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). Each of these alleles carries point mutations within the calmodulin (CaM) binding site of Spc110p which affect CaM binding in vitro; two of the mutant proteins fail to bind CaM detectably (spc110-111, spc110-118) while binding to the third (spc110-124) is temperature-sensitive. All three alleles are suppressed to a greater or lesser extent by elevated dosage of the CaM gene (CMD1), suggesting that disruption of CaM binding is the primary defect in each instance. To determine the consequences on Spc110p function of loss of effective CaM binding, we have therefore examined in detail the progression of synchronous cultures through the cell division cycle at the restrictive temperature. In each case, cells replicate their DNA but then lose viability. In spc110-124, most cells duplicate and partially separate the SPBs but fail to generate a functional mitotic spindle, a phenotype which we term 'abnormal metaphase'. Conversely, spc110-111 cells initially produce nuclear microtubules which appear well-organised but on entry into mitosis accumulate cells with 'broken spindles', where one SPB has become completely detached from the nuclear DNA. In both cases, the bulk of the cells suffer a lethal failure to segregate the DNA.
Stirling
D A
DA
Department of Biochemistry, The University, Dundee, UK.
Rayner
T F
TF
Prescott
A R
AR
Stark
M J
MJ
D000483:Alleles; D001665:Binding Sites / Q000235:genetics; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D004261:DNA Replication; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D007150:Immunohistochemistry; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D008938:Mitosis / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D008941:Spindle Apparatus / Q000378:metabolism / Q000648:ultrastructure; D013696:Temperature
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; C073277:SPC110 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins