Mod20p appears to act by recruiting components of the gamma-tubulin complex to non-spindle pole body MTOCs. The identification of mod20p-related proteins in higher eukaryotes suggests that this may represent a general mechanism for the organization of noncentrosomal MTOCs in eukaryotic cells.

Microtubules have a central role in eukaryotic cell polarity, in part through interactions between microtubule end-binding proteins and the cell cortex. In the fission yeast Schizosaccharomyces pombe, microtubules and the polarity modulator tea1p maintain cylindrical cell shape and strictly antipodal cell growth. The tea1p protein is transported to cell tips by association with growing microtubule plus ends; once at cell tips, tea1p releases from microtubule ends and associates with the cell cortex, where it coordinates polarized growth. Here we describe a cortical protein, mod5p, that regulates the dynamic behaviour of tea1p. In mod5Delta cells, tea1p is efficiently transported on microtubules to cell tips but fails to anchor properly at the cortex and thus fails to accumulate to normal levels. mod5p contains a signal for carboxy-terminal prenylation and in wild-type cells is associated with the plasma membrane at cell tips. However, in tea1Delta cells, although mod5p remains localized to the plasma membrane, mod5p is no longer restricted to the cell tips. We propose that tea1p and mod5p act in a positive-feedback loop in the microtubule-mediated regulation of cell polarity.

From an insertional mutagenesis screen, we isolated a novel gene, mto2؉, involved in microtubule organization in fission yeast. mto2⌬ 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⌬ 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 ␥-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 mto2⌬ 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⌬ mutants, localization of mto2p to all of these MTOCs is strongly reduced or absent. We also find that in mto2⌬ mutants, cytoplasmic forms of the ␥-tubulin complex are mislocalized, and the ␥-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 ␥-tubulin complex. INTRODUCTIONThe ␥-tubulin complex is a large, conserved multisubunit protein complex involved in microtubule nucleation in eukaryotic cells (Stearns and Kirschner, 1994;Zheng et al., 1995; for reviews, see Gunawardane et al., 2000;Schiebel, 2000;Job et al., 2003). In a variety of cell types, the ␥-tubulin complex is found both localized to microtubuleorganizing centers (MTOCs) and also in significant levels in cytoplasmic pools. Although much of the characterization of ␥-tubulin function has involved work on soluble complexes, most MTOCs in vivo are associated with largely insoluble compartments or subcellular organelles, and as a result we currently understand very little of the mechanisms controlling the intracellular localization and activity of the ␥-tubulin complex and how these mechanisms might be regulated. Recently, a small number of proteins have been identified that may interact with the ␥-tubulin complex and recruit it to structures such as the centrosome, the Golgi apparatus, or other MTOCs (Takahashi et al., 2002;Terada et al., 2003;Kawaguchi and Zheng, 2004;Rios et al., 2004;Thompson et al., 2004;Venkatram et al., 2004; Zimmerman et al., 2004a,b). However, in this area much still remains to be learned.The fission yeast Schizosaccharomyces pombe has recently become a useful model system for understanding microtubule nucleation and dynamics. Three distinct patterns of microtubule nucleation take place during the mitotic cell cycle in fission yeast (Hagan, 1998). During interphase, dynamic microtubules tend to run along the long axis of the cylindrically shaped cells and terminate at cell tips. These microtubules are nucleated from cytoplasmic interphase MTOCs (iMTOCs)...