A Novel Role for the GTPase-Activating Protein Bud2 in the Spindle Position Checkpoint

Abstract

The spindle position checkpoint (SPC) ensures correct mitotic spindle position before allowing mitotic exit in the budding yeast Saccharomyces cerevisiae. In a candidate screen for checkpoint genes, we identified bud2Δ as deficient for the SPC. Bud2 is a GTPase activating protein (GAP), and the only known substrate of Bud2 was Rsr1/Bud1, a Ras-like GTPase and a central component of the bud-site-selection pathway. Mutants lacking Rsr1/Bud1 had no checkpoint defect, as did strains lacking and overexpressing Bud5, a guanine-nucleotide exchange factor (GEF) for Rsr1/Bud1. Thus, the checkpoint function of Bud2 is distinct from its role in bud site selection. The catalytic activity of the Bud2 GAP domain was required for the checkpoint, based on the failure of the known catalytic point mutant Bud2R682A to function in the checkpoint. Based on assays of heterozygous diploids, bud2R682A, was dominant for loss of checkpoint but recessive for bud-site-selection failure, further indicating a separation of function. Tem1 is a Ras-like protein and is the critical regulator of mitotic exit, sitting atop the mitotic exit network (MEN). Tem1 is a likely target for Bud2, supported by genetic analyses that exclude other Ras-like proteins.

Spindle pole body-associated Atg11, an autophagy-related protein, regulates microtubule dynamics essential for high-fidelity chromosome segregation

“…An ATG11-add back strain in the atg11∆ background complemented the TBZ-induced growth defects suggesting a specific role of ATG11 towards kinetochore-microtubule interactions (Figure 1B). Atg11 displayed three primary interactions in the interaction network including Spc72 and Bud2 which plays a critical role in mitotic spindle alignment and spindle position checkpoint (SPOC) respectively (31,32) (Figure 1C). While atg11∆ mutant cells did not show any growth defects at a lower temperature of 14°C (Figure S1B), a condition that destabilizes microtubules (33), growth of these mutant cells is severely affected at an elevated temperature of 37°C (Figure S1B), that promotes faster microtubule dynamics (34).…”

“…While one pathway compensates for the other, loss of both is lethal for the cell (Miller and Rose, 1998). Misaligned spindles lead to the activation of spindle position checkpoint (SPOC) (Bloecher et al, 2000, Nelson et al, 2012, Pereira et al, 2000, Wang et al, 2000, Yeh et al, 1995, a surveillance mechanism that halts the cell cycle and prevents the exit from mitosis by inhibiting the mitotic exit network (MEN) signalling.…”

“…On the other hand, the cytosolic face of the SPBs nucleates aMTs which aid in the alignment and positioning of the mitotic spindle along the polarity axis, critical for proper nuclear division during asymmetric cell division in S. cerevisiae . Misaligned spindles lead to the activation of the spindle position checkpoint (SPOC) (Bloecher, Venturi, & Tatchell, 2000; Nelson, Sanson, Park, & Cooper, 2012; Pereira, Hofken, Grindlay, Manson, & Schiebel, 2000; Wang, Hu, & Elledge, 2000; Yeh, Skibbens, Cheng, Salmon, & Bloom, 1995), a surveillance mechanism that halts the cell cycle and prevents exit from mitosis by inhibiting the mitotic exit network (MEN) signalling pathway (Caydasi, Ibrahim, & Pereira, 2010).…”

“…Such asymmetry leads to the nucleation of more aMTs from the dSPB necessary for the mitotic spindle positioning along the mother-bud axis and ensures non-random SPB inheritance (12)(13)(14)(15)(16)(17)(18). Misaligned spindles activate the spindle position checkpoint (SPOC) (19)(20)(21)(22)(23), a surveillance mechanism that halts the cell cycle and prevents exit from mitosis by inhibiting the mitotic exit network (MEN) signaling pathway (24).…”

Transfer of Septin Rings to Cytokinetic Remnants Directs Age-Sensitive ER stress Surveillance Cell Cycle Re-entry

“…We next examined the Bud1-activating components Bud5 (a GEF for Bud1) and Bud2 (a GTPase-activating protein, GAP, for Bud1) ( Figure 2B) (Marston et al, 2001;Nelson et al, 2012). Specifically, we tested the Bud1/Rsr1-associated population of Bud5 ( Figure S2D-S2E) and Bud2 ( Figure S2F-S2G) using split YFP assays between Bud1-Bud5 and Bud1-Bud2, respectively.…”