Figure 6.

SIC1 is ubiquitinated by chimeric SCF complexes. (A) Hi5 insect cells were infected with various baculoviruses expressing ^PHACDC4, CDC53^PHA (53), ^PHis6hCUL1 (C), ySKP1^His6 (Y), or hSKP1 (H) as indicated. At 72 hr post-infection, lysates were prepared and SCF complexes were affinity purified on an anti-Polyoma matrix, and eluted complexes were incubated for 2 hr at 25°C with MBP-SIC1^MHis6 in the presence of ^His6yUBA1 (E1), yCDC34 (E2), purified CLN2/GST-CDC28^HA/CKS1, ubiquitin, and an ATP-regenerating system. At the end of the incubation, the samples were fractionated by SDS/PAGE and were immunoblotted with anti-myc antibodies to detect MBP-SIC1^MHis6. Bound antibodies were visualized by ECL. (B) The indicated SCF complexes were purified from baculovirus-infected insect cell lysates and were incubated with the full set of ubiquitination components (lanes 1 and 4) or in the absence of CLN2/GST-CDC28^HA/CKS1 (lanes 2 and 5) or ubiquitin (lanes 3 and 6). (C) Purified SCF complexes containing CDC53^PHA (53), ^PHis6hCUL1 (CUL1), ySKP1^His6 (Y), or hSKP1 (H) subunits were incubated with ubiquitination components containing either yCDC34 (Y) or hCDC34 (H).