Characterization of the human homologue of the yeast spc98p and its association with gamma-tubulin.
A trimeric complex formed by Tub4p, the budding yeast gamma-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 gamma-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 gamma-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 gamma-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.
Tassin
A M
AM
Institut Curie, Section Recherche, Unité Mixte de Recherche 144 du Centre National de la Recherche Scientifique, 75248 Paris Cedex 05, France. atassin@curie.fr
Celati
C
C
Moudjou
M
M
Bornens
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D018385:Centrosome; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D007136:Immunoglobulins / Q000378:metabolism; D008297:Male; D008869:Microtubule-Associated Proteins / Q000032:analysis* / Q000235:genetics; D008969:Molecular Sequence Data; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013094:Spermatozoa / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
D007136:Immunoglobulins; D008869:Microtubule-Associated Proteins; C112306:TUBGCP3 protein, human; D014404:Tubulin