Fission yeast Cut2 required for anaphase has two destruction boxes

Abstract

The fission yeast Schizosaccharomyces pombe cut2+ gene is essential for sister chromatid separation. Cut2 protein, which locates in the interphase nucleus and along the metaphase spindle, disappears in anaphase with the same timing as mitotic cyclin destruction. This proteolysis depends on the APC (Anaphase‐Promoting Complex)–cyclosome which contains ubiquitin ligase activity. The N‐terminus of Cut2 contains two stretches similar to the mitotic cyclin destruction box. We show that both sequences (33RAPLGSTKQ and 52RTVLGGKST) serve as destruction boxes and are required for in vitro polyubiquitination and proteolysis. Cut2 with doubly mutated destruction boxes inhibits anaphase, whereas Cut2 with singly mutated boxes can suppress cut2 mutations. Strong expression of the N‐terminal 73 residues containing the destruction boxes leads to the accumulation of endogenous cyclin and Cut2, and arrests cells 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.

Drosophilasecurin destruction involves a D-box and a KEN-box and promotes anaphase in parallel with Cyclin A degradation

“…After low-level expression in pim-mutant embryos, residual mitotic degradation of singlemutant proteins might free some separase activity sufficient for sister chromatid separation. Similar results have been observed with the fission yeast securin Cut2, which is completely stabilized in a Xenopus extract destruction assay by mutations in either of the two D-boxes, and yet, low-level expression of single-but not double-mutant proteins is able to complement growth of cut2-ts strains at the restrictive temperature (Funabiki et al, 1997). We emphasize that even in wild-type cells, mitotic PIM degradation appears to be far from complete, and it can be speculated that it is the PIM protein of a special pool of separase complexes that is more efficiently degraded, perhaps on kinetochores or during transport on spindles towards kinetochores.…”

An E2 enzyme Ubc11 is required for ubiquitination of Slp1/Cdc20 and spindle checkpoint silencing in fission yeast

“…37 The B-type cyclin/Cdc13 and securin/Cut2 cause a mitotic arrest if not properly degraded in the APC/C-dependent manner. [38][39][40][41] We therefore attempted to examine how the ubc11-P93L mutation affects the stability of proteins normally degraded in the APC/C-dependent manner. The cellular levels of Slp1, Cdc13 and Cut2 were first monitored in the ubc11-P93L mutant.…”

Cell cycle mechanisms of sister chromatid separation; Roles of Cut1/separin and Cut2/securin

“…By way of comparison, a top2 mutant cell which is defective in condensation and separation is shown (Uemura et al 1987). (Zou et al 1999;Nagase et al 1996) Yamada et al 1997) in a manner which resembles highly that of mitotic cyclins (Funabiki et al 1996a(Funabiki et al , 1997. The destruction modes of Cut2 and Cdc13 (a ®ssion yeast mitotic cyclin) are shown in Fig.…”