Budding YeastBFA1Has Multiple Positive Roles in Directing Late Mitotic Events

Abstract

The proper regulation of cell cycle transitions is paramount to the maintenance of cellular genome integrity. In Saccharomyces cerevisiae, the mitotic exit network (MEN) is a Ras-like signaling cascade that effects the transition from M phase to G1 during the cell division cycle in budding yeast. MEN activation is tightly regulated. It occurs during anaphase and is coupled to mitotic spindle position by the spindle position checkpoint (SPoC). Bfa1 is a key component of the SPoC and functions as part of a two-component GAP complex along with Bub2. The GAP activity of Bfa1-Bub2 keeps the MEN GTPase Tem1 inactive in cells with mispositioned spindles, thereby preventing inappropriate mitotic exit and preserving genome integrity. Interestingly, a GAP-independent role for Bfa1 in mitotic exit regulation has been previously identified. However the nature of this Bub2-independent role and its biological significance are not understood. Here we show that Bfa1 also activates the MEN by promoting the localization of Tem1 primarily to the daughter spindle pole body (dSPB). We demonstrate that the overexpression of BFA1 is lethal due to defects in Tem1 localization, which is required for its activity. In addition, our studies demonstrate a Tem1-independent role for Bfa1 in promoting proper cytokinesis. Cells lacking TEM1, in which the essential mitotic exit function is bypassed, exhibit cytokinesis defects. These defects are suppressed by the overexpression of BFA1. We conclude that Bfa1 functions to both inhibit and activate late mitotic events.

A noncanonical GTPase signaling mechanism controls exit from mitosis in budding yeast

“…4H), we propose that the likely candidate for the inhibitor of Tem1-Cdc15 interaction is Bfa1. In support of this hypothesis, overexpression of Bfa1 without its partner Bub2, arrests cells in anaphase 39,40 , presumably through inhibiting Tem1-Cdc15 interaction.…”

A Novel Hyperactive Nud1 Mitotic Exit Network Scaffold Causes Spindle Position Checkpoint Bypass in Budding Yeast

“…It was previously shown that the overexpression of BFA1 using a galactose-inducible promoter caused inhibition of mitotic exit in a BUB2 -independent manner [ 51 ]. We demonstrated that this defect is due to the role of Bfa1 in Tem1-localization to the SPB and that the defect could be suppressed by concomitant overexpression of TEM1 [ 32 ]. We sought to uncover new modes of MEN regulation by selecting for and characterizing spontaneous suppressors of GAL-BFA1 lethality.…”

“…One possible mechanism of action would be early or increased recruitment of Tem1 to the SPBs, as this has been shown to hyperactivate the MEN [ 17 , 29 , 31 ]. The nud1-A308T allele was discovered as a suppressor of lethality in GAL-BFA1 cells, in which Tem1 localization is disrupted ( Figure 1 B; [ 32 ]). Therefore, we looked at Tem1 localization in GAL-BFA1 nud1-A308T cells to determine whether Tem1 recruitment was responsible for the suppression phenotype.…”

“…Prior work has shown that tethering Tem1 to both SPBs by creating fusions with SPB outer plaque components such as SPC72 or CNM67 or to the dSPB by tethering with BFA1 leads to SPoC bypass and can accelerate anaphase [ 28 , 31 ]. On the other hand, removal of Tem1 from SPBs by the attachment of a CAAX plasma membrane-targeting signal or by overexpression of its SPB receptor BFA1 can inhibit MEN activation [ 28 , 32 ]. Therefore, restriction of Tem1′s SPB association to anaphase cells with correct spindle position is essential for cell cycle control and ploidy maintenance ( Figure 1 A).…”

Moonlighting at the Poles: Non-Canonical Functions of Centrosomes

“…Given the temporal pairing of late mitotic events and cytokinesis, many MEN components are also required for the completion of the cytokinetic process. Amongst them, SPB-bound Tem1 and the Bfa1/Bub2 complex were shown to be crucial for successful cytokinesis ( Whalen et al, 2018 ) and the activity of the SPB-enriched Cdc5 kinase required to complete cytokinesis. In late anaphase, Bfa1 maintains Cdc5 mainly on the cytoplasmic side of the daughter SPB ( Park et al, 2003 ).…”