Department of Biological Infomation, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology* Center for Biological Resources and Informatics, Tokyo Institute of Technology**
○Yoshiki Nagano* Shuya Fukai** Osamu Nureki*
A single cell divides into mother and daughter cells during proliferation in a budding yeast, Saccharomyces cerevisiae. In this process, vacuole is fragmented and distributed in each cells. This vacuole inheritance process requires the MyosinV family protein, Myo2p. Myo2p and its adaptor proteins, Vac8p and Vac17p, form a complex and transport the vacuoles along cytoskeletal actin filaments. Vac8p contains an armadillo repeat motif and has been found to be necessary for the vacuolar inheritance, cytoplasm-to-vacuole protein targeting pathway, formation of the nucleus-vacuole junction, vacuole-vacuole fusion, and caffeine resistance. Armadillo repeat is a typical structural motif to mediate protein-protein interactions. For instance, β-catenin is essential for the Wnt signaling pathway and cadherin-based cell adhesion. A nuclear transport factor, Importin-a, recognizes its cognate nuclear localization signals to achieve selective nuclear import. Similarly, the armadillo repeats of Vac8p might be involved in the vacuole/cargo recognition. Here, we report the result from the preliminary crystallographic analysis of S. cerevisiae Vac8p. Vac8p was overproduced in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The GST-tag was cleaved after purification by a glutathione column, and purified by anion-exchange, and size-exclusion columns. The purified Vac8p protein was crystallized using the vapor-diffusion method at 293 K. The Vac8p crystal diffracted X-ray up to 3.8 Å at BL41XU, SPring8. The crystal belongs to the space group P2, with unit-cell parameters, a = 300.6 Å, b = 130.1 Å, c = 300.9 Å, β = 118.7. Refinement of the crystallization condition is in progress.