To isolate genes involved in sister chromatid
separation, we generated a temperature sensitive collection of yeast
mutants in a strain containing a GFP system that allows visualization of
a single chromosome locus. Seven complementation groups that do not
separate sister chromatids at anaphase were identified by microscopy.
The loc mutants (loss of cohesion) die rapidly at the non-permissive temperature. Although sister chromatid separation fails in
loc2 mutants, the spindle elongates normally resulting in paired
sister chromatids randomly segregating to a single spindle pole. The
sister chromatid separation defect is not due to activation of the
spindle assembly checkpoint because loc2 mad2 double mutants
exhibit the loc2 mutant phenotype. Clb2p proteolysis is normal in
loc2 mutants indicating that loc2 does not have a general
defect in ubiquitin-mediated proteolysis. Since degradation of the Pds1
protein is essential for sister chromatid separation, we analyzed the
phenotype of pds1 loc2 double mutants to determine the relative
order of gene function. The pds1 loc2 mutants exhibit the
loc2 phenotype, suggesting that LOC2 acts downstream of
PDS1 to separate sister chromatids. The loc2 mutants
display decreased permissive temperatures when combined with mutations
in genes that regulate sister chromatid separation, the MCD1 and
ESP1 genes. Loc2 protein levels are cell cycle regulated and peak
at the time of sister chromatid separation when Loc2p is localized to
the mitotic spindle.
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