PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF TUBULIN FROM THE BUDDING YEAST SACCHAROMYCES-CEREVISIAE

We describe a method for isolating milligram quantities of assembly-co mpetent tubulin from the budding yeast Saccharomyces cerevisiae. The t ubulin is >95% purified and free of contaminating enzyme activities. A s a result, it has been possible to determine the yeast tubulin equili brium-binding constant for Mg-GTP and the tubulin GTPase activity unde r nonassembling and assembling conditions. We also determined the crit ical concentration for yeast tubulin polymerization and found it to be significantly lower than that for bovine brain tubulin under identica l conditions. Similarly, the dynamic properties both of individual yea st microtubules and of bulk microtubule suspensions were significantly different from those of bovine brain microtubules free of microtubule -associated proteins. The data suggest that the properties of the yeas t tubulin may reflect the particular functional requirements of the ye ast cell. With this method, it is now possible to introduce any desire d tubulin gene mutation into the budding yeast and correlate the pheno typic effects of the mutation in cells with the effects of the mutatio n on the biochemical and polymerization properties of the tubulin.