Cdc5-Dependent Asymmetric Localization of Bfa1 Fine-Tunes Timely Mitotic Exit

다국어 초록 (Multilingual Abstract)

<▼1><P>In budding yeast, the major regulator of the mitotic exit network (MEN) is Tem1, a GTPase, which is inhibited by the GTPase-activating protein (GAP), Bfa1/Bub2. Asymmetric Bfa1 localization to the bud-directed spindle pole body (SPB) during metaphase also controls mitotic exit, but the molecular mechanism and function of this localization are not well understood, particularly in unperturbed cells. We identified four novel Cdc5 target residues within the Bfa1 C-terminus: <SUP>452</SUP>S, <SUP>453</SUP>S, <SUP>454</SUP>S, and <SUP>559</SUP>S. A Bfa1 mutant in which all of these residues had been changed to alanine (Bfa1<SUP>4A</SUP>) persisted on both SPBs at anaphase and was hypo-phosphorylated, despite retaining its GAP activity for Tem1. A Bfa1 phospho-mimetic mutant in which all of these residues were switched to aspartate (Bfa1<SUP>4D</SUP>) always localized asymmetrically to the SPB. These observations demonstrate that asymmetric localization of Bfa1 is tightly linked to its Cdc5-dependent phosphorylation, but not to its GAP activity. Consistent with this, in kinase-defective <I>cdc5-2</I> cells Bfa1 was not phosphorylated and localized to both SPBs, whereas Bfa1<SUP>4D</SUP> was asymmetrically localized. <I>BFA1<SUP>4A</SUP></I> cells progressed through anaphase normally but displayed delayed mitotic exit in unperturbed cell cycles, while <I>BFA1<SUP>4D</SUP></I> cells underwent mitotic exit with the same kinetics as wild-type cells. We suggest that Cdc5 induces the asymmetric distribution of Bfa1 to the bud-directed SPB independently of Bfa1 GAP activity at anaphase and that Bfa1 asymmetry fine-tunes the timing of MEN activation in unperturbed cell cycles.</P></▼1><▼2><P><B>Author Summary</B></P><P>During mitosis the replicated chromosomes are distributed equally to the daughter cells. Once the chromosomes have segregated properly, a pathway called the mitotic exit network (MEN) becomes activated to complete mitosis. How MEN activation is coordinated with segregation of the chromosomes is currently a focus of interest. In budding yeast, Tem1 initiates MEN activation and Bfa1 negatively regulates Tem1 with Bub2. The polo kinase Cdc5 also activates MEN by directly phosphorylating and inhibiting Bfa1. The spindle pole body (SPB), which corresponds to the mammalian centrosome, acts as a platform for these MEN components. The Bfa1/Bub2 complex localizes to SPBs and regulates the association of Tem1 with the SPBs. When the spindle aligns correctly along the mother-bud axis, Bfa1/Bub2 is restricted to the bud-oriented SPB. Conversely, when the spindle is misaligned, Bfa1/Bub2 is present on both SPBs and mitotic exit is delayed, suggesting that the spatial distribution of Bfa1/Bub2 controls the timing of mitotic exit. In this study, we identified new Cdc5 target phosphorylation residues in Bfa1 that function in its asymmetric distribution on SPBs and showed that the asymmetric Bfa1 distribution was required for timely mitotic exit during unperturbed cell cycle of the budding yeast.</P></▼2>