Preprotein recognition by the Toc complex

Abstract

The Toc core complex consists of the pore-forming Toc75 and the GTPases Toc159 and Toc34. We confirm that the receptor form of Toc159 is integrated into the membrane. The association of Toc34 to Toc75/Toc159 is GTP dependent and enhanced by preprotein interaction. The N-terminal half of the pSSU transit peptide interacts with high affinity with Toc159, whereas the C-terminal part stimulates its GTP hydrolysis. The phosphorylated C-terminal peptide of pSSU interacts strongly with Toc34 and therefore inhibits binding and translocation of pSSU into Toc proteoliposomes. In contrast, Toc159 recognises only the dephosphorylated forms. The N-terminal part of the pSSU presequence does not influence binding to the Toc complex, but is able to block import into proteoliposomes through its interaction with Toc159. We developed a model of differential presequence recognition by Toc34 and Toc159.

Figure 1

Isolation of Toc159 out of the soluble cell extract. (A) Leaf extract was prepared according to the materials and methods. The pellet (lanes 1–3) or supernatant (lanes 4, 5) of the 500g (lane 1), 10⁵g (lanes 2, 4) and 3 × 10⁵g (lanes 3, 5) centrifugation step were loaded in equivalent amounts onto the chloroplast pellet on SDS–PAGE and immunodecorated with indicated antibodies. (B) The lipid content of the supernatant fractions (see (A)) was analysed by separation over TLC plate. Standard lipids were applied: sulphochinovosyldiglyceride (SL), phosphatidylinositol (PI), phosphatidylglyceride (PG), MGDG, DGDG and phosphatidylcholine (PC). The 10⁵g supernatant was used for coimmunoprecipitation with Toc159 (C, lane 2), NDPK (F, lane 2), MGDG (G up, lane 2) or DGDG-antiserum (G down, lane 2) and their corresponding preimmunsera (C, F, G lane 1). The precipitate was analysed for the presence of chloroplast envelope proteins (C) or Toc159 (G) by immunostaining and lipids by extraction (D, E). (H) Proteoliposomes containing Toc75 were centrifuged at 10⁵g, 3 × 10⁵g and 6 × 10⁵g. Supernatants (lanes 1–3) and pellets (lanes 4–6) were immunodecorated with Toc75 antiserum. (I) The soluble extract from 10⁵g spin (see (A)) was incubated in the absence (lanes 1, 4) or presence of lipase X (lanes 2, 5) or lipase mixture (lanes 3, 6) and subsequently subjected to a 6 × 10⁵g spin centrifugation. Pellets (lanes 1–3) and supernatants (lanes 4–6) were immunodecorated with Toc75 antiserum. (J) An electron micrograph shows an enlargement of the chloroplast periphery of an isolated chloroplast. Arrows mark membrane disturbances of the envelope membranes.

Figure 2

Membrane-bound Toc159. (A) Ultra thin sections of OEVs were incubated with gold-labelled antiserum against Toc159 (left) or Toc34 (right). The staining is visualised by electron microscopy and marked by arrows. The quantification of the distribution of the signals of three independent experiments is depicted as percentages of total signals. (B) Purified OEVs (30μg protein) were incubated with 4M urea for 30min on ice. After separation of pellet (P) and supernatant (S), both fractions were analysed by immunoblotting. (C) Purified OEVs (120μg protein) were treated with trypsin (lanes 2, 7, 12). After the digestion, OEVs were extracted by 100mM Na₂CO₃ (lanes 8, 13) followed by centrifugation (lanes 3, 8, 13), and subjected to post trypsin incubation (lanes 4–6, 9–11, 14–16). The samples were analysed by immunoblotting with the indicated antisera. For comparison, nontreated OEVs were loaded (lanes 1, 17). Typical proteolytic products of Toc159 (circle and triangle), Toc75 (square) and Toc34 (asterisk) are marked. (D) A model of the topology of membrane-bound Toc159 is shown.

Figure 3

Recognition of nonphosphorylated pSSU by Toc159 is GTP dependent. (A) OEVs (50μg protein) were separated by SDS–PAGE and transferred to the nitrocellulose membrane. Nonphosphorylated (lanes 1, 2) and phosphorylated (lanes 3, 4) pSSU in the absence (lanes 1, 3) or presence (lanes 2, 4) of GMP-PNP was bound and visualised by autoradiography. The same blot was immunodecorated with the indicated antisera (below). (B) Proteoliposomes containing Oep16 (lanes 1,2), Toc159 (lanes 3,4) or Toc34 (lanes 5,6) were incubated with in vitro phosphorylated pSSU (P³²-pSSU, up), wheat germ lysate in vitro translated and phosphorylated pSSU (S³⁵-pSSU-WG, middle), or reticulocyte lysate in vitro translated and nonphosphorylated pSSU (S³⁵-pSSU-Ret, down) in the absence (lanes 1, 3, 5) or presence of GMP-PNP (lanes 2, 4, 6). TP indicates 10% of the protein used. (C, D) pSSU (20μg) was coupled to 20μl Toyopearl AF-tresyl 650M column material and subsequently incubated with 250ng Toc159_f. The incubation was performed in the absence or presence of guanine (C) or adenine nucleotides (D). The amount of bound Toc159 in percentage of input was quantified after immunostaining using AIDA software. One representative result of three experiments is depicted.

