Fig. 1: Identification of the kinetochore interactome in Cryptococcus neoformans.

a Conservation of kinetochore proteins across the mentioned species. The cladogram represents the relationship between the species; each subphylum is color-coded. The presence (green boxes) or absence (yellow boxes) of each kinetochore protein in every species is shown. Pink arrows indicate loss events of CCAN proteins, excluding CENP-C^Mif2. Orange lines in the cladogram refer to basidiomycete lineages that have lost most CCAN components. The gray arrow points to C. neoformans. Right, schematic of a typical kinetochore ensemble. FLAG-labeled proteins of kinetochore subcomplexes (CENP-C, Mis12C, and Ndc80C) in C. neoformans are indicted and used for the immunoprecipitation (IP)–mass-spectrometry (MS) identification of the kinetochore interactome. Protein complexes identified (solid borders) or those that remained unidentified (dotted borders) by the IP–MS experiment in C. neoformans are shown. b Domain architectures of identified CENP-T^Cnn1 homologs among basidiomycetes. Color coding of subphyla is followed as in a. c Proteins eluted after FLAG-IP, of CENP-C^Mif2 in the C. neoformans strain SHR896 (CENP-C::CENP-C^MIF2-3×FLAG), Dsn1 in SHR824 (DSN1::DSN1-3×FLAG), Spc25 in SHR823 (SPC25::SPC25-3×FLAG), and an untagged wild-type (WT) control (H99) after thiabendazole (TBZ) treatment, were separated on a gradient PAGE gel and silver-stained. Blue and green arrows mark the inner and outer kinetochore bait proteins, respectively. d Micrographs of C. neoformans cells at the M phase expressing GFP-tagged proteins identified by the IP–MS screen. Kinetochores are marked by inner kinetochore proteins, CENP-C^Mif2-mCherry for Bgi1^Bkt1 in SHR876 (BGI1::BGI1-V5-GFP), Bkt2 in SHR897 (BKT2::BKT2-V5-GFP), and Yta7 in SHR842 (YTA7::YTA7-V5-GFP) or mCherry-CENP-A^Cse4 for Mcm6 in SHR905 (MCM6::MCM6-V5-GFP) and Sos7 in SHR845 (SOS7::GAL7p-GFP-SOS7). Scale bar, 3 μm. Source data are available as a Source Data file.