Fig. 5.

VSR1 interacts with itself or with MID via an N-terminal domain. (A–D) Yeast two-hybrid experiments with schematics of bait and prey constructs shown on the left, and dilutions of yeast expressing indicated construct pairs spotted on the right. Results are summarized as - (no interaction), + (weak interaction), or ++ (strong interaction). The RWP-RK domains are depicted as diamonds and the protein dimerization domains (DDs) are depicted as rectangles. VSR1 is shown in pink (Chlamydomonas) or red (Volvox), and MID is shown in light blue (Chlamydomonas) or dark blue (Volvox). Empty indicates empty vector controls. Growth of two independent cotransformants spotted in 1:5 serial dilutions on permissive media (-LW) or interaction-dependent media (-AHLW) are shown. White colonies on -LW and growth on -AHLW indicate the interaction between introduced bait and prey proteins. (E and F) Coimmunoprecipitation (IP) of VSR1 and MID in Chlamydomonas (E) and Volvox (F). (E) Co-IPs for Chlamydomonas were done in two strains, both expressing a FLAG epitope–tagged transgenic MID protein rescuing a mid-2 deletion allele (8). One strain also expressed a transgene encoding a HA-epitope–tagged VSR1 protein as described in SI Appendix, Fig. S4A. Immunoblots (IBs) with indicated antibodies are shown in each row with the Ponceau S-stained membrane below. Input and unbound lanes were loaded with 1/60 of the total sample. Pellet lanes were loaded with 1/10 of the total sample. (F) Co-IP with αFLAG from sexually induced Volvox pseudomales expressing OLLAS-tagged MID with and without FLAG-tagged VSR1. Loading amounts were the same as in E for input, unbound, and pellet fractions. (G) Model for bipotential gamete differentiation based on VSR1-VSR1 homodimers binding to and activating promoters of plus/female genes and VSR1-MID heterodimers binding to and activating minus/male genes. See main text for additional details.