IDENTIFICATION OF AN SPC110P-RELATED PROTEIN IN VERTEBRATES

Although varying in size and complexity, centrosomes have conserved fu nctions throughout the evolutionary range of eukaryotes, and thus may display conserved components, In this work, we took advantage of the r ecent advances in the isolation of the budding yeast spindle pole body , the development of specific immunological probes and the molecular c haracterisation of genes involved in spindle pole body duplication or assembly, Screening a monoclonal antibody library against Saccharomyce s cerevisiae spindle pole body components, we found that two monoclona l antibodies, directed against two different parts of the yeast Spc110 p, decorate the centrosome from mammalian cells in an asymmetrical man ner. Western blot experiments identified a 100 kDa protein specificall y enriched in centrosome preparations from human cells, This protein i s phosphorylated during mitosis and is tightly associated with the cen trosome: only denaturing conditions such as 8 M urea were able to solu bilise it. Purified immunoglobulins directed against Spc110p inhibit m icrotubule nucleation on isolated human centrosomes, using brain phosp hocellulose-tubulin or Xenopus egg extract tubulin, This result sugges ted that the centrosomal 100 kDa protein could be involved in a microt ubule nucleation complex, To test this hypothesis, we turned to Xenopu s species, in which mAb anti-Spc110p decorated centrosomes from somati c cells and identified a 116 kDa protein in egg extract, We performed a partial purification of the gamma-tubulin-ring complex from egg extr act, Sucrose gradient sedimentation, immunoprecipitation and native ge ls demonstrated that the Xenopus 116 kDa protein and gamma-tubulin wer e found in the same complex. Altogether, these results suggest the exi stence of an yeast Spc110-related protein in vertebrate centrosomes wh ich is involved in microtubule nucleation.