FIG. 6.

PIP box of CDT-1 is required for degradation in S phase. (A) Representation of wild-type and mutant CDT-1 proteins that were ectopically expressed as GFP fusions. The top panel is a diagram of wild-type CDT-1 with PIP box (green), cyclin binding sites (red), and potential CDK phosphorylation sites (lines on top). The lower diagram is CDT-1^PIP(6A), a mutant protein in which six conserved residues of the PIP box have been mutated to alanine (sequence below diagrams). (B to D) Expression of CDT-1^WT::GFP with no treatment (B) or with ddb-1 RNAi treatment (C), and CDT-1^PIP(6A)::GFP with no RNAi treatment (D). CDT-1::GFP epifluorescence image is on top and the Prnr-1::RFP epifluorescence image is below. G₁ phase seam cells (90 min posthatching) are shown on the left, and S-phase seam cells (180 min posthatching) are shown on the right. Seam cells are labeled. Note that CDT-1^WT::GFP is absent from S-phase seam cells, while CDT-1^PIP(6A)::GFP is present in S-phase seam cells, similar to CDT-1^WT::GFP in ddb-1(RNAi) larvae. The cells surrounding the seam cells are hypodermal hyp7 cells. In hyp7 cells, Prnr-1::RFP expression arises during embryonic cell divisions and perdures through 180 min posthatching, due to the stability of RFP. Therefore, because of the perdurance, Prnr-1::RFP expression cannot be used as an S-phase marker in hyp7 cells at 90 or 180 min posthatching. Scale bar, 10 μm.