CHARACTERIZATION OF THE MEIOTIC SPINDLE POLE IN BUDDING YEAST

University Of Colorado, Boulder CO

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In budding yeast, the spindle pole body (SPB) is the sole microtubule organizing center of the cell. During meiosis, however, the cytoplasmic face of the SPB is converted from a site of microtubule nucleation to a site of membrane formation. This transformation is accomplished by the recruitment of at least four meiosis-specific proteins that displace the gamma-tubulin complex and form a novel structure, termed the meiotic outer plaque (MOP), which serves as a site of de novo membrane formation. The MOP is a highly ordered structure and several proteins that localize to the MOP have been identified, but how they are arranged and how they promote membrane formation is not known. We have immuno labeled strains of S. cerevisiae that have different spindle pole body proteins fused with GFP. Both Ady4p and Spo74p localize to the MOP and have been shown to promote prospore membrane growth. The goal of this project is to develop a high-resolution structural model of this membrane organizing center. The small size of the complex (300-400nm in diameter) make Immuno-EM and EM tomography essential for this analysis. By combining these techniques with mutants lacking individual protein components of the MOP, as well as results from fluorescence-based analysis, a robust model of the organization of the MOP will be generated. Such a model will be an important step in the long-term goal of understanding how the MOP promotes the formation of intracellular membranes. We have begun to characterize the first stages of pro-spore membrane (PSM) formation in wild type cells as well as the lack of PSM formation in Spo14 deletion mutants.