CYCLIN-LIKE ACCUMULATION AND LOSS OF THE PUTATIVE KINETOCHORE MOTOR CENP-E RESULTS FROM COUPLING CONTINUOUS SYNTHESIS WITH SPECIFIC DEGRADATION AT THE END OF MITOSIS

CENP-E is a kinesin-like protein that binds to kinetochores through th e early stages of mitosis, but after initiation of anaphase, it reloca lizes to the overlapping microtubules in the midzone, ultimately conce ntrating in the developing midbody. By immunoblotting of cells separat ed at various positions in the cell cycle using centrifugal elutriatio n, we show that CENP-E levels increase progressively across the cycle peaking at similar to 22,000 molecules/cell early in mitosis, followed by an abrupt (>10 fold) loss at the end of mitosis. Pulse-labeling wi th [S-35]methionine reveals that beyond a twofold increase in synthesi s between G1 and G2, interphase accumulation results primarily from st abilization of CENP-E during S and G2. Despite localizing in the midbo dy during normal cell division, CENP-E loss at the end of mitosis is i ndependent of cytokinesis, since complete blockage of division with cy tochalasin has no affect on CENP-E loss at the M/G1 transition. Thus, like mitotic cyclins, CENP-E accumulation peaks before cell division, and it is specifically degraded at the end of mitosis. However, CENP-E degradation kinetically follows proteolysis of cyclin B in anaphase. Combined with cyclin A destruction before the end of metaphase, degrad ation of as yet unidentified components at the metaphase/anaphase tran sition, and cyclin B degradation at or after the anaphase transition, CENP-E destruction defines a fourth point in a mitotic cascade of time d proteolysis.