Figure 6: M-Cdk phosphorylates Bfa1.
From: Temporal and compartment-specific signals coordinate mitotic exit with spindle position
(a) Bfa1-3HA migration profile on SDS–PAGE during late anaphase (Tem1 depletion). Arrow indicates the slow migrating form of Bfa1. Note the presence of faster migrating forms of Bfa1 in SPO12-containing strains (the first and the third lanes). (b) In vitro phosphorylation of Bfa1 fused to Maltose-binding-protein (MBP-Bfa1). The analogue-sensitive (as) mitotic M-Cdk complex (Cdc28-as/Clb2-TAP) purified from yeast was incubated with bacterially purified MBP-Bfa1, MBP-Bfa1-6A or MBP in the presence of DMSO or the ATP analogue 1NM-PP1. Incorporation of γ32P-ATP into MBP-Bfa1 and the levels of MBP-Bfa1 are shown on the left and right, respectively. (c) Immunoblot showing migration profile of Bfa1 mutants. Cultures were released from G1-block under Tem1-depleting conditions. Arrow indicates the slow migrating form of Bfa1. Samples were collected 105 min after release (see supplementary Fig. 9e for the entire time course). (d) SAC proficiency of bfa1Δ, BFA1, bfa1-6A and bfa1-6D cells. Cells were released from G1-block (t=0) in nocodazole containing medium. Nocodazole depolymerizes microtubules, provoking metaphase arrest. Note that all cell types except bfa1Δ accumulated as large budded mitotic cells, indicating cell-cycle arrest. Per strain, 100 cells were counted per time point. Graph is a representative of three independent experiments. (e) Percentage of SPOC-deficient phenotypes for the indicated strain backgrounds. Graphs show an average of three independent experiments. Error bars indicate s.d. Per cell type, 100 cells were counted. Asterisk indicates significant differences according to two-tailed Student’s t-test (P<0.05). (f,g) Growth analysis of Bfa1 mutants. (f) cdc5-10 bfa1Δ temperature-sensitive strains without or with integrated BFA1, bfa1-6A, bfa1-6D were grown at permissive (30 °C) and semipermissive (35 °C) temperatures. (g) rts1Δ bfa1Δ cells without or with integrated BFA1, bfa1-6A, bfa1-6D were spotted on glucose (glu) or galactose (Raf/Gal) containing agar plates. Cells contained either empty or Gal1-KIN4 containing URA3-based 2 μm plasmid (p416). Note that deletion of RTS1 rescues the lethality of KIN4 overexpression46,69. (h,i) Percentage of SPOC-deficient phenotypes. Each bar is an average of three independent experiments. Error bars show s.d. Per experiment, 100 cells were counted per strain. Asterisks indicate difference according to two-tailed Student’s t-test (P<0.05).
