Regulation of kinetochore function by post-translational modifications in Saccharomyces cerevisiae

Abstract

Aneuploidy, characterized by cells with an incorrect number of chromosomes, is linked to genetic disorders such as cancer, Down’s syndrome, and miscarriage. In cancer, studies show that aneuploidy is associated with cancer aggressiveness and chemotherapy resistance. Aneuploidy in yeast is common and can be beneficial. Yeast rely on genetic heterogeneity to survive in fluctuating environments and toxic stressors. It remains unclear what drives chromosome segregation errors that lead to aneuploidy. The fidelity of chromosome segregation relies on proper kinetochore (KT)- microtubule (MT) attachments, and errors in attachments lead to aneuploidy.

The Dam1 protein is a component of the outer kinetochore Dam1 complex (Dam1C), and has a critical function in chromosome segregation by maintaining stable binding to microtubules. It is assembled along with nine other subunits, including Duo1, to form Dam1C. Oligomerization of multiple Dam1Cs around MTs result in stable KT-MT attachments. Dam1 is also a major target that is regulated to correct errors in KT-MT attachments, that left uncorrected could result in chromosome missegregation. Phosphorylation of Dam1 serines by the Aurora Kinase B (Ipl1), promotes KT-MT dissociation. The phosphorylation of Dam1 has been postulated to act by decreasing electrostatic interactions between KT and MT. It remains unclear if these phosphorylation events might modulate another aspect of KT-MT binding. The glucose signaling kinase Protein Kinase A also functions in chromosome segregation by phosphorylating Dam1 S31, yet it is not fully understood how this modification impacts the chromosome segregation process and the implications of being linked to carbon utilization or sensing. Additionally, it remains unclear how much phosphorylation is compatible for optimal (normal) chromosome segregation fidelity.

Here, we report that serine phosphorylation reduces Dam1 interaction with Duo1. Dam1 S20 and 31 phosphorylation by PKA and Ipi1 have a limited effect on binding to Duo1, but the C-terminal residues S257, 265, and 292 have a significant impact on Duo1 binding. Modeling and genetic assay, demonstrate that a short alpha helix in Dam1 (from S257 to N278) regulates the interaction with Duo1. We also demonstrate that yeast grown on galactose, glycerol, and maltose all display increased chromosome loss rates. Alterations in chromosome segregation fidelity by growing cells on suboptimal carbon sources were associated with greater survival on antifungals. Fluctuation assays show that 50% or more, but less than 100%, of Dam1 S31 phosphorylation, is associated with optimal chromosome segregation. On the other hand, less than 50% or 100% Dam1 S31 phosphorylation is associated with higher chromosome segregation loss rates. Overall, the results highlight how phosphorylation on the C-terminal tail of Dam1 impacts the formation of the alpha helical structure, which influences the interactions between Dam1- Duo1 and is crucial for binding of the Dam1C to MTs. This brings new insight into how phosphorylation functions to modulate parts of the Dam1C structure. This suggests the possibility that phosphorylation may not only reduce electrostatic interactions between KTs and MTs but also modulate Dam1 binding to Duo1 and together alter KT-MT stability. Finally, we provide evidence that acetylation of Dam1 contributes to regulation of outer kinetochore function raising the possibility that this is another pathway to connect kinetochore activity to glucose availability and chromosome segregation fidelity.