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First published online 15 January 2003
doi: 10.1242/jcs.00291

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Membrane insertion of the chloroplast outer envelope protein, Toc34: constrains for insertion and topology

¹ Department of Botany, Ludwig-Maximilian University Munich, 80368 Munich, Germany
² Department of Botany, University Kiel, 24098 Kiel, Germany

View larger version (16K): [in a new window]   Fig. 1. Toc34 and mutants. Toc34, (2-230)Toc34, Toc34C++ and (2-230)Toc34C++ are shown, indicating the deletions and the charge distribution of the 15 amino acids flanking the transmembrane domain. The amino acid sequence of the short constructs is given and the exchanged amino acids are presented in bold. The position of the [35S]-labelled methionines and [3H]-labelled leucines is also shown.

View larger version (34K): [in a new window]   Fig. 2. Insertion of Toc34 and Toc34C++ into chloroplasts under various conditions. (A) [3H]-Leucine-labelled Toc34 was incubated for 5 minutes with chloroplasts (lanes 2-9) then treated with thermolysin (Thr, lanes 3, 5, 7 and 9). Before incubation, chloroplasts were incubated for 10 minutes with 10 µg of purified preSSU (pSSU, lanes 4 and 5) or 10 mM spermine (spm, lanes 6 and 7). Lane 1 shows 10% translation product (TP). (B) [3H]-Leucine-labelled translation product minus RNA (lanes 2 and 3), or Toc34C++ (lanes 5 and 6), was used for insertion into chloroplasts, which was followed by thermolysin treatment (Thr, lanes 3 and 6). Lanes 1 and 4 show 10% translation product. A model of the orientation of the wt protein and the charge mutant C++ is presented between A and B. (C) [3H]-Leucine-labelled Toc34 or [35S]-methionine-labelled precursor of AOX (10% translation product in lane 1) were incubated with mitochondria for 15 minutes (lanes 2-4); mitochondria were then treated with 120 µg/ml thermolysin (lane 3, Th) or 100 mM Na2CO3 (lane 4, M) for 30 minutes at 4°C. Thermolysin activity was stopped by the addition of EDTA (lane 3) and membranes were isolated by centrifugation at 200,000 g (lane 3).

View larger version (19K): [in a new window]   Fig. 3. Insertion of Toc34 into protein-free membranes depends on the transmembrane region. (A) [3H]-Leucine-labelled Toc34 (lane 1) was treated with thermolysin (Thr, lanes 2, 4 and 6) before (lane 2) and after insertion into free liposomes (1 mM final lipid concentration, lanes 3-6), followed by incubation in 100 mM Na2CO3 (lanes 5 and 6) for 30 minutes at 4°C and pelleting of the membrane fraction. (B) [3H]-Leucine-labelled Toc34-252 and [35S]-methionine-labelled Tic40 (10% TP, lane 1) were incubated with liposomes (lanes 2-4) without (lane 2) and with (lanes 3, 4) competition with N-liposomes, followed by thermolysin treatment (lane 4).

View larger version (22K): [in a new window]   Fig. 4. Insertion of Toc34 into protein-free liposomes under different conditions. (A) [3H]-Leucine-labelled Toc34 was incubated with protein-free liposomes (1 mM final lipid concentration, lanes 2-11) of different lipid composition (for nomenclature, see Table 1) or of outer envelope lipids (OEL, lanes 12-14), followed by thermolysin treatment (Thr, lanes 3, 5, 7, 9, 11, 13 and 14). In lane 14 the membrane was solubilised by TX-100 before thermolysin treatment. Binding (before thermolysin treatment, black bar) and insertion (8 kDa fragment after thermolysin treatment corrected for the number of leucine residues, grey bar) was quantified as described in Materials and Methods and is shown as a histogram. The binding of Toc34 to liposomes of composition C3 was set to 100%. The results represent an average of at least three independent experiments. (B) [3H]-Leucine-labelled Toc34 was incubated with protein-free liposomes (1 mM final lipid concentration, composition C3, lanes 1 and 2) in the presence of 1 mM MgCl2 (lanes 1-10) and 1 mM GTP (lane 3 and 4), 1 mM GMP-PNP (lanes 5 and 6), 1 mM GDP (lanes 7 and 8) or 1 mM ATP (lanes 9 and 10) followed by quantification of binding and insertion as for Fig. 3. Binding of Toc34 to liposomes in the absence of nucleotides was set to 100%. The results represent an average of at least three independent experiments.

View larger version (42K): [in a new window]   Fig. 5. Insertion of Toc34 and variants into chloroplasts outer envelope membrane. (A) [3H]-Leucine-labelled Toc34 (upper, left), (2-230)Toc34 (lower, left), Toc34C++ (upper, right) and (2-230)Toc34C++ (lower, right) were incubated with chloroplasts as described and shown in the legend for Fig. 2. Binding and insertion was quantified as for Fig. 3. The binding of Toc34 was set to 100% and the numbers in brackets indicate the s.e.m. The models indicate the orientation of the proteins and the location of the C-terminus. The results represent an average of at least three independent experiments. (B) Insertion of [3H]-leucine-labelled (lane 1-3) and [35S]-methionine-labelled (lanes 4-6) (2-230)Toc34 (upper panel) and (2-230)Toc34C++ (lower panel) into chloroplasts (lanes 2, 3, 5 and 6) was performed as described in Fig. 2 and followed by thermolysin treatment (Thr, lanes 3 and 6). In lanes 1 and 4, 10% of the translation product is shown.

View larger version (42K): [in a new window]   Fig. 6. Insertion of Toc34 and variants into protein-free liposomes. [3H]-Leucine-labelled Toc34 (upper, left), (2-230)Toc34 (lower, left), Toc34C++ (upper, right) and (2-230)Toc34C++ (lower, right) were incubated with liposomes (1 mM final lipid concentration, composition C3), and binding and insertion was quantified as described in the legend for Fig. 5. The binding of Toc34 to protein-free liposomes was set to 100%. The results represent an average of at least three independent experiments and the numbers in brackets indicate the s.e.m. The models indicate the orientation of the proteins, and C the location of the C-terminus. (B) Insertion of [3H]-leucine-labelled (lanes 1-3) and [35S]-methionine-labelled (lanes 4-6) (2-230)Toc34 (upper panel) and (2-230)Toc34C++ (lower panel) into liposomes (lanes 2, 3, 5 and 6) was performed as described in Fig. 2 and followed by thermolysin treatment (Thr, lanes 3 and 6). In lanes 1 and 4, 10% of the translation product is shown.

View larger version (47K): [in a new window]   Fig. 7. Insertion of Toc34 and variants into protein-free liposomes lacking PG. (A) Insertion of [3H]-leucine-labelled Toc34 (upper, left), (2-230)Toc34 (lower, left), Toc34C++ (upper, right) and (2-230)Toc34C++ (lower, right) into liposomes of the composition C5 (Table 1) was performed, quantified and presented as described in the legend for Fig. 6. (B) Insertion of [3H]-leucine-labelled (lanes 1-3) and [35S]-methionine-labelled (lanes 4-6) (2-230)Toc34 (upper panel) and (2-230)Toc34C++ (lower panel) into protein-free liposomes not containing PG (lanes 2, 3, 5 and 6) was performed as described in Fig. 2 and followed by thermolysin treatment (Thr, lanes 3 and 6). In lanes 1 and 4, 10% of the translation product is shown.