Figure 4.
Toc75 Was Cross-Linked to OEP14-[125I]-ADP.
(A) Schematic representation of OEP14-His6 mutant V8C. The targeting/insertion signal (open box) and the hexa-histidine tag (filled box) are shown.
(B) Label-transfer cross-linking experiment with V8C-[125I]-ADP. Chloroplasts were incubated with V8C-[125I]-ADP, and cross-linking was activated by UV irradiation. Chloroplasts were pelleted, and a small portion was analyzed by SDS-PAGE and viewed by autoradiography (lane 1) or by immunobloting decorated with anti-OEP14 antibodies (lane 2). Asterisks indicate the V8C-[125I]-ADP multimers, which were also recognized by the anti-OEP14 antibodies. Closed circles indicated nonspecific cross-hybridization by the antibodies. Envelope membranes (Env) were isolated from the rest of the chloroplasts. A small portion of the membranes was analyzed by SDS-PAGE and autoradiography (lane 3). The open circle indicates the 55-kD protein of unknown identity. The rest of the membranes were solubilized and immunoprecipitated (IP) with antibodies against Toc34, Toc75, and Toc159 (lanes 4 to 6). Lane 7 shows an immunoblot of total chloroplast proteins probed with anti-Toc75 antibodies. The position of the IgG heavy chain is marked by a bracket. Molecular masses of marker proteins are labeled at left.