Cell cycle-dependent proteolysis in plants: Identification of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor MG132

It is widely assumed that mitotic cyclins are rapidly degraded during anaph ase, leading to the inactivation of the cell cycle-dependent protein kinase Cdc2 and allowing exit from mitosis. The proteolysis of mitotic cyclins is ubiquitin/26S proteasome mediated and requires the presence of the destruc tion box motif at the N terminus of the proteins. As a first attempt to stu dy cyclin proteolysis during the plant cell cycle, we investigated the stab ility of fusion proteins in which the N-terminal domains of an A-type and a a-type tobacco mitotic cyclin were fused in frame with the chloramphenicol acetyltransferase (CAT) reporter gene and constitutively expressed in tran sformed tobacco BY2 cells. For both cyclin types, the N-terminal domains le d the chimeric cyclin-CAT fusion proteins to oscillate in a cell cycle-spec ific manner. Mutations within the destruction box abolished cell cycle-spec ific proteolysis. Although both fusion proteins were degraded after metapha se, cyclin A-CAT proteolysis was turned off during S phase, whereas that of cyclin B-CAT was turned off only during the tate G(2) phase. Thus, we demo nstrated that mitotic cyclins in plants are subjected to post-translational control (e.g., proteolysis). Moreover, we showed that the proteasome inhib itor MG132 blocks BY2 cells during metaphase in a reversible way. During th is mitotic arrest, both cyclin-CAT fusion proteins remained stable.