The C-terminus of Bfa1p in budding yeast is essential to induce mitotic arrest in response to diverse checkpoint-activating signals

During mitosis, genomic integrity is maintained by the proper coordination of anaphase entry and mitotic exit through mitotic checkpoints. In budding yeast, exit from mitosis is triggered by the activation of the small GTPase Tem1p. Bfa1p in association with Bub2p negatively regulates Tem1p in response to spindle damage, spindle misorientation, and DNA damage, resulting in cell cycle arrest. To delineate the Bfa1p domains that respond to distinct checkpoint signals, we constructed 13 Bfa1 deletion mutants. The C-terminal 184 amino acids of Bfa1p (Bfa1-D8(391-574)) contained the entire capacity of Bfa1p to generate mitotic arrest in response to spindle damage, spindle misorientation, and DNA damage. This domain was also enough to interact with the mitotic exit network proteins Tem1p, Bub2p, and Cdc5p, and to localize to the spindle pole body (SPB). Over-expression of Bfa1-D8(391-574) induced late anaphase arrest as efficient as the full-length Bfa1p in a Bub2p-dependent manner. In contrast, the N-terminal portion of Bfa1p (Bfa1-D16(1-376)) could not localize to SPB and did not block mitotic exit in response to diverse checkpoint signals. Bfa1-D16(1-376) interacted with Tem1p but not with Bub2p and its over-expression partially arrested cells in mitosis in the absence of Bub2p. By random mutagenesis of Bfa1-D8(391-574) with hydroxylamine, we isolated a point mutant of D8, D8(E438K), which interacts with both Tem1p and Bub2p but cannot respond to checkpoint signals. This mutant also showed reduced efficiency in the localization to SPB. Taken together, our study demonstrated that various checkpoint signals are transmitted to the C-terminal domain of Bfa1 (Bfa1-D8(391-574)) and that Bfa1p localization to SPB is necessary but not sufficient to regulate mitotic exit in response to various checkpoint signals.