Temperature-sensitive ipl1-2/Aurora B mutation is suppressed by mutations in TOR complex 1 via the Glc7/PP1 phosphatase

Abstract

Ipl1/Aurora B is the catalytic subunit of a complex that is required for chromosome segregation and nuclear division. Before anaphase, Ipl1 localizes to kinetochores, where it is required to establish proper kinetochore-microtubule associations and regulate the spindle assembly checkpoint. The protein phosphatase Glc7/ PP1 opposes Ipl1 for some of these activities. To more thoroughly characterize the Glc7 phosphatase that opposes Ipl1, we have identified mutations that suppress the thermosensitivity of an ipl1-2 mutant. In addition to mutations in genes previously associated with ipl1 suppression, we recovered a null mutant in TCO89, which encodes a subunit of the TOR complex 1 (TORC1), the conserved rapamycin-sensitive kinase activity that regulates cell growth in response to nutritional status. The temperature sensitivity of ipl1-2 can also be suppressed by null mutation of TOR1 or by administration of pharmacological TORC1 inhibitors, indicating that reduced TORC1 activity is responsible for the suppression. Suppression of the ipl1-2 growth defect is accompanied by increased fidelity of chromosome segregation and increased phosphorylation of the Ipl1 substrates histone H3 and Dam1. Nuclear Glc7 levels are reduced in a tco89 mutant, suggesting that TORC1 activity is required for the nuclear accumulation of Glc7. In addition, several mutant GLC7 alleles that suppress the temperature sensitivity of ipl1-2 exhibit negative synthetic genetic interactions with TORC1 mutants. Together, our results suggest that TORC1 positively regulates the Glc7 activity that opposes Ipl1 and provide a mechanism to tie nutritional status with mitotic regulation.

Suppressors ofipl1-2in Components of a Glc7 Phosphatase Complex, Cdc48 AAA ATPase, TORC1, and the Kinetochore

“…Ipl1 activity in this mutant, as measured by the phosphorylation levels of histone H3Ser10, is reduced at 24° but growth rate is not affected at 24° (Hsu et al 2000). However, ipl1-2 cells lose chromosomes and viability rapidly at 30° (Tatchell et al 2011). To survey in an unbiased way for mutations that suppress the temperature sensitivity of ipl1-2 , we mutagenized an ipl1-2 strain (KT1963) with UV light and isolated revertants that grew at 33° (see Materials and Methods for details).…”

“…*bFrameshift after codon 122 (underlined) resulting in stop after 15 missense codons: …GSG N N… →…GSG N KPQVYELFGYGKRSS*cFrameshift after codon 291 (underlined) resulting in stop after 2 missense codons: …NVYK K LD… →…NVYK K IK*dTatchell et al 2011.…”

“…We identified this cold and caffeine-sensitive suppressor as a null mutant in TCO89 , which encodes a component of TORC1. A preliminary characterization of this mutant has been published (Tatchell et al 2011). …”

The TOR Complex 1 Is a Direct Target of Rho1 GTPase

“…In this regard, the recent finding that Glc7. a type 1 protein phosphatase, is an effector of TORC1 may represent a potential mechanism responsible for Rho1-induced Sch9 dephosphorylation (Tatchell et al, 2011). …”

Environmental signaling through the mechanistic target of rapamycin complex 1

“…Recent findings from the Tatchell laboratory have further reinforced the link between mTORC1 signaling and mitotic regulation. A genetic screen for yeast mutants that could suppress a temperature-sensitive mutation within the essential mitotic Aurora kinase, Ipl1, identified tco89 Δ 116 . Loss of Tco89 resulted in reduced nuclear accumulation of the PP1 phosphatase, Glc7, which opposes Ipl1-mediated substrate phosphorylation during mitosis.…”