Fission yeast mto2p regulates microtubule nucleation by the centrosomin-related protein mto1p

From an insertional mutagenesis screen, we isolated a novel gene, mto2+, involved in microtubule organization in fission yeast. mto2 Delta strains are viable but exhibit defects in interphase microtubule nucleation and in formation of the postanaphase microtubule array at the end of mitosis. The mto2 Delta defects represent a subset of the defects displayed by cells deleted for mto1+ (also known as mod20+ and mbo1+), a centrosomin-related protein required to recruit the gamma-tubulin complex to cytoplasmic microtubule-organizing centers (MTOCs). We show that mto2p colocalizes with mto1p at MTOCs throughout the cell cycle and that mto1p and mto2p coimmunoprecipitate from cytoplasmic extracts. In vitro studies suggest that mto2p binds directly to mto1p. In mto2A mutants, although some aspects of mto1p localization are perturbed, mto1p can still localize to spindle pole bodies and the cell division site and to satellite particles on interphase microtubules. In mto1 Delta mutants, localization of mto2p to all of these MTOCs is strongly reduced or absent. We also find that in mto2A mutants, cytoplasmic forms of the gamma-tubulin complex are mislocalized, and the gamma-tubulin complex no longer coimmunoprecipitates with mto1p from cell extracts. These experiments establish mto2p as a major regulator of mto1p-mediated microtubule nucleation by the gamma-tubulin complex.