Figure 5.

Import ability of chloroplasts isolated from the pale-green tic56-3 mutant. A, In vitro chloroplast protein import assay with chloroplasts isolated from 27-d-old wild-type and tic56-3 seedlings. For the import reactions, the chloroplast suspensions of the two plant types were adjusted to equal protein levels. The chloroplasts were incubated with two different radiolabeled import substrates, preE1α (top) and preSSu (bottom), and import was allowed to proceed for 0, 7.5, or 15 min. The samples were analyzed by SDS-PAGE and autoradiography. As a loading control, part of the Coomassie Blue-stained SDS-PAGE gel is shown underneath the corresponding autoradiograph. IVT, Product of in vitro translation. The photographs above the gels show the phenotypes of the tic56-3 Arabidopsis mutant in comparison with the wild type (ecotype Wassilewskija) grown on Murashige and Skoog (MS) agar supplemented with 0.8% (w/v) Suc for 14 d under an 8-h photoperiod. B, Quantification of the imported, processed form of the substrates at 15 min of import. The amount of imported substrate in the wild-type sample was set to 100%. Data were derived from three independent experiments. C, Chloroplasts isolated from 27-d-old tic56-3 seedlings have strongly reduced levels of Tic56 or the truncated form of Tic56 as monitored by western blotting. Fifty micrograms of chloroplast protein of tic56-3 and wild-type chloroplasts from two independent preparations was analyzed by western blotting with anti-Tic56 antiserum. D, Chloroplast were lysed hypotonically and separated into soluble (S) and membrane protein (P) fractions by centrifugation. Chloroplasts (cp) and subfractions were subjected to western-blot analysis with anti-Tic56 antiserum. The 130-kD protein and the truncated form of Tic56 (Tic56-TF) became apparent in the membrane protein fraction of tic56-3 chloroplasts (asterisks; compare with Fig. 1B, 16-d-old plants).