Identifying Functional Links Between Kap121 and Chromosome Segregation in Yeast.

• Author / Creator

  Cairo, Lucas V

• Nuclear pore complexes (NPCs) form aqueous portals in the nuclear envelope (NE) and are the sole sites for molecular exchange between nuclear and cytoplasmic compartments. While ions, small metabolites and molecules under ~40 kDa can freely passage through NPCs, most macromolecular transport across the NE is facilitated by a family of structurally related mobile transport receptors termed Karyopherins (Kaps). Kaps confer the ability to translocate through NPCs by establishing a series of low affinity interactions with Nups that contain phenylalanine-glycine repeat motifs (FG-Nups). While this general transport mechanism describes how the vast majority of macromolecular transport events are governed, far less is known about how nuclear transport is modulated in response to changes in cellular physiology. In budding yeast, which undergoes a closed mitosis, nuclear import mediated by Kap121 is inhibited in a regulated manner following arrest in metaphase with the microtubule-destabilizing drug nocodazole. The inhibition of Kap121-mediated import is facilitated by molecular rearrangements within the NPC, which prevent Kap121 and its cargos from moving through the NPC central channel. Here, we have investigated the underlying signaling mechanism required for triggering activation of the Kap121-transport inhibitory pathway (KTIP). We have discovered that the KTIP is activated in response to the loss of kinetochore-microtubule (KT-MT) attachments during mitosis. A key regulator of KTIP activation is the spindle assembly checkpoint (SAC) signaling protein, Mad1. Following KT detachment from spindle MTs, Mad1 participates in a signaling event and dynamically cycles between NPCs and KTs to initiate changes in the NPC driving the arrest of Kap121-mediated import. Activation of this signaling pathway requires the activity of the Aurora B-kinase Ipl1, and the resulting changes to transport prevent the nuclear accumulation of the Kap121 cargo Glc7p, a phosphatase that functions as an antagonist of Ipl1-kinase activity. Thus, through reducing the nuclear accumulation of Glc7p, the KTIP fosters the establishment of a nuclear environment that promotes Ipl1-function and the eventual reorganization and proper assembly of the KT-MT interface. We have also uncovered a novel function for Kap121 in regulating KT bi-orientation and chromosome segregation during cell division distinct from its role in transporting proteins through the NPC. We present results showing that Kap121 physically interacts with Dam1 and Duo1, two key structural components of the Dam1 complex, which is an essential MT-bound assembly required for linking KTs to dynamic MT ends in yeast. We demonstrate that the interaction between Kap121 and the Dam1 complex is RanGTP-insensitive, suggesting this interaction occurs within the interior of the nucleus, potentially at the KT-MT interface. Kap121 binding to Dam1 and Duo1 promotes their stability, as a mutant version of Kap121 that can no longer bind these proteins leads to a significant decrease in their cellular levels. Our experimental results support a model where Kap121 confers a “chaperone-like” function within the nucleus where, through its physical association with Dam1/Duo1, contributes to the structural stability of the Dam1 complex by preventing Dam1 and Duo1 degradation.

• Subjects / Keywords

  • Chromosome Segregation
  • Nuclear Transport
  • Karyopherin

• Graduation date

  Spring 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3W950Z9T

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.