Cell Cycle Regulation In Budding Yeast

Diabetes, Digestive, Kidney Diseases

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Abstract

Chromosome segregation is a complex process that requires multiple levels of regulation. Our goal is to understand the regulation of various mitotic processes and to uncover the molecular function of proteins that have been identified as mitotic regulators but whose exact role is unknown. In the past year, we pursed the following topics:[unreadable] [unreadable] 1. Nuclear positioning in budding yeast. [unreadable] In budding yeast, chromosome segregation takes place within an intact nucleus. Consequently, as cells progress through the cell cycle the nucleus, which is originally in the mother cell, has to be repositioned in order to ensure that half of the chromosomes find their way into the bud, or daughter cell. We previously discovered a regulatory pathway that is responsible for generating a force that repositions the dividing nucleus as cells go through mitosis (Ross and Cohen-Fix, Dev Cell 2004). In an attempt to identify the underlying mechanism of this process, we screened through a collection of mutants to determine if any were defective in this force. One of the mutations isolated was in the ASE1 gene, which codes for a spindle associated protein. The previously known function of Ase1p, a homolog of the mammalian PRC1, is to stabilize the spindle midzone. Thus, the observation that Ase1p could affect nuclear positioning, a process that is thought to occur outside the nucleus, was surprising. We also found that the Ase1 protein plays an important role in spindle formation during early mitosis. In addition, we observed that Ase1p is involved in regulating the number and orientation of cytoplasmic microtubules, which are the vehicle through which force is exerted on the nucleus. Since Ase1 is not detected outside the nucleus, we hypothesize that in an ase1 mutant, the abundance of tubulin due to the inability to form a spindle leads to an increase in the number of cytoplasmic mitorubules, which in turn promote unregulated nuclear movement. This is a novel function for Ase1p that is likely be relevant to the mechanism by which nuclei are positioned in higher eukaryotes. The results of this study were published in the journal Cell Cycle.[unreadable] [unreadable] 2. Apq12p, a novel protein involved in cell cycle regulation. [unreadable] Cells have regulatory mechanisms, called checkpoints, that monitor the presence of intracellular damage or structural errors, and arrest cell cycle progression until the damage or error is repaired. Consequently, mutations in various cell cycle genes do not lead to a detectable phenotype because checkpoint pathways compensate for defects that would otherwise be deleterious. To uncover novel cell cycle genes using a genetic approach, we previously conducted a screen for mutations that lead to cell death when checkpoint pathways are inactive (Sarin et al, Genetics 2004). Through this screen we isolated APQ12, a gene that codes for an ER-associated protein of unknown function. The involvement of the ER protein in cell cycle regulation is intriguing, as this is one of the first examples that links ER function with cell cycle progression. Interestingly, the absence of Apq12p leads to intracellular damage that activated the spindle assembly checkpoint pathway, suggesting that Apq12p is involved in spindle or kinetochore function. We have been able to discern the orientation of Apq12p within the ER membrane and we are in the process of isolating Apq12- interacting proteins. Furthermore, we have identified several genes whose over-expression suppresses the lethality associated with the absence of both Apq12 and a functional spindle assembly checkpoint pathway. We are currently focusing our efforts on understanding the mechanism by which these genes promote viability and on the molecular function of Apq12.