Expression of Amino- and Carboxyl-terminal g- and a-Tubulin Mutants in Cultured Epithelial Cells*

Three distinct tubulin proteins are essential for microtubule function: a-, b-, and g-tubulin. After translation, aand b-tubulin proteins combine into a soluble, 7 S heterodimer that is multimerized to form the microtubule filament. Conversely, g-tubulin combines with several proteins into a soluble, 25 S multi-protein particle, the gammasome that is essential for nucleating microtubule filaments at the centrosome. The proteins that assist tubulins in executing their specific functions are largely unknown. As an initial approach to address this issue, we first decided to identify domains of mammalian aand g-tubulin necessary for their function by creating mutant mammalian aand g-tubulin (both deletion and hybrid mutants) and assaying their behavior in stably transfected Chinese hamster ovary epithelial cells. First, we demonstrated that addition of a carboxylterminal epitope tag had no effect on the subcellular localization of either aand g-tubulin. Second, we found that both the amino and carboxyl termini of g-tubulin were essential for its incorporation into the gammasome. Third, we found that the amino and carboxyl termini of a-tubulin were necessary for incorporation of the a-b-tubulin heterodimer into the microtubule filament network. In general, a-tubulin sequences could not replace those of g-tubulin and vice versa. Taken together, these results suggest that the amino and carboxyl termini of aand g-tubulin and perhaps regions throughout these proteins were necessary for their specific functions.