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. 2010 Jul 13;285(39):29738–29749. doi: 10.1074/jbc.M109.099069

FIGURE 5.

FIGURE 5.

The thioredoxin-like encoding gene complements the ccs5 mutant. For ABC, the ccs5 mutant (T78.15b−) carries the PmR cassette, and the CCS5 -complemented transformants result from the co-transformation of the PmR cassette and the CCS5 gene. A, fluorescence kinetics indicate restoration of cytochrome b6f in the complemented ccs5 mutant. Fluorescence induction and decay kinetics of the ccs5, wild type, and ccs5 complemented with the wild type CCS5 gene. One representative transformant (lane a) is shown. Fluorescence transients were measured on colonies grown for 2 days on solid TAP medium after a short dark adaptation using FluorCam 700 from Photon Systems Instruments. The fluorescence is in arbitrary units (A.U.) and recorded over a 3-s illumination period. B, the CCS5 gene restores phototrophic growth to the ccs5 mutant. 10-Fold dilution series of ccs5 and a representative ccs5 transformant (lane a) carrying the CCS5 gene have been plated on acetate (heterotrophic conditions, 20 μmol/m2/s of light) and minimal medium (phototrophic conditions, 500 μmol/m2/s of light) and incubated for 1 week at 25 °C. C, plastid c-type cytochromes (cyt) accumulation is restored in the CCS5-complemented ccs5 strain. Wild type (CC124), ccs5, and two independent ccs5 transformants (lanes a and b) carrying the CCS5 gene were analyzed for cytochrome f and cytochrome c6 accumulation via heme stain and immunoblot analyses. Samples corresponding to 18 μg of chlorophyll were separated in 12% SDS-acrylamide gel to detect cytochrome f, CCS5, and CF1 of the ATPase that serves as a loading control. Samples corresponding to 16 μg of chlorophyll were separated in 15% native acrylamide gel to detect cytochrome c6. For an estimation of the protein abundance in the ccs5 complemented strain, dilutions of the wild type sample were loaded on the gel. Gels were transferred to polyvinylidene difluoride membranes before heme staining by chemiluminescence and immunodetection with antisera against Chlamydomonas cytochrome f, cytochrome c6, CCS5, or CF1.