Figure 1.

Association of Toc159 with Tic56, a component of the 1-MD TIC complex. A, SyproRuby stain of an eluate obtained after the purification of TAP-Toc159 from Triton X-100-solubilized membrane proteins of TAP-Toc159:ppi2 plants (159). As a control, the same purification procedure was performed with wild-type plants (c). Subsequent mass spectrometric analysis of gel slices A to F revealed the presence of Tic56 peptides in gel slice D (data not shown). B, Specificity of the anti-Tic56 serum. Fifty micrograms of protein of total protein extracts of the wild type (WT) and of two tic56 T-DNA insertion mutants (tic56-1 on the left and tic56-3 in the center) was analyzed by SDS-PAGE and immunoblotting with anti-Tic56 antiserum. Each x indicates a 70-kD cross reaction of the serum with a protein present in wild-type and mutant samples. To further analyze the additional bands (asterisks) appearing in tic56-3, chloroplasts of 16-d-old wild-type and tic56-3 seedlings were isolated, treated or not with thermolysin (50 µg mL⁻¹), and subjected to the same immunoblotting procedure. C, Confirmation of the copurification of Tic56 with Toc159 by immunoblotting of fractions from a TAP-Toc159 purification. Fifty micrograms of protein of Triton X-100-solubilized membrane proteins loaded to Homo sapiens (Hs)IgG beads (load), 50 µg of the column flow through (ft), and 25% of two different wash fractions (w1 and w6) and the tobacco etch virus protease eluates (elu) were probed with antisera as indicated.