FIGURE 5.

Ras1 interacts with Cac1 and Gib2. A, C. neoformans var. neoformans (serotype D) (C. n. (D)), var. grubii Cac1 (serotype A) (C. n. (A)), and C. gattii (C. g.) contain putative RA domains that are homologous to those of S. cerevisiae (S. c.) and C. albicans (C. a.). Numbers mark the amino acid locations of the RA domains. B, Ras1 interacts with Cac1 through the conserved RA domain in a yeast two-hybrid assay. C, Ras1 and paralog Ras2 interact with Gib2. D, co-immunoprecipitation confirms the interactions between Gib2 and Ras1, Ras1 and Cac1 (RA domain), and Gib2 and eIF4A. Anti-GST and Anti-Xpress antibodies were used to detect the respective fusion proteins following PAGE and Western blot analysis. E, Gib2 interacts with eukaryotic initiation factor eIF4A homolog. The Y2H assay conditions and media used were the same as that described in Fig. 3. DD indicates dropout medium lacking leucine and tryptophan, and QD indicates dropout medium lacking leucine, tryptophan, histidine, and adenine. co-IP was performed following the previously published method (17). AD, pGADT7; BD, pGBKT7.