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J. Cell Biol.,
Volume 141, Number 3, May 4, 1998 689-701
-Tubulin
Institut Curie, Section Recherche, Unité Mixte de Recherche 144 du Centre National de la Recherche Scientifique, 75248 Paris
Cedex 05, France
A trimeric complex formed by Tub4p, the
budding yeast 
-tubulin, and the two spindle pole body
components, Spc98p and Spc97p, has recently been
characterized in Saccharomyces cerevisiae. We reasoned that crucial functions, such as the control of microtubule nucleation, could be maintained among divergent species. SPC98-related sequences were
searched in dbEST using the BLASTN program. Primers derived from the human expressed sequence tag
matching SPC98 were used to clone the 5' and 3'
cDNA ends by rapid amplification of cDNA ends
(RACE)-PCR. The human Spc98 cDNA presents an
alternative splicing at the 3' end. The deduced protein
possesses 22% identity and 45% similarity with the
yeast homologue. We further report that the human
Spc98p, like -tubulin, is concentrated at the centrosome, although a large fraction is found in cytosolic
complexes. Sucrose gradient sedimentation of the cytosolic fraction and immunoprecipitation experiments
demonstrate that both -tubulin and HsSpc98p are in the same complex. Interestingly, Xenopus sperm centrosomes, which are incompetent for microtubule nucleation before their activation in the egg cytoplasm,
were found to contain similar amounts of both Spc98p
and -tubulin to human somatic centrosomes, which are competent for microtubule nucleation. Finally, affinity-purified antibodies against Spc98p inhibit microtubule nucleation on isolated centrosomes, as well as in
microinjected cells, suggesting that this novel protein is
indeed required for the nucleation reaction.
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