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. 2001 Sep 3;154(5):925–936. doi: 10.1083/jcb.200102093

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Copyright © 2001, The Rockefeller University Press

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Figure 7. — Chromosomes inhibit APC/C. (A) Chromosomes prolong the inhibition of the APC/C activity in mitotic lysates. APC/C activity in lysates prepared from interphase (○) and mitotic (⋄) cells were monitored for up to 60 min. The same lysates were assayed for ACP/C activity in the presence of chromosomes (• and ♦). (B) Chromosomes inhibit APC/C fraction directly. Partially purified mitotic APC/C (gel filtration form of the APC/C was prepared as described above), MCC and in vitro–translated CDC20 were preincubated, respectively, in the presence or absence of chromosomes at 300°C. Upon completion of the preincubation, the APC/C inhibitory assays were assembled as described below. After 30 min, E1, E2-C, and radio- labeled substrate were added to initiate the ubiquitin ligase reaction and APC/C activity was determined over the course of 30 min. (1) APC. APC/C was preincubated alone; (2) APC+CR. APC/C was preincubated with chromosomes; (3) APC+CR SUP. Chromosomes were preincubated, the mix was centrifuged for 10 min at 14,000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity; (4) APC+CDC20/CR. In vitro–translated CDC20 was preincubated with chromosomes, the mix was centrifuged for 10 min at 14.000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity; (5) APC+MCC. MCC was preincubated alone; (6) APC+MCC/CR. MCC was preincubated with chromosomes, the mix was centrifuged for 10 min at 14,000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity. All reactions were supplemented with CDC20 to maintain the same concentration with the samples where preincubation of CDC20 with chromosomes has been tested. (C) Chromosomes inhibit highly purified APC/C. Gel filtration fraction of the APC/C was further purified on MonoQ anion exchange FPLC column as described above. The APC/C was preincubated with or without chromosomes and the ubiquitination reaction was initiated upon addition of all necessary ingredients as described above. 0, 5, 10, and 30 min time points were taken to determine the APC/C activity. In all experiments presented in the figure the Ub–substrate conjugates were visualized and quantified as described above.