FISSION YEAST CUT2 REQUIRED FOR ANAPHASE HAS 2 DESTRUCTION BOXES

The fission yeast Schizosaccharomyces pombe cut2(+) gene is essential for sister chromatid separation, Cut2 protein, which locates in the in terphase nucleus and along the metaphase spindle, disappears in anapha se with the same timing as mitotic cyclin destruction, This proteolysi s depends on the APC (Anaphase-Promoting Complex)-cyclosome which cont ains ubiquitin ligase activity, The N-terminus of Cut2 contains two st retches similar to the mitotic cyclin destruction box. We show that bo th sequences (33RAPLGSTKQ and 52RTV-LGGKST) serve as destruction boxes and are required for in vitro polyubiquitination and proteolysis, Cut 2 with doubly mutated destruction boxes inhibits anaphase, whereas Cut 2 with singly mutated boxes can suppress cut2 mutations, Strong expres sion of the N-terminal 73 residues containing the destruction boxes le ads to the accumulation of endogenous cyclin and Cut2, and arrests cel ls in metaphase, whereas the same fragment with the mutated boxes does not, Cut2 proteolysis occurs in vitro using Xenopus mitotic extracts in the presence of functional destruction boxes, Furthermore, Cut2 is polyubiquitinated in an in vitro system using HeLa extracts, and this polyubiquitination requires the destruction boxes.