The ␥-tubulin complex, via its ability to organize microtubules, is critical for accurate chromosome segregation and cytokinesis in the fission yeast, Schizosaccharomyces pombe. To better understand its roles, we have purified the S. pombe ␥-tubulin complex. Mass spectrometric analyses of the purified complex revealed known components and identified two novel proteins (i.e., Mbo1p and Gfh1p) with homology to ␥-tubulin-associated proteins from other organisms. We show that both Mbo1p and Gfh1p localize to microtubule organizing centers. Although cells deleted for either mbo1 ؉ or gfh1 ؉ are viable, they exhibit a number of defects associated with altered microtubule function such as defects in cell polarity, nuclear positioning, spindle orientation, and cleavage site specification. In addition, mbo1⌬ and gfh1⌬ cells exhibit defects in astral microtubule formation and anchoring, suggesting that these proteins have specific roles in astral microtubule function. This study expands the known roles of ␥-tubulin complex components in organizing different types of microtubule structures in S. pombe.

Summary Cdc14-family phosphatases play a conserved role in promoting mitotic exit and cytokinesis by dephosphorylating substrates of cyclin dependent kinase (Cdk). Cdc14-family phosphatases have been best studied in yeast (for review see [1] [2]), where budding yeast Cdc14 and its fission yeast homolog Clp1 are regulated in part by their localization, with both proteins thought to be sequestered in the nucleolus in interphase. Cdc14/Clp1 are released from the nucleolus in mitosis, and in late mitosis a conserved signaling pathway termed the MEN/SIN acts through an unknown mechanism to keep Cdc14 and Clp1 respectively out of the nucleolus [3-6]. Here we show that the most downstream SIN component, the Ndr-family kinase Sid2, acts to maintain Clp1 in the cytoplasm in late mitosis by phosphorylating Clp1 directly and thereby creating binding sites for the 14-3-3 protein Rad24. Mutation of the Sid2 phosphorylation sites on Clp1 disrupts the interaction between Clp1 and Rad24, and causes premature return of Clp1 to the nucleolus during cytokinesis. Loss of Clp1 from the cytoplasm in telophase renders cells sensitive to perturbation of the actomyosin ring, but does not affect other functions of Clp1. Because all components of this pathway are conserved, this might be a broadly conserved mechanism for regulation of Cdc14-family phosphatases.

Proteomic, localization, and enzymatic activity screens in fission yeast reveal how deubiquitinating enzyme localization and function are tuned.