Control of programmed cyclin destruction in a cell-free system

To ask what controls the periodic accumulation and destruction of the mitotic across the cell cycle, we have developed a cell-free system from clam embryos that reproduces several aspects of cyclin behavior. One or more rounds of cyclin proteolysis and resynthesis occur in vitro, and the destruction of the cyclins is highly specific. The onset, duration, and extent of cyclin destruction and the appropriately stagered disappearance of cyclin A and cyclin B are correctly regulated during the first cycle in the cell-free system. Just as in intact cells, lysates made from early interphase cells require further protein synthesis to reach the cyclin destruction point, and lysates made from later stages do not. Using the cell-free system we show that cyclin disappearance requires ATP and Mg2+. By combining lysates from different cell cycle stages, we show that (a) interphase lysates do not contain a dominant inhibitor of cyclin destruction and (b) the timing of cyclin destruction is determined by the cell cycle stage of the cytoplasm rather than the cell cycle stage of the substrate cyclins themselves. Among a large variety of agents tested, only a few affect cyclin destruction. Tosyl-lysine chlormethyl ketone (TLCK, a protease inhibitor), 6-dimethylaminopurine (6-DMAP, a kinase inhibitor), certain sulfhydryl-blocking agents, ZnCl2 and EDTA (but not EGTA) completely block cyclin destruction in vitro. Addition of 1 mM Ca2+ to the cell-free system has no effect on cyclin stability, but 5 mM Ca2+ leads to the rapid destruction of cyclins and a small number of other proteins.