CALMODULIN LOCALIZES TO THE SPINDLE POLE BODY OF SCHIZOSACCHAROMYCES-POMBE AND PERFORMS AN ESSENTIAL FUNCTION IN CHROMOSOME SEGREGATION

The essential calmodulin genes in both Saccharomyces cerevisiae and Sc hizosaccharomyces pombe were precisely replaced with genes encoding fu sions between calmodulin and the green fluorescent protein (GFP), In l iving budding yeast the GFP-calmodulin fusion protein (GFP-Cmd1p) loca lized simultaneously to sites of cell growth and to the spindle pole b ody (SPB), the yeast analog of the centrosome, Having demonstrated pro per localization of GFP-calmodulin in budding yeast, we examined the l ocalization of a fusion between GFP and calmodulin (GFP-Cam1p) in fiss ion yeast, where calmodulin had not been localized by any method. We f ind GFP-Cam1p also localizes both to sites of polarized cell growth an d to the fission yeast SPB, The localization of calmodulin to the SPB by GFP fusion was confirmed by indirect immunofluorescence. Antiserum to S. pombe calmodulin labeled the ends of the mitotic spindle stained with anti-tubulin antiserum. This pattern was identical to that seen using antiserum to Sad1p, a known SPB component. We then characterized the defects in a temperature-sensitive S. pombe calmodulin mutant, Mu tant cam1-E14 cells synchronized in S phase completed DNA synthesis, b ut lost viability during transit of mitosis, Severe defects in chromos ome segregation, including hypercondensation, fragmentation, and unequ al allocation of chromosomal material were observed, Immunofluorescenc e analysis of tubulin revealed a population of cells containing either broken or mislocalized mitotic spindles, which were never observed in wild-type cells, Taken together with the subcellular localization of calmodulin, the observed spindle and chromosome segregation defects su ggest that calmodulin performs an essential role during mitosis at the fission yeast SPB.