CHARACTERIZATION OF THE HUMAN HOMOLOG OF THE YEAST SPC98P AND ITS ASSOCIATION WITH GAMMA-TUBULIN

A trimeric complex formed by Tub4p, the budding yeast gamma-tubulin, a nd the two spindle pole body components, Spc98p and Spc97p, has recent ly been characterized in Saccharomyces cerevisiae. We reasoned that cr ucial functions, such as the control of microtubule nucleation, could be maintained among divergent species. SPC98-related sequences were se arched in dbEST using the BLASTN program. Primers derived from the hum an expressed sequence tag matching SPC98 were used to clone the 5' and 3' cDNA ends by rapid amplification of cDNA ends (RACE)-PCR. The huma n Spc98 cDNA presents an alternative splicing at the 3' end. The deduc ed protein possesses 22% identity and 45% similarity with the yeast ho mologue. We further report that the human Spc98p, like gamma-tubulin, is concentrated at the centrosome, although a large fraction is found in cytosolic complexes. Sucrose gradient sedimentation of the cytosoli c fraction and immunoprecipitation experiments demonstrate that both g amma-tubulin and HsSpc98p are in the same complex. Interestingly, Xeno pus sperm centrosomes, which are incompetent for microtubule nucleatio n before their activation in the egg cytoplasm, were found to contain similar amounts of both Spc98p and gamma-tubulin to human somatic cent rosomes, which are competent for microtubule nucleation. Finally, affi nity-purified antibodies against Spc98p inhibit microtubule nucleation on isolated centrosomes, as well as in microinjected cells, suggestin g that this novel protein is indeed required for the nucleation reacti on.