Characterization of KRE31 protein and its possible role in y-tubulin related processes

Accurate chromosome segregation in eukaryotic cells requires proper functioning of the mitotic spindle. Improper distribution of chromosomes during cell division can lead to genetic disorder such as Down syndrome or contribute to cancer. The Mitotic spindle is composed of protein fiber called microtubules, which are polymers of alpha- and beta- tubulin. These cytoskeletal elements in eukaryotic cells perform essential functions during chromosome segregation in mitosis and meiosis and are essential in organelle positioning, secretion, cellular movement, and the establishment and maintenance of cell polarity. The microtubule-organizing center (MTOC) assembles and organizes microtubules and thus its function is essential to the process of cell division. y-Tubulin is a universal component of all microtubule-organizing centers (MTOCs) and plays an essential role in the formation of a functional, bipolar mitotic spindle. Because y-tubulin is an important component of the MTOC and appears to be intimately involved in MTOC function, an understanding of the proteins associated with y-tubulin may provide insights into how MTOCs function. The objective this research is the molecular characterization of the Saccharomyces cerevisiae KRE31 gene and its protein product. KRE31 was identified through a yeast two-hybrid screen as a novel, previously uncharacterized, gene encoding a protein suspected of physically interacting with y-tubulin. The research presented here provides an initial characterization of the KRE31 gene and its protein product, Kre3lp, including determination of whether the gene is essential for viability, cellular localization of Kre31p, and a system to test whether Kre3lp physically associates with budding yeast y-tubulin, Tub4p, under normal cellular conditions. Meiotic analysis revealed the KRE31 gene to be essential for viability indicating a crucial role for Kre31p in yeast. Kre31p was found to be localized to nucleus, likely the nucleolus, by fluorescence microscopy. In addition, a system has been developed for testing the physical interaction between Kre31 and Tub4 proteins under normal cellular conditions. This initial characterization leads to many interesting possibilities. The apparent nucleolar localization suggests a function independent of MTOC formation and function; however it does not rule out its involvement in MTOC related or dependent functions. The system developed for testing in vivo association between Kre31p and Tub4p will aid in evaluating in which cellular process(es) the essential function(s) of Kre31p are involved.