Analysis of MPS2 in the budding yeast Saccharomyces cerevisiae

It is crucial to the eukaryotic cell cycle that the centrosome undergo precise duplication to generate the two poles of the mitotic spindle. In the budding yeast Saccharomyces cerevisiae, centrosomal functions are provided by the spindle pole body (SPB). Our laboratory had isolated a specific mutant, mps2-1 (monopolar spindle 2-1), which, at the nonpermissive temperature, prevents proper SPB duplication and leads to the formation of a monopolar spindle plus a defective SPB. Prior to the research conducted during the course of this thesis project, nothing was known about the MPS2 gene itself.; This thesis describes the characterization of the budding yeast gene MPS2, and the protein that it encodes. The 1161 base pair MPS2 gene has been cloned and sequenced, and is predicted to encode a polypeptide of 387 amino acids containing two coiled-coils and a putative C-terminal hydrophobic transmembrane. Deletion series and subclones have identified the minimal MPS2 gene fragments necessary for rescuing mps2 cells from their mutant defects, and suggest that the N-terminal-most 20% of the MPS2 gene is not necessary for wild-type cell growth. The mps2-1 mutant lesion is located in this N-terminal region. The MPS2 gene has been completely deleted from the genome to demonstrate that MPS2 is essential for normal cell cycle progression and ploidy maintenance in yeast. Although the plasmid-shuffle genetic test indicates that MPS2 is essential for yeast cell viability, it has been observed that spores that do not contain MPS2 can germinate and grow, albeit poorly. These mps2{dollar}Delta{dollar} null cells exhibit a mutant phenotype that significantly differs from the phenotype that is characteristic of mps2-1 cells. Most notably, these mps2{dollar}Delta{dollar} null cells do not exhibit a cell cycle arrest, and appear to contain normal bipolar spindles, even at nonpermissive temperature.; The predicted motifs of the Mps2 protein, as well as MPS2's mutant phenotype and functional similarity to another gene necessary for SPB duplication, NDC1 (nuclear division cycle 1), fueled the hypothesis that Mps2p might be a structural protein that anchor the SPB in the nuclear envelope. To test this hypothesis, the Mps2 protein was examined. The 6xHis::Mps2 fusion protein was produced in bacteria for the purpose of inducing anti-Mps2p antibodies in rabbits that could be used to localize the Mps2p in yeast cells. This polyclonal serum was affinity purified and used successfully on protein blots of fractionated yeast cells to show that Mps2p signal is strongest in the fractions containing crudely purified nuclear pore complexes and spindle pole bodies. However, the polyclonal antibody proved itself unworthy in whole cell localization studies, so epitope-tagged versions of Mps2p were constructed. Immunofluorescence studies using antibodies directed against the epitope tags showed that the ProA::Mps2 fusion protein localizes to the nuclear envelope in a punctate fashion. The aforementioned findings might help validate the hypothesis that MPS2 encodes a membrane-bound protein that is necessary for anchoring the spindle pole body into the nuclear envelope.