FIGURE 6.

Cr-cpSRP54 is not involved in transit complex formation in Chlamydomonas. A–D, protein complex formation using the following equimolar combinations of recombinant proteins was analyzed by size exclusion chromatography: Cr-His-cpSRP43 and Cr-LHCP (A) and Cr-His-cpSRP43 (43), Cr-LHCP (LHCP), and Cr-His-cpSRP54M (54M) (B). As controls, the single proteins Cr-His-cpSRP43 (C) and Cr-His-cpSRP54M (D) were analyzed. Elution fractions ranging from 8 to 18.5 ml were analyzed by SDS-PAGE and Coomassie staining (A–D). Cr-His-cpSRP54M was added to the combination of Cr-His-cpSRP43 and Cr-LHCP after dilution and removal of SDS (B). The same elution profiles were obtained when Cr-His-cpSRP54M was added to this combination before SDS removal.³ Single proteins were always treated in the same way as in assays analyzing complex formation. E, pulldown assays were conducted by incubation of recombinant Cr-GST-cpSRP43 and the Cr-LHCP in vitro translation product (TP) with the indicated combinations of recombinant Cr-cpSRP54-His and a stromal extract from Chlamydomonas. Control reactions were performed with recombinant GST. Proteins were enriched using glutathione-Sepharose. Samples of the load and the eluted fractions were analyzed by Western blotting using antibodies directed against the GST tag, His tag, or LHCP. The asterisk (*) marks signals caused by an unspecific cross-reaction of the α-Cr-LHCP antibody with the eluted GST protein. To detect endogenous stromal Cr-cpSRP54 (Cr-54) in the load and eluted fractions, the samples from pulldown assays conducted in the presence of Cr-GST-cpSRP43, Cr-LHCP, and stromal extract were blotted and analyzed using an antibody directed against Cr-cpSRP54M. mAU, milliabsorbance units.