Identification and characterization of EMA1/Ema1p. (A) Conjugation. In the sexual process of conjugation, two cells mate (1); the Mic undergoes meiosis to form two haploid pronuclei, one of which is reciprocally exchanged between the two conjugating cells (2); the migratory and stationary pronuclei then fuse to create a zygotic nucleus (3); the zygotic nucleus divides mitotically twice to produce the next generation of new Macs and Mics (4); paired cells separate and one of the two new Mics and the parental Mac are destroyed (5); and cells resume vegetative growth with Mic mitosis, followed by cytokinesis (6). (B) Copurification of Ema1p with Flag-HA-Twi1p. Two wild-type strains (No-tag) or two Flag-HA-TWI1 strains were mated and were harvested at 9 h post-mixing. Flag-HA-Twi1p-containing complexes were enriched by gel filtration and immuno-affinity purified first with anti-Flag antibody and then with anti-HA antibody. The purified proteins were separated by SDS-PAGE and analyzed by silver staining. The arrow and the asterisk indicate the positions of Flag-HA-Twi1p and the Twi1p-associated proteins identified by mass spectrometry analysis, respectively. (C) Twi1p coimmunoprecipitates with Ema1p. An EMA1-HA strain and a wild-type strain were mated and lysed at 6 h post-mixing. (Right) The Ema1p-HA-containing complex was pulled down with an anti-HA antibody. (Left) As a control, two wild-type strains were crossed and processed similarly. Coimmunoprecipitated proteins (anti-HA IP) or total proteins (Input) used for immunoprecipitation were analyzed on Western blot using anti-Twi1p antiserum. (D) Expression of EMA1 mRNA. Total RNAs from exponentially growing (E), starved (S), and conjugating (2, 4, 6, 8, 10, 12, and 14 h post-mixing) wild-type cells were analyzed by Northern hybridization. RPL21 was used as a loading control. (E–L) Localization of Ema1p. Mating pairs of wild-type cells in the early (E) and late (F) premeiosis, pronuclear exchange (G), Mac Anlagen (H,I), Nuclear Alignment (J), Pair Separation (K), and Mic Elimination (L) stages were processed for immunostaining. See Supplemental Figure S1 for the conjugation stages. Ema1p was localized using anti-Ema1p antiserum (left) and DNA was stained by DAPI (right). Arrows, arrowheads, and arrowheads marked with “An” indicate Macs, Mics, and developing new Macs, respectively. The staining detected at the junction of cells (double arrowheads) was also observed in ΔEMA1 strains (see ) and thus represents cross-reaction of the antiserum with other proteins.