Isolation and characterization of calmodulin-binding centrosome components related to Saccharomyces cerevisiae Spc110p from the fission yeast Schizosaccharomyces pombe and humans
Accurate segregation of chromosomes during eukaryotic, cell division depends on the integrity of the mitotic spindle apparatus. Nucleation of mitotic spindle microtubules is a highly regulated process that requires linking the soluble gamma-tubulin complex to the centrosome. In the budding yeast Saccharomyces cerevisiae, the calmodulin-binding protein Spc110p links the gamma-tubulin complex to the spindle pole body (SPB), a centrosome equivalent.To complement work on Spc110p I examined whether centrosomal calmodulin targets might exist in divergent organisms. The localization of calmodulin to the SPB of the fission yeast Schizosaccharomyces pombe and the mitotic defects exhibited by a fission yeast calmodulin mutant suggested that a calmodulin-binding Spc110p homologue is present in fission yeast.I identified a calmodulin-binding fission yeast Spc110p homologue based on its homology with a related protein in the filamentous fungus Aspergillus nidulans. I named this fission yeast protein Pcp1p for p̱ole target of c̱almodulin in S. p̱ombe. A fusion of Pcp1p to GFP localizes to the SPB throughout the cell cycle. Overexpression of Pcp1p in S. pombe cells causes abnormal SPB aggregates, ectopic and broken mitotic spindles, and DNA missegregation. Localization of Pcp1p to the SPB, and to abnormal SPB aggregates in S. pombe cells overexpressing Pep1p, was confirmed by immunoelectron microscopy.I subsequently used the calmodulin-binding site sequences of these fungal homologues to identify kendrin as the human orthologue of Spc110p. Kendrin is a large centrosomal protein with a predicted central coiled-coil region and a C-terminal calmodulin-binding domain and localizes specifically to centrosomes throughout the cell cycle. In mitotic human breast carcinoma cells containing abundant centrosome-like structures, kendrin is found only at centrosomes associated with microtubules. Kendrin shares striking sequence homology with mouse pericentrin, a centrosome component known to interact with gamma-tubulin. Kendrin additionally contains a unique calmodulin-binding C-terminal domain not found in mouse pericentrin.Analysis of public sequence data indicates that the human and mouse genomic regions encoding kendrin and pericentrin, respectively, share synteny, revealing that kendrin and pericentrin are direct homologues. Together my data demonstrate the conservation from yeast to vertebrates of Spc110p-related molecules that bind calmodulin and localize to the SPB/centrosome.