A Study of sumoylation as a novel mechanism that regulates Kar9p function and spindle positioning in Saccharomyces cerevisiae

Thesis (Ph. D.)--University of Rochester. Dept. of Biology, 2008.Spindle positioning is an essential process during cell division. To ensure that the chromosomes segregate properly, the spindle needs to be oriented along the long axis of cell division. In asymmetrically dividing cells, positioning and orientation of the spindle are coordinated with the plane of cytokinesis to produce two cells with different properties. The budding yeast, Saccharomyces cerevisiae divides asymmetrically. In this organism, the mitotic spindles are positioned and oriented along the long axis of cell division which is determined by the site of bud emergence. This process is accomplished by the functions of various proteins to bring about the communication between cell polarity signals and cytoplasmic microtubules. In yeast, spindle positioning is mediated by two pathways, the KAR9 pathway and the dynein pathway. The KAR9 pathway is responsible for orienting the cytoplasmic microtubules and positioning the spindle prior to the onset of anaphase. The dynein pathway generates forces to pull the spindle across the plane of cytokinesis. Proteins functioning in these pathways include those associated with microtubules and actin. The interactions between these proteins are essential for regulating the process of spindle positioning. In this report, I evaluate the hypothesis that spindle positioning proteins of S. cerevisiae are regulated by the process of sumoylation. Sumoylation is accomplished post-translationally by attaching the SUMO moiety to the target protein. I found that Kar9p and Bim1p, key players of the KAR9 pathway, interact with the sumoylation machinery in yeast. I demonstrated that Kar9p can be conjugated by the yeast SUMO, Smt3p in vitro. Using kar9-L304P, a mutant that disrupted the Kar9p-Smt3p two-hybrid interaction, I found that sumoylation may be involved in restricting Kar9p to the daughter bound spindle pole body. Data in this report also suggest that phosphorylation which is known to regulate its asymmetric localization might affect the sumoylation of Kar9p. In addition, I also found that other spindle positioning proteins such as Stu2p, Bik1p and Pac1p interact with the sumoylation machinery. Using the in vitro sumoylation assay, Stu2p and Bik1p were found to be conjugated by Smt3p. I also discovered a mutation in Pac1p that might indicate that Pac1p can bind to Smt3p non-covalently. Furthermore by two-hybrid analysis, I found that the interaction between Bik1p and Pac1p might be important for each of their interaction with Smt3p. Although further studies are needed to elucidate how sumoylation might regulate spindle positioning, the present study supports the hypothesis that sumoylation plays a role in the spindle positioning in budding yeast. Because many of the molecular mechanisms used in spindle positioning are conserved, these studies suggest the possibility that sumoylation might also be involved in regulating this process in other systems