Figure 4

Toc159 recognises the N-terminal part of pSSU transit peptide with high affinity. (A) An outline of pSSU presequence and its peptides A1, B2 and E2 used in assays is presented. (B) Thiol-activated sepharose (20μl) with bound peptides (0.6mg/ml) was incubated with 250ng Toc159_f without (lanes 1–3) or with GTP (lanes 4–6), GDP (lanes 7–9) or GMP-PNP (lanes 10–12). A BSA-coated column was used as control (lane 13). The quantification of one representative example of three independent experiments is shown. The Toc159 binding to A1 in the presence of GMP-PNP was set to 1. (C) Toyopearl material (20μl) coated with pSSU (1mg/ml) was incubated with 250ng Toc159 in the absence (lanes 2–5) or presence of 0.5mM GMP-PNP (lanes 7–10). The binding was competed by addition of peptides to a final concentration of 5μM. A BSA-coated column was used as control (lanes 1, 6). The quantification of one representative example of three independent experiments is shown. The Toc159 binding to pSSU in the presence of GMP-PNP was set to 1. (D) Same experiment as in (C), but with expressed Toc34. (E) A model of interaction of the presequence of pSSU is depicted.

Figure 5

The nonphosphorylated C-terminal part of the pSSU presequence stimulates GTP hydrolysis by Toc159. (A) Toc159 was incubated with α³²P-GTP for 1h at 20°C (lane 2) followed by spotting on a PEI cellulose plate. GTP alone without any protein was loaded as a control (lane 1). For measurements of GTP hydrolysis of α³²P-GTP by Toc159 (B, solid line), Toc34 (C, solid line) or Toc complex (D, solid line) was determined in the presence of pSSU (dashed-single dotted line), A1 (dashed line), B2 (dotted line), E2 (dashed-double dotted line), A1 and E2 (grey solid line), and B2 and E2 (dashed grey line). At indicated time points, GTP and GDP were separated on a PEI cellulose plate and hydrolysis was quantified. Data are shown as depicted on the right side and represent the average of at least three independent measurements. Lines represent the least-square analysis using an exponential equation.

Figure 6

The stability of the Toc complex is guanine nucleotide dependent. Coimmunoprecipitation of purified OEVs (150μg protein) with antisera against Toc34 (A, left), Toc159 (A, right), preimmunsera (A, lanes 4, 8) or Oep24 (B) was performed in the absence (A, lanes 1, 4, 5, 8; B, lanes 1–3) and presence (A, lanes 2–3, 6–7; B, lanes 4, 5) of guanine nucleotides. In (B), the flow-through (lane 1) and wash (lane 2) of the immunoprecipitation using Oep24 antibodies is shown. The bound proteins were identified by immunoblotting. (C) OEVs (150μg protein) were incubated with EDTA (left), GDP (middle) or GMP-PNP (right), solubilised with 1.5% n-decylmaltoside and lipase treatment and separated by linear sucrose gradient centrifugation. Fractions of the gradient were collected and the protein content was analysed by silver staining. The identified Toc components and the lipase (^*) are marked. (D) The Toc complex from a linear gradient in the absence of nucleotides was incubated with EDTA (left), GDP (middle) or GMP-PNP (right), and subjected to a sucrose step-gradient centrifugation. The protein content of the fractions was analysed by immunoblotting with the indicated antisera. (E) Toc34 (2μg) was preincubated with GMP-PNP (lanes 1–3, 7, 8) or GDP (lanes 4–6, 9, 10) followed by incubation with 250ng Toc159_f (lanes 1, 4) preincubated with GMP-PNP (lanes 3, 6–8) or GDP (lanes 4–6, 9, 10) and pSSU presequence peptides (lanes 7–10). The amount of bound Toc159 is depicted as the percentage of total input. (F) A model of GTP-dependent association of Toc34 to Toc159/Toc75 is presented.

Figure 7

Import of pSSU is reduced by the N-terminal and phosphorylated C-terminal part of pSSU. (A) Isolated chloroplasts were incubated with ³⁵S-labelled pSSU translated in reticulocyte lysate for 10min at 20°C in the absence (lane 1) or presence of 5μM peptides of pSSU presequence (lanes 2–4) or pSSU (lane 5). Samples were separated via SDS–PAGE and visualised by a phospho-imager. The import rate into isolated chloroplasts is given as a percentage of the input. (B) Proteoliposomes with reconstituted Toc complex were incubated with ³⁵S-labelled pSSU translated in reticulocyte lysate in the absence (lanes 1–8) or presence of GMP-PNP (lanes 9–16) or GTP (lanes 17–24). The binding and import reaction was performed in the absence (lanes 7–8, 15–16, 23–24) or presence of peptides of the pSSU presequence (lanes 1–6, 9–14, 17–22). Surface-bound preproteins were digested by trypsin treatment (even lanes). (C) Import of ³⁵S-labelled pSSU translated in reticulocyte lysate into proteolipsomes with enclosed stromal fraction containing reconstituted Toc complex (upper part) or co-reconstituted Toc159 and Toc75 (lower part) was performed in the absence (lanes 1–2) or presence of peptides of the pSSU presequence (lanes 3–8). (D) Toc complex proteoliposomes were incubated with in vitro translated pSSU using reticulocyte lysate (S³⁵-pSSU-Ret, up), wheat germ lysate (S³⁵-pSSU-WG, middle) or in vitro phosphorylated pSSU (P³²-pSSU, down), in the absence (lanes 1,2) or presence of GTP (lanes 3, 4). Surface-bound preproteins were digested by trypsin treatment (lanes 2, 4).

Figure 8

Model of preprotein recognition and translocation by the Toc complex.