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

Junwon Kim,Young Yil Bahk,Kiwon Song,Guangming Luo,Orna Cohen-Fix

doi:10.1371/journal.pgen.1002450

Junwon Kim, Young Yil Bahk + Show 3 more

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https://doi.org/10.1371/journal.pgen.1002450

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Journal: PLoS Genetics	Publication Date: Jan 12, 2012
Citations: 49	License type: CC BY 4.0

Affiliation: Yonsei University, Konkuk University

Abstract

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: 452S, 453S, 454S, and 559S. A Bfa1 mutant in which all of these residues had been changed to alanine (Bfa14A) 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 (Bfa14D) 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 cdc5-2 cells Bfa1 was not phosphorylated and localized to both SPBs, whereas Bfa14D was asymmetrically localized. BFA14A cells progressed through anaphase normally but displayed delayed mitotic exit in unperturbed cell cycles, while BFA14D 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.

Highlights

In eukaryotes, mitotic entry is driven by a rise in cyclindependent kinase (Cdk) activity, which is required for the formation of a bipolar spindle and chromosome segregation (For a review, see [1])
The spindle pole body (SPB), which corresponds to the mammalian centrosome, acts as a platform for these mitotic exit network (MEN) components
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

Summary

Introduction

Mitotic entry is driven by a rise in cyclindependent kinase (Cdk) activity, which is required for the formation of a bipolar spindle and chromosome segregation (For a review, see [1]). For cells to subsequently undergo cytokinesis and enter the G1 phase of the cell cycle, Cdk-mediated phosphorylation events are reversed and Cdk activity declines (For reviews, see [2,3]). In budding yeast, this transition, called mitotic exit, is triggered by a signaling cascade known as the mitotic exit network (MEN). The MEN is a crucial target of various checkpoints that keep mitotic Cdk activity high until the daughter chromosomes have segregated properly. The MEN coordinates spindle position and mitotic progression in asymmetrically dividing cells such as budding yeast, where the division plane is predetermined. When spindles misalign relative to the division plane, mitotic exit is delayed by preventing the MEN activation [6]

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