Identification of protein transport complexes in the chloroplastic envelope membranes via chemical cross-linking

Abstract

Transport of cytoplasmically synthesized proteins into chloroplasts uses an import machinery present in the envelope membranes. To identify the components of this machinery and to begin to examine how these components interact during transport, chemical cross-linking was performed on intact chloroplasts containing precursor proteins trapped at a particular stage of transport by ATP limitation. Large cross-linked complexes were observed using three different reversible homobifunctional cross-linkers. Three outer envelope membrane proteins (OEP86, OEP75, and OEP34) and one inner envelope membrane protein (IEP110), previously reported to be involved in protein import, were identified as components of these complexes. In addition to these membrane proteins, a stromal member of the hsp100 family, ClpC, was also present in the complexes. We propose that ClpC functions as a molecular chaperone, cooperating with other components to accomplish the transport of precursor proteins into chloroplasts. We also propose that each envelope membrane contains distinct translocation complexes and that a portion of these interact to form contact sites even in the absence of precursor proteins.

Figure 1

Cross-linking scheme. (i) Precursor proteins are bound to chloroplasts in the presence of low levels of ATP. (ii) Chloroplasts containing precursor proteins are treated with a cleavable cross-linker. (iii) Cross-linked complexes are solubilized with detergent and purified. (iv) Complexes are analyzed by SDS-PAGE either with (iv-b) or without (iv-a) cleavage of cross-links.

Figure 2

Chloroplast-bound precursor protein treated with various cross-linkers. In vitro translated ³⁵S-labeled prSS and chloroplasts were incubated in the dark with 75 μM ATP for 20 min at 4°C. Intact chloroplasts were repurified through a cushion of 40% Percoll and then treated with indicated concentration of DST (A), DSP (B), or DTSSP (C) for 15 min at 4°C. Cross-linking reactions were quenched with 50 mM glycine. Chloroplasts were recovered by centrifugation and dissolved in electrophoresis sample buffer. Crosslinked products were analyzed by SDS-PAGE under reducing (DST) or non- reducing (DSP and DTSSP) conditions, followed by fluorography. Major cross-linked complexes generated by DST (T), DSP (P), and DTSSP (S) are shown with numbers 1 to 4 in order of decreasing size. Arrowhead, prSS; SG, stacking gel.

Figure 3

Fractionation of cross-linker–treated chloroplasts. After cross-linking with 2.5 mM DSP (lanes 2–6) or 10 mM DTSSP (lanes 7–11), intact chloroplasts containing bound prSS (lanes 2 and 7) were hypertonically lysed, and soluble (lanes 3 and 8) and insoluble (lanes 4 and 9) fractions were separated by centrifugation. The pellet was resuspended in solution containing 1% LDS and subjected to another centrifugation. Solubilized (lanes 5 and 10) and insoluble (lanes 6 and 11) fractions were analyzed by SDS-PAGE and fluorography. Lane 1 shows a sample before cross-linking treatment. Arrowhead, prSS; SG, stacking gel.

Figure 4

Immunoprecipitation of cross-linked products. Cross-linked complexes generated with DSP (A) and DTSSP (B) were solubilized with 1% LDS before immunoprecipitation by antibodies raised against OEP86 (lane 3), OEP75 (lane 4), OEP34 (lane 5), SS (lane 7), LS (lane 8), IEP110 (lane 11), and IEP35 (lane 12). Controls included immunoprecipitation by preimmune sera for OEP86 (lane 2), SS (lane 6), and IEP110 (lane 10). 20% of the sample subjected to immunoprecipitation is shown in lanes 1 and 9. Arrowhead, prSS.

Figure 5

Immunoprecipitation of cross-linked complexes with antibodies against chloroplastic chaperones. Cross-linked complexes generated with DSP (lanes 1–5) and DTSSP (lanes 6–10) were solubilized with 1% LDS before immunoprecipitation by antibodies (I) raised against S78 (lanes 3 and 8) and ClpC (lanes 5 and 10). Controls included immunoprecipitation by preimmune sera (P) for S78 (lanes 2 and 7) and ClpC (lanes 4 and 9). 20% of the samples subjected to immunoprecipitation are shown (T) in lanes 1 and 6. Arrowhead, prSS.

Figure 6

Sephacryl S-500 gel filtration of cross-linked complexes generated with DSP and DTSSP. Cross-linked complexes generated with DSP or DTSSP were solubilized with 1% LDS before being loaded onto a Sephacryl S-500 size exclusion column (1.5 × 42 cm). The elution patterns of radioactivity (solid line) and absorbance at 280 nm (dashed line) are shown in A (DSP) and B (DTSSP). Three consecutive fractions were combined to generate pools as shown on the bottom of A and B. Each pool was analyzed by SDSPAGE under nonreducing conditions, followed by fluorography, as shown in C (DSP) and D (DTSSP).

Figure 7

Analysis of complexes purified by immunoprecipitation. Cross-linked complexes generated with DSP (lanes 1–4) or DTSSP (lanes 5–8) and partially purified by gel filtration on Sephacryl S-500 were further purified by immunoprecipitation with anti-OEP75 antibodies. The immunoprecipitates were treated with β-mercaptoethanol to cleave the cross-links. The resulting proteins were resolved by SDS-PAGE, followed by detection via immunoblotting probed with anti-IEP110, -OEP86, -OEP75, -OEP34, -ClpC, -S78, -LS, and -SS antibodies. Two consecutive pools from the Sephacryl S-500 column were combined and used for immunoprecipitation. The immunoprecipitates from combined pools 7 and 8 (7–8) are shown in lanes 1, 2, 5, and 6; the immunoprecipitates from combined pools 9 and 10 (9–10) are shown in lanes 3, 4, 7, and 8. The samples before and after immunoprecipitation are shown (T and IP). 20% of the samples subjected to immunoprecipitation were loaded on T lanes.

Figure 8

Comparison of components in the complexes generated in the presence of prSS or in the absence of prSS. Cross-linked complexes were generated with DSP in the presence of prSS (lanes 1–4) or in the absence of prSS (lanes 5–10). The complexes were partially purified by gel filtration on Sephacryl S-500 and purified by immunoprecipitation with antibodies against OEP75 (lanes 6 and 9) or IEP110 (lanes 2, 4, 7, and 10). The immunoprecipitates were treated with β-mercaptoethanol and analyzed as described in Fig. 7. Two consecutive pools from the Sephacryl S-500 column were combined and used for immunoprecipitation. The immunoprecipitates from combined pools 7 and 8 (7–8) are shown in lanes 1, 2, 5, 6, and 7; the immunoprecipitates from combined pools 9 and 10 (9–10) are shown in lanes 3, 4, 8, 9, and 10. Samples before immunoprecipitation are shown (T). The volume used was 20% of that subjected to immunoprecipitation.