MUTATIONS WHICH BLOCK THE BINDING OF CALMODULIN TO SPC110P CAUSE MULTIPLE MITOTIC DEFECTS

We have generated three temperature-sensitive alleles of SPC110, which encodes the 110 kDa component of the yeast spindle pole body (SPB), E ach of these alleles carries point mutations within the calmodulin (Ca M) binding site of Spc110p which affect CaM binding in vitro; two of t he mutant proteins fail to hind CaM detectably (spc110-111, spc110-118 ) while binding to the third (spc110-124) is temperature-sensitive, Al l three alleles are suppressed to a greater or lesser extent by elevat ed dosage of the CaM gene (CMD1), suggesting that disruption of CaM bi nding is the primary defect in each instance, To determine the consequ ences on Spc110p function of loss of effective CaM binding, we have th erefore examined in detail the progression of synchronous cultures thr ough the cell division cycle at the restrictive temperature, In each c ase, cells replicate their DNA but then lose viability, In spc110-124, most cells duplicate and partially separate the SPBs but fail to gene rate a functional mitotic spindle, a phenotype which we term 'abnormal metaphase', Conversely, spc110-111 cells initially produce nuclear mi crotubules which appear well-organised but on entry into mitosis accum ulate cells with 'broken spindles', where one SPB has become completel y detached from the nuclear DNA, In both cases, the bulk of the cells suffer a lethal failure to segregate the DNA.