Figure 2: Degradation of Ndd1 is APC/C^Cdh1 dependent.

(a) Levels of endogenous TAP-tagged Ndd1, as well as Clb2, were probed in the absence and presence of Cdh1^m11 induction. Tubulin served as a loading control. The level of Ndd1 mRNA was assayed by real-time PCR under both conditions and are shown as the mean of three independent experiments run in duplicates each. Error bars represent s.d. of six samples. (b) Endogenous TAP-tagged Ndd1 was probed in wild-type or cdh1Δ-cycling cells (UT) or cells arrested in prometaphase by Nocodozole (Noc) or in G1 by α factor. Clb2 served as a control for APC/C^Cdh1 activity and actin as a loading control. FACS analysis of cells harvested in parallel shows that cells were indeed arrested. (c) GST-NDD1 was overexpressed for 2 h by galactose in wild-type and cdh1Δ cells containing the pGAL-NDD1 plasmid and stopped by addition of glucose at time 0. (d) Acm1 expression was induced by galactose in cells harbouring the GAL-ACM1::LEU2 construct. Levels of endogenous Ndd1–Tap and Cdc5 were examined at the indicated time points after Acm1 induction. FACS analysis of cells harvested in parallel shows that Acm1 overexpression does not lead to cell cycle arrest or significantly perturb the cell cycle. (e) Cells expressing HA-tagged wild-type Ndd1 or the Ndd ³¹⁸KTPA³²¹ (Ndd1^KA) putative destruction box mutant were grown in raffinose, Expression was induced for 2 h with galactose and stopped by addition of glucose at time 0. (f) Bacterially expressed GST-Ndd1 was incubated with APC/C and APC/C+Cdh1, resolved by gel electrophoresis and immunoblotted either with anti ubiquitin (left) or anti GST (right) antibodies. The background ubiquitination observed in the left blot in the absence of Ndd1 (right lane) is presumably auto ubiquitination of Cdh1 (ref. 60).