These 47 residues appear largely unstructured in the OE17 crystal structure (Calderone translated Tp-GS15-47 (lane 1) was assayed for thylakoid transport with chloroplast lysate for 30 min in the light. a long linker peptide (Fincher, 2001). Although this chimeric precursor stalled midway across them membrane, its linker contained short ZD-0892 hydrophobic segments that might be misidentified by the translocase as transmembrane domains (Summer were used as the folded protein domains. GS repeats have served as flexible linkers and shown to be unstructured (Kortt (Musser and Theg, 2000). translated OE17 was shown to be folded by partial resistance to limited proteinase K treatment (Supplementary Figure 1), as described (Musser and Theg, 2000). translated protein A was partially resistant to limited proteinase K treatment (Supplementary Figure 1), and also efficiently bound to IgG Sepharose (data not shown), further supporting that it is folded. Our first objective was to determine if a defined unstructured linker between pOE17 and protein A would result in translocation arrest, as had previously been seen (Fincher, 2001). Constructs containing three (GS3). Nine (GS9), and 15 (GS15) GS repeats were prepared (Figure 1A). pOE17-GS3-protA was efficiently imported into chloroplasts (Figure 1B, lanes 2 and 3), localized to thylakoids (lane 5), and processed to mature size. If the import reaction was conducted in the presence of ionophores to dissipate proton gradients, the imported protein accumulated in the stromal fraction (lanes 8C11), indicating that transport was mediated by the cpTat system. Thermolysin treatment of recovered thylakoids gave rise to a smaller degradation product (Figure 1B, lane 6, asterisk) that was larger than mature OE17 (mOE17) and smaller than protein A, suggesting that it contained the linker and mOE17. The ZD-0892 degradation product was not inherently resistant to protease, because thermolysin treatment completely degraded mOE17-GS3-protA if the membrane barrier was disrupted with Triton X-100 (lane 7), or by sonication (Supplementary Figure 2). The precursor, in the absence of thylakoids, was also completely degraded by a comparable amount of thermolysin (Supplementary Figure 2). Open in a separate window Figure 1 Chimeric precursor proteins containing pOE17, linkers consisting of repeats of the peptapeptide GGGGS, and protein A are arrested mid transport by the cpTat pathway. (A) Diagrammatic representation of the chimeric precursor proteins used in this experiment. (B) Import of pOE17-GS3-protA into intact chloroplasts. translated pOE17-GS3-protA (lane 1) ZD-0892 was incubated with intact chloroplasts in an import assay, in the absence (lanes 2C7) or presence (lanes 8C11) of nigericin and valinomycin (Materials and methods). Recovered chloroplasts were either treated with thermolysin (lane 3) or not (lane 2), and intact chloroplasts repurified. The untreated chloroplasts were lysed and fractionated into a stromal fraction (lanes 4 and 9) and a thylakoid fraction (lanes 5 and 10). Aliquots of the ZD-0892 thylakoid fraction were treated with thermolysin in the absence (lanes 6 and 11) or presence of 1% Triton X-100 Rabbit Polyclonal to HBP1 (lane 7). (C) Transport of pOE17-GS(3C15)-protA into washed thylakoids. translated precursors (lanes 1, 5 and 9) were incubated with washed thylakoids in a transport assay (Materials and methods). Recovered thylakoids were mock treated (lanes 2, 6 and 10) or treated with thermolysin, in the absence (lanes ZD-0892 3, 7 and 11) or presence of 1% Triton X-100 (lanes 4, 8 and 12). Translation products (tp) represent 5% of assays; all other samples represent 100% of assays. Designations: p, precursor; m, mature form; DP, degradation product; mk, mock treatment. Asterisks indicate the protease resistant degradation products. Transport properties of constructs with the three different linkers were compared with a washed thylakoid transport assay. Figure 1C shows that the three precursors were processed by thylakoids to mature size (lanes 2, 6 and 10) and gave thermolysin degradation products of increasing size as the linker size increased (lanes 3, 7, 11 and asterisks). Time-course analysis showed that the partially transported proteins are end products of the transport reaction rather than intermediates (Supplementary Figure 3). The identity of degradation products was determined by immunoprecipitation (Figure 2). Thylakoids.