Marco Conceptual

In addition to the strong binding of Toc159 to the N-terminal peptide it was shown that the C-terminal non-phosphorylated peptide induces the GTPase activity of the receptor (Jelic, personal communication). The impact of the observed interactions on protein import across the Toc translocon was further analysed. Isolated chloroplasts were preincubated with the different peptides and the import was initiated by addition of radio-labelled pSSU (Fig. 6A). Preincubation with B2 and E2 lead to a 50% decrease in import, whereas A1 had no effect (Fig. 6A, lane 2-4). These findings support the earlier result that B2 and E2 strongly bind to Toc34 or Toc159, respectively, whereas A1 reveals a low affinity for Toc159 (Fig. 5C and Fig. 5D). After preincubation with expressed full-length pSSU the import of radio-labelled pSSU was almost completely abolished (Fig. 6A, lane 5).

To manifest an effect of the peptides on the Toc core translocon, isolated Toc complex was reconstituted into liposomes as previously described (Schleiff et al., 2003b). Proteoliposomes were incubated with radio-labelled pSSU in the presence of GMP-PNP to stimulate binding (Fig. 6B, lane 15 and 16) or GTP to promote import (Fig. 6B, lane 23 and 24). To distinguish bound and pSSU imported into proteoliposomes a trypsin treatment was performed in order to digest surface bound preproteins, whereas imported pSSU remains unaffected (Schleiff et al., 2003b). In the presence of GMP-PNP the precursor remains protease sensitive indicating its presence on the surface of the liposomes (Fig. 6B, lane 16), while in the presence of GTP pSSU was protease resistant indicating its translocation into the proteoliposomes (Fig. 6B, lane 24). The binding of the preprotein in the presence of GMP-PNP or GTP was enhanced compared to the control conditions with no nucleotides (Fig. 6B, lane 7, 15 and 23). Proteoliposomes were subsequently preincubated with the peptides A1, B2 or E2

before addition of precursor protein (Fig. 6B, lane 1-6, 9-14 and 17-22). A1 slightly reduces the binding of pSSU in the absence or presence of GMP-PNP in comparison to the amount of bound pSSU in the absence of peptides (Fig. 6B, lane 3 and 11). However, A1 does not impair import of pSSU into proteoliposomes since the protease protected form of pSSU was detected (Fig. 6B, lane 20). In contrast, E2 reduces pSSU binding in the absence of nucleotides (Fig. 6B, lane 5) and like B2 inhibits import of pSSU (Fig. 6B, lane 18 and 22). Under binding conditions in the presence of GMP- PNP B2 is the only peptide, which leads to a reduction of pSSU association to the proteoliposomes (Fig. 6B, lane 9). This observation can be explained by a preceded binding of B2 to Toc34 while the interaction of Toc159 with E2 takes place at a later stage and inhibits import.

Figure 6. Import of pSSU is reduced by the N-terminal and phosphorylated C-terminal part of pSSU. (A)

Isolated chloroplasts were incubated with radio-labelled pSSU translated in reticulocyte lysate for 10min at 20°C in the absence (lane 1) or presence of 5 µM peptides of pSSU presequence (lane 2-4) or pSSU (lane 5). Samples were separated via SDS-PAGE and visualised by phosphor-imager. The import rate into isolated chloroplasts is given in percentage of input. (B) Proteoliposomes with reconstituted Toc complex were incubated with radio-

labelled in reticulocyte lysate translated pSSU in the absence (lane 1-8) or presence of GMP-PNP (lane 9-16) or GTP (lane 17-24). The binding and import reaction was performed in the absence (lane 7-8, 15-16, 23-24) or presence of peptides of pSSU presequence (lane 1-6, 9-14, 17-22). Surface bound preproteins were digested by trypsin treatment (even lanes). (C) Import of radio-labelled pSSU translated in reticulocyte lysate into

proteoliposomes with enclosed stromal fraction containing either reconstituted Toc complex (upper panel) or co- reconstituted Toc159 and Toc75 (lower panel) was performed in the absence (lane 1-2) or presence of peptides of the pSSU presequence (lane 3-8). (D) Toc complex proteoliposomes were incubated with in vitro translated

pSSU using reticulocyte lysate (35_{S-pSSU-Ret, up) or wheat germ lysate (}35_{S-pSSU-WG, middle) or with}

in vitro

phosphorylated pSSU (32_{P-pSSU, down), in the absence (lane 1, 2) or presence of GTP (lane 3, 4). Surface}

bound preproteins were digested by trypsin treatment (lane 2, 4).

In a parallel approach proteoliposomes were loaded with stromal extract containing the precursor processing peptidase (Schleiff et al., 2003b). In this system a processing event of pSSU to mSSU (mature form of pSSU) indicates a successful import reaction (Fig. 6C, lane 1). To reveal the specificity of the peptides the assays were conducted with proteoliposomes containing the Toc complex (Toc159, Toc75 and Toc34; upper panel) or Toc159 and Toc75 as a minimal import competent unit (lower panel, Schleiff et al., 2003b). The proteoliposomes were now incubated with A1, B2 or E2 before adding the full-length precursor. The B2 and E2 peptides are able to inhibit translocation of pSSU in proteoliposomes containing the Toc complex (Fig. 6C, lane 5 and 7, upper panel), whereas only E2 prevents pSSU import into proteoliposomes containing Toc159 and Toc75

(Fig. 6C, lane 7-8, lower panel). This result supports the observation that B2 is strongly bound by Toc34 but not by Toc159, whereas E2 is only recognised by Toc159 with high affinity (Fig. 5C and 5D). In contrast, import of pSSU into proteoliposomes with the reconstituted Toc complex is only slightly reduced by the presence of A1 (Fig. 6C, lane 4, upper part). This can be explained by the low affinity binding of A1 to Toc34 (Schleiff et al., 2002). Preincubation with A1 does not affect import into proteoliposomes with reconstituted Toc159 and Toc75. This result indicates a transient interaction of Toc159 to A1 since it is capable to stimulate GTP-hydrolysis of Toc159 but it does not block the import event. To reveal the activity of the signal peptidase in all cases membranes were solubilised with the detergent Triton X-100 to release the signal peptidase, which results in processing of pSSU (Fig. 6C, lane 2, 4, 6 and 8).

It was shown that Toc34 recognises the C-terminal phosphorylated peptide, where Toc159 interacts with the non-phosphorylated C-terminal part. This indicates that dephosphorylation before translocation across the envelope membranes occurs already at the Toc complex. To support this conclusion, pSSU was translated either in reticulocyte lysate (resulting in no phosphorylation) or in wheat germ lysate (resulting in phosphorylation (Waegemann and Soll, 1996)). In addition purified pSSU was phosphorylated in vitro. All three different kinds of pSSU were used for import assays into

proteoliposomes with reconstituted Toc complex (Fig. 6D). In line with previous results translocation of pSSU that was translated in reticulocyte lysate was only achieved in the presence of GTP (Fig. 6D, lane 4, Schleiff et al., 2003b) as judged by the protease resistant form. When in vitro phosphorylated

or in wheat germ lysate translated pSSU was used (Fig. 6D, middle, lower panel) no protease resistant pSSU could be observed. The results indicate that phosphorylated pSSU cannot be translocated across the membrane.