Figure 5. HsSAS-6 Degradation Is Mediated by the 26S Proteasome and Depends on a KEN Box.
(A and B) HeLa cells 10 hr after release from a thymidine block were treated with MG132 or DMSO alone (Control) for 30 min, fixed, and stained with antibodies against HsSAS-6 (red). DNA is shown in blue. Telophase cells were picked based on DNA staining and imaged, and the average cytoplasmic HsSAS-6 signal was quantified (n = 10; arbitrary units; errors represent 95% confidence intervals). Scale bar, 10 μm.
(C) Schematic representation of HsSAS-6 fragments: N-ter (aa 1–173), C-ter (aa 148–657), and C-ter-ΔKEN (aa 148–657; K589A, E590A, N591A). The putative D boxes are indicated in black; the predicted coiled-coil domain, in gray; and the KEN box, in red.
(D–F) Dual GFP fluorescence and phase-contrast time lapse microscopy of HeLa cells expressing GFP-HsSAS-6N-ter (D), GFPHsSAS-6C-ter (E), or GFP-HsSAS-6C-ter-ΔKEN (F). Time 0 min corresponds to the metaphase to anaphase transition. See also Movies S1–S3. Scale bar, 10 μm.
(G) HeLa cells were transfected with Myc-Cdh1 and treated with aphidicolin (for 48 hr) starting 24 hr after transfection. The average cytoplasmic HsSAS-6 signal was quantified for randomly picked transfected cells and their nontransfected neighbors (n = 10; arbitrary units; errors represent 95% confidence intervals). Scale bar, 10 μm. Note that we performed staining with PCNA antibodies (data not shown) to verify that cells transfected with Myc-Cdh1 are arrested in S phase, as previously reported (Sorensen et al., 2000). Note also that the arbitrary units in (G) cannot be compared to those in (A) and (B), because the imaging was performed under different conditions in the two experiments.