nonbinding conditions the gold appeared to be randomly distributed over the carbon surface. (G) Representative complexes with one to four (from left to right) gold particles are shown (top), increasing the contrast to show the gold particles (bottom).

Figure 5.

Figure 5.

Two-dimensional single particle analysis. (A) A silver-stained gel of the sample used is shown. (B) An electron micrograph of the sample recorded as described using the two-layer carbon technique is shown. A gallery of selected Toc complex particles (C) and the average of 3,207 aligned images is shown (D). (E) At very high contrast, only the densest regions of the image are visible. (F) Particles were classified into eight classes. The numbers in the lower right corner represent the number of images comprising each class. See also Fig. S1 available at

http://www.jcb.org/cgi/content/full/jcb.200210060/DC1

.

Figure 6.

Figure 6.

Symmetry of the particles by comparing the two-layer carbon and deep stain technique. Size and shape of the Toc complex particles in deep stain (A, C, and E) and two-layer carbon stain (B, D, and F) were determined at a calibrated magnification of 58,300×. A and B show the average of centered particle images before rotational alignment (4,028 images in A and 2,058 images in B). C and D are the averages of the rotationally and translationally aligned two-dimensional images, respectively. E and F show the average of the three-dimensional reconstructions. (G) Results of rotational symmetry analysis of the two-dimensional particles are listed.

Figure 7.

Figure 7.

Three-dimensional volume of the core complex. The three-dimensional volume of a class comprising 679 particles shown as rendered volume surface (A–C, F, and G) as a series of slices perpendicular to the z axis. (A) A threshold series of the top view is given starting at low threshold (1). (B) The side view (top left) and top view (top right) of the volume at threshold similar to 4 in A is shown. The bottom part shows the volume rotated by 180° along the z axis. C shows the same views at a lower threshold. (D) The Fourier ring correlation and noise correlation reference curves indicate a resolution of 32 Å. (E) A series of z slices spaced by 3.5 Å. (F) The top view at the same threshold as in 4 (A) is shown enlarged. (G) The left half of the volume is removed to reveal the interior structure.

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