Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts

Abstract

The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.

Figure 1. Identifying a suitable time point during active import for identification of the translocon complex

(a) [³⁵S]prRBCS was incubated with isolated chloroplasts under import conditions at 20°C. At each time point, a portion of the import mixture was removed and the import reaction terminated by diluting with cold import buffer. An equal amount of protein was loaded in each lane. (b) Chase after 20-min import. [³⁵S]prRBCS was incubated with isolated chloroplasts under import conditions at 20°C for 20 min. Chloroplasts were isolated and resuspended in import buffer containing 3 mm ATP. A portion of the chloroplasts were immediately re-isolated (chase time 0 min), or the reaction was incubated further at 20°C for 10 or 20 min (chase time 10 and 20 min). An equal amount of protein was loaded in each lane.

Figure 2. Sucrose density gradient analyses of solubilized chloroplast membranes after prRBCS import and chase

(a) Chloroplasts were incubated with prRBCS for 20 min under import conditions, then re-isolated and further chased for 0–20 min. Membrane fractions from chloroplasts of each time point were solubilized and analyzed by sucrose density gradients. Filled triangles indicate the four fractions quantified in (b). An immunoblot of the large subunit of Rubisco (RBCL) is shown to indicate the position of endogenous Rubisco holoenzyme. Underlines mark the mature RBCS at the supercomplex fractions. (b) Quantification of fractions representing distinct complexes from gels shown in (a). For each gel, the amount of prRBCS in fraction 19 (supercomplex) was set to 100% and the relative amounts of prRBCS in fractions 5 and 7 and mature RBCS in fraction 13 were then plotted.

Figure 3. Blue-native PAGE analyses of fractions from the two precursor peaks

Fractions 11–19 (a) or fractions 4–9 (b) of the 20-min import samples as shown in Figure 2(a) were analyzed by BN-PAGE. Molecular masses (in kDa) are labeled according to the migration positions of ferritin monomer (440), dimer (880), and trimer (1320). Asterisks indicate the position of Rubisco holoenzyme.

Figure 4. Antibody-shift BN-PAGE analyses of the composition of the C1 and C2 complexes

(a) The sucrose-density gradient fraction containing C1 was incubated with various antibodies, as labeled at the bottom, to remove possible components in C1 (pre., pre-immune serum of the respective antibody). The sharp band between 880 and 1320 kDa was formed due to the presence of a major serum protein at the position marked by the asterisk. (b) Same as in (a) and the experiment was repeated with a second anti-Toc159 serum (αToc159-2). (c) The sucrose-density gradient fraction containing C2 was incubated with various antibodies, as labeled at the bottom, to remove possible components in C2.

Figure 5. Translocon complexes associated with importing prPORB

(a) [³⁵S]prPORB was incubated with chloroplasts under import conditions for 20 min. Total membranes from lysed chloroplasts were analyzed by sucrose density gradient as in Figure 2(a). (b) Fractions 3–10 from (a) were analyzed by BN-PAGE. The asterisk indicates the position of Rubisco holoenzyme. (c and d) Antibody-shift BN-PAGE analyses of the composition of the C1 and C2 complexes, respectively. Sucrose-density gradient fractions containing C1 or C2 were incubated with various antibodies, as labeled at the bottom, to remove possible components (pre., pre-immune serum of the respective antibody). The sharp bands between 880 and 1320 kDa were formed due to the presence of major serum proteins at the positions marked by asterisks. Anti-Tic110 antibodies were affinity purified and therefore did not contain the serum proteins.

Figure 6. Model of translocon complex assembly states during precursor import into chloroplasts

During import, precursors first associate with the C1 complex composed of the Toc components. A population of C1 may contain Toc159 in a different conformation. Another distinct complex, C2, composed of the Toc components, Tic110, Hsp93, and hsp70 exists. However, whether C2 is an intermediate step between C1 and the Tic/Toc supercomplex is not clear. The presence of Tic22 and Tic20 in C2 has also not yet been demonstrated. Other additional translocon components, like Tic40, then assemble to form the Tic/Toc supercomplex. The scissor represents the stromal transit-peptide processing peptidase and its position indicates that processing most likely occurs at the supercomplex.