(a) Cartoon depiction of the assay (GL, glycine-linker; P, phosphate group). (b) Graph showing the percentage of surviving cells ± s.d. of the indicated strains after nocodazole treatment relative to untreated cells (t = 0 h; n = 3). (c) M-Track analysis of Myc-HKMT-Cdc55 and H3-HA-Net1^1-600 in a cdc55Δ/net1Δ strain. The immunoblots show Myc, methylation and HA signals over time after galactose (Gal) induction of HKMT-Cdc55 expression. The plots show methylation signals normalized to HA levels and plotted as a percentage of the highest normalized methylation signal (100%) ± s.d. (n = 3). 2μ = high copy number yeast vector with 2μ origin of replication (d) Immunoblots showing M-Track analysis of Myc-HKMT-Cdc55 and either H3-HA-Net1^1-600 (left) or H3-HA-Net1^1-600(3Cdk) (right) in a cdc55Δ/net1Δ strain. The plots (below) were generated as in c (n = 3). (e) Immunoblots showing M-Track analysis of H3-HA-Net1^1-600 and either Myc-HKMT-Cdc55 in a cdc55Δ strain (left), a Myc-HKMT in a BY4741 strain (center) or a Myc-HKMT-Rts1 in an rts1Δ strain (right). The plots were generated as in c; n = 3.

(a) Cartoon depiction of the M-Track assay. Bait protein 'Y' is fused to the catalytic domain of a human histone lysine (K) methyltransferase (HKMT), and prey protein 'X' is fused to the N terminus of histone H3 and the hemagglutinin epitope tag (HA). Upon interaction with the bait, the prey is stably marked by methylation (M, methyl group). (b) M-Track analysis of rapamycin-induced dimerization of FKBP12-rapamycin binding (FRB)-HKMT and FK506 binding protein (FKBP)-H3-HA in a TOR1-1 fpr1Δ yeast strain. A schematic of the assay and immunoblots with the indicated antibodies are shown. The plot shows fold changes of the trimethylation signal normalized to HA levels; plotted values are the mean ± s.d. (n = 3). Basal signal at t = 0 min after rapamycin treatment was set to 1. CEN = centromeric, low copy number yeast vector. (c) M-Track analysis of Pbs2-HKMT and Sho1-H3-HA mutants. In vitro dissociation constants of the interactions are indicated. Immunoblots with the indicated antibodies are shown for samples from an ssk2Δ/ssk22Δ (bottom) or ssk2Δ/ssk22Δ/sho1Δ/pbs2Δ (top) strain. The cartoons (right) depict the interaction between Pbs2 and the Sho1ΔSH3 mutant in the absence (top) or presence (bottom) of endogenous Sho1. (d) M-Track analysis of Pbs2-HKMT and Ste11-H3-HA. Schematics depicting the interaction (P, phosphate group) and immunoblots of samples from the ste11Δ/pbs2Δ strain in the absence or presence of NaCl-induced osmotic stress are shown. Fold changes of the trimethylation signal are plotted as in b; n = 3.