CENTROSOMES ISOLATED FROM SPISULA-SOLIDISSIMA OOCYTES CONTAIN RINGS AND AN UNUSUAL STOICHIOMETRIC RATIO OF ALPHA-TUBULIN BETA-TUBULIN

Centrosome-dependent microtubule nucleation involves the interaction o f tubulin subunits with pericentriolar material. To study the biochemi cal and structural basis of centrosome-dependent microtubule nucleatio n, centrosomes capable of organizing microtubules into astral arrays w ere isolated from parthenogenetically activated Spisula solidissima oo cytes. Intermediate voltage electron microscopy tomography revealed th at each centrosome was composed of a single centriole surrounded by pe ricentriolar material that was studded with ring-shaped structures sim ilar to 25 nm in diameter and <25 nm in length. A number of proteins c opurified with centrosomes including: (a) proteins that contained M-ph ase-specific phosphoepitopes (MPM-2), (b) alpha-, beta-, and gamma-tub ulins, (c) actin, and (d) three low molecular weight proteins of <20 k D. gamma-Tubulin was not an MPM-2 phosphoprotein and was the most abun dant form of tubulin in centrosomes. Relatively little alpha- or beta- tubulin copurified with centrosomes, and the ratio of alpha- to beta-t ubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, sugge sting that centrosomes contain beta-tubulin that is not dimerized with alpha-tubulin.