PURIFICATION AND CHARACTERIZATION OF ASSEMBLY-COMPETENT TUBULIN FROM ASPERGILLUS-NIDULANS

We have developed a procedure for purifying assembly-competent tubulin from Aspergillus nidulans. To our knowledge, this is the first report of the purification of assembly-competent tubulin from a filamentous fungus, and the procedure should be of great value in analyzing the la rge number of alpha- and beta-tubulin mutations that have been isolate d and characterized in A, nidulans. Our procedure consists of overprod uction of alpha- and beta-tubulin, partial purification by ion-exchang e chromatography, and final purification by rounds of assembly and dis assembly. We have found that taxol promotes the assembly of A. nidulan s tubulin into microtubules, but a higher concentration of taxol is re quired for maximal assembly of A. nidulans tubulin than is required fo r brain tubulin, The critical concentration for assembly in the presen ce of taxol is also significantly higher for A, nidulans tubulin than for brain tubulin. In addition, A. nidulans microtubules that were ass embled and maintained in the presence of taxol depolymerized in condit ions in which taxol-stabilized mammalian microtubules remain intact. T hese results suggest that A. nidulans tubulin has a lower affinity for taxol than mammalian tubulin.