Cnn1 inhibits the interactions between the KMN complexes of the yeast kinetochore - PubMed (original) (raw)

Cnn1 protein–protein interactions and localization to the inner kinetochore. (a) SDS–PAGE gels depicting Cnn1 and co-purifying proteins. Cnn1 was tagged with a 3FLAG or GST epitope and expressed from PGAL1, which replaced the endogenous promoter of CNN1 (PGAL1–CNN1–3FLAG) or was placed on a 2 μ vector (p_PGAL1–GST–CNN1_). Both strains and the untagged parent (negative control) were grown in 2% galactose YP medium (23 °C). For purification details, see Methods. The left, central and right panels indicate peptides corresponding to Cnn1 (red) and enriched proteins (blue) that were identified by mass spectrometry following five, two or three washes of the affinity beads, respectively. The number of peptides and MASCOT scores of the enriched proteins were: left panel: Cnn1 (6, 5, 4, 4, 3, 2, 3, 2, 2; 384, 272, 195, 193, 148, 141, 126, 113, 80), Tub1 (6; 394), Tub2 (5; 346); central panel: Cnn1 (15, 10, 10, 6, 6, 6, 5, 4, 3, 3; 891, 602, 500, 343, 295, 288, 199, 182, 159, 126), Tub2 (4; 174), Spc25 (2; 90), Sgt1 (3, 134), Mtw1 (3; 125), Bik1 (3; 118); and right panel: Cnn1 (7, 7, 6, 4, 4; 423, 319, 298, 223, 213), Spc24 (3; 203) and Spc25 (8; 346). (b) Identification of Cnn1 interactors in Gal4-based Y2H screens. Cnn1 fragments (purple bars) were chosen from the predicted secondary structure of Cnn1 (plotted,

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) and tagged N- or C-terminally with the Gal4 DNA-binding domain (DBD). The bait constructs were screened against all S. cerevisiae ORFs tagged C-terminally with the Gal4 activation domain. The construct containing the first 150 residues of Cnn1 consistently interacted with Spc24, Spc25 and Ndc80 (Ndc80 complex). Cnn1 interacted only once with Duo1 (Dam1 complex; n.d., not determined). (c) Cnn1 interactors identified in affinity purifications (blue arrows, this study; dashed blue arrows, published,) and Y2H screens (red arrows: this study; dashed red arrows, published,). (d) Kinetochore localization of Cnn1 (purple ellipse) as revealed by CENIV ChIP (Supplementary Fig. S4). The blue arrows indicate the kinetochore recruitment pathways. Recruitment code: green, total dependence; orange, partial dependence; red, independence.