Ama1p ubiquitylation by APC/CCdc20
A: in vitro ubiquitylation of Ama1p and mutant derivatives as indicated using the APC/C prepared from mnd2∆ cdh1∆ CDC16 ::TAP strain (RSY1381, see Methods for details). In vitro transcription coupled translation produced Cdc20p was added to all extracts except for lane 12. 35S labeled Ama1p harboring the following mutations:- lanes 1, 2 and 3 CB∆/IR∆, lanes 4, 5 and 6 CB∆/IR∆/GxEN, lane 7 CB∆/IR∆/GxEN/Db1/Db2, lanes 8 and 9 CB∆/IR∆/Db1 and lanes 10,11 and 12 CB∆/IR∆/GxEN/Db1 was prepared by in vitro transcription coupled translation. B: Doc1p is not required for APC/CCdc20 mediated ubiquitylation of Ama1p. In vitro ubiquitylation assays on Ama1pCB∆/IR∆ using APC/C purified from mnd2∆, cdh1∆ CDC16::TAP (RSY1381, lanes 1, 2 and 3) or mnd2∆ cdh1∆ doc1∆ CDC16::TAP (RSY1748 lanes 4, 5 and 6). Time after the addition of Cdc20p to the reactions (minutes at 37°C) is given.