Figure 3. The SAF-degrons are adjacent to domains required for spindle assembly.

A. Schematic overview of Bard1 (RING: RING-domain; ANK: ankyrin repeats; BRCT: Brct-domain; D: D-box). B. Mutation of D-boxes stabilizes Bard1 in mitotic extracts. ³⁵S-labeled mutants were tested for APC/C-dependent degradation in mitotic extracts with active APC/C^Cdc20. The C50G/H68A-mutant ablates the function of the RING-domain, but has no effect on APC/C-dependent degradation. C. Mutation of D-boxes stabilizes Bard1 in vivo. 293T cells were co-transfected with Bard1-mutants and Cdc20, and Bard1-levels were determined by Western blot. D. Schematic overview of Hmmr. (MT: microtubule-binding domain; ZIP: leucine zipper; T: TEK-box; K: KEN-box) E. Mutation of a D- and KEN-box stabilizes Hmmr in G1-extracts. The indicated ³⁵S-labeled point mutants were tested for APC/C-dependent degradation in G1-extracts as described above. Reaction products were analyzed by autoradiography. F. Mutation of D-, KEN-, and TEK-boxes stabilize Hmmr in vivo. ^mycHmmr or the indicated mutants were co-expressed in 293T cells with Cdh1, and protein levels were analyzed by Western blot using αmyc-antibodies. G. Schematic overview of HURP. (MT: microtubule binding domain; GKAP: GKAP-domain). H. The degradation of HURP in G1-extracts depends on D-, KEN-, and TEK-boxes. To ensure degradation of wt-HURP, G1-extracts were treated with Ran^Q69L (see later). The indicated ³⁵S-labeled mutants were incubated in G1-extracts before being analyzed by autoradiography. I. Degradation of ^HAHURP and its mutants in HeLa cells after release from nocodazole arrest. Samples were taken at the indicated times and analyzed for HURP-levels by Western blot. J. Schematic overview of NuSAP. The SAP- and ChHD-domains were defined by Rijmaekers et al., 2003. K. Degradation of NuSAP in mitotic extracts depends on D- and KEN-boxes. If indicated, the mitotic extract was supplemented with Ran^GTP. ³⁵S-labeled mutants were incubated in the extracts for different times, before being analyzed by autoradiography. In NuSAP^KR, a stretch of Lys residues has been changed to alanines, leading to its degradation in the absence of Ran^GTP. M. The degradation of NuSAP in cells depends on D- and KEN-boxes. The respective ^HANuSAP mutants (wt; DK: mutation of D- and KEN-box; KR: mutation of a Lys-rich cluster) were co-expressed with Cdh1 in 293T cells, as indicated. The levels of NuSAP and its mutants were detected by Western blot.