The major non-tubulin polypeptide found associated with microtubules purified from unfertilized sea urchin eggs by cycles of pH-dependent assembly

Microtubule-associated proteins (MAPs) are the major accessory proteins on microtubules, the prominent, cylindrical component of the cytoskeleton. Despite the essential roles performed by microtubules in eukaryotic cells, little is known regarding the function of the filamentous MAPs, other that those that serve as motor proteins. Structural and biochemical analysis has revealed a great deal about the in vitro behavior of neuronal MAPs (Olmsted, 1986; Wiche et al., 1991). In addition, recent transfection experiments have suggested that brain MAPs promote microtubule assembly and stabilize microtubule arrays in vivo (Lewis et al., 1989; Kanai et al., 1989), as well as alter the morphogenesis of neuronal cells (Dinsmore and Solomon, 1991; Baas et al., 1991). Yet, the deletion of the DNA sequence encoding a 205,000 Mr MAP from Drosophila results in viable animals with no detectable phenotype (Pereira et al., 1992). These results indicate that we are only beginning to sort out the functional complexity of these MAPs. Mammalian brain has been a major source of microtubule protein for the past 20 years because it contains large quantities of tubulin and MAPs that readily assemble into microtubules in vitro. However, the bulk of this microtubule protein originates from cells that have stopped dividing. To identify MAPs and other regulatory factors that may regulate microtubule function during mitosis, we have developed methods for the purification of tubulin (Suprenant and Rebhun, 1983) and microtubule protein (Suprenant and Marsh, 1987; Suprenant et al. 1989) from unfertilized sea urchin egg extracts in the absence of taxol (Vallee and Bloom, 1983; Bloom et al., 1985; Vallee and Collins, 1986; Scholey et al. 1984, 1985; Hirokawa and Hisanaga, 1987; Hosoya et al., 1990). The unfertilized sea urchin egg contains a large store of tubulin (Raff et al., 1971) that becomes incorporated into the first cleavage mitotic apparatus (Bibring and Baxandall, 1977) and ciliated blastula (Bibring and Baxandall, 1981). Although there are no detectable microtubules in unfertilized sea urchin eggs (Harris et al., 1980), microtubules readily assemble in cytosolic extracts prepared at alkaline pH (Suprenant and Marsh, 1987). When purified by further cycles of assembly and disassembly, these microtubules are found to be composed principally of tubulin and a 77,000 Mr polypeptide that coassembles stoichiometrically. Although this polypeptide has not been characterized, its abundance in these microtubule prepara445 Journal of Cell Science 104, 445-456 (1993) Printed in Great Britain © The Company of Biologists Limited 1993