Figure 3.

Accumulation of soluble and membrane-bound aCRY at different stages of the Chlamydomonas sexual cycle. A, The expression of soluble aCRY in vegetative cells (V), pG, and gametes (G) of mating type plus and minus (mt⁺ and mt⁻) as well as in early zygotes (eZ) and zygotes (Z). V, pG, G, and eZ were prepared as described in “Materials and Methods.” +N or −N represents the presence or the absence of a nitrogen source in the medium. Black bars on the top indicate darkness and white bars light. Seventy-five-microgram proteins of each sample were separated by 9% SDS-PAGE and immunoblotted with anti-aCRY antibodies. Unspecified protein bands from the PVDF membrane stained with Coomassie Brilliant Blue R250 after immunochemical detection were used as loading control (LC). B, Expression of aCRY in a soluble protein fraction of late zygotes under darkness (D), and 6 h (L6), 12 h (L12), and 24 h (L24) illumination, respectively. The modified version of aCRY (upper band in D, L6, L12, and L24) was quantified in combination with aCRY (indicated as dark plus light gray portion) as well as alone (light gray portion). C, The expression of aCRY in the membrane fractions of V, pG, and G of mt⁺ and mt⁻ wild-type (WT) strains, as well as of early zygotes (eZ). D, The expression of aCRY in the membrane fraction of Z under darkness (D) as well as after 6 h (L6), 12 h (L12), and 24 h (L24) illumination. A to D, Quantified aCRY protein levels of three biological replicates relative to the protein level of V cells of the mt⁻ strain, harvested at LD24 in the dark, are shown in each diagram with standard deviations.