Molecular analysis of the cytosolic Didycostelium γ-tubulin complex

gamma-Tubulin plays an essential role in microtubule nucleation and organization and occurs, besides its centrosomal localization, in the cytosol, where it forms soluble complexes with other proteins. We investigated the size and composition of gamma-tubulin complexes in Dictyostelium, using a mutant cell line in which the endogenous copy of the gamma-tubulin gene had been replaced by a tagged version. Dictyostelium gamma-tubulin complexes were generally much smaller than the large gamma-tubulin ring complexes found in higher organisms. The stability of the small Dictyostelium gamma-tubulin complexes depended strongly on the purification conditions, with a striking stabilization of the complexes under high salt conditions. Furthermore, we cloned the Dictyostelium homolog of Spc97 and an almost complete sequence of the Dictyostelium homolog of Spc98, which are both components of gamma-tubulin complexes in other organisms. Both proteins localize to the centrosome in Dictyostelium throughout the cell cycle and are also present in a cytosolic pool. We could show that the prevailing small complex present in Dictyostelium consists of DdSpc98 and gamma-tubulin, whereas DdSpc97 does not associate. Dictyostelium is thus the first organism investigated so far where the three proteins do not interact stably in the cytosol.