Figure 3. S293 of the SRM is the Critical DRE Residue for Slimb Recognition.

(A) 13 hydroxyl amino acids (red) within the DRE of full-length fly Plk4 were individually mutated to alanines and used to evaluate the impact of each residue on Plk4 stability and centriole duplication. S293 and T297 reside within the SRM (yellow highlight).

(B) (Top) Anti-GFP immunoblot of lysates prepared from S2 cells transiently co-expressing the indicated Plk4-EGFP construct and Nlp-EGFP (loading control). All Plk4 mutants are single alanine mutants except SBM (a double mutant of S293A/T297A). (Bottom) Plk4-EGFP intensities were measured by densitometry of the anti-GFP immunoblot and normalized with their respective Nlp-EGFP loading controls. The plotted values are the normalized Plk4 intensities relative to the WT-Plk4 treatment.

(C) Anti-GFP immunoprecipitates of lysates prepared from S2 cells transiently expressing 3xFLAG-ubiquitin and the indicated Plk4-EGFP construct were probed with anti-GFP, FLAG, and Slimb antibodies. Short and long exposures of the anti-FLAG immunoblot are shown.

(D) Amounts of associated endogenous Slimb were determined by densitometry of the anti-Slimb immunoblot and then normalizing the measurements with the amounts of Plk4-EGFP present in the IPs. The plotted values are relative to the WT-Plk4 treatment.

(E) The relative amounts of total Plk4 FLAG-Ubi were calculated using the densitometry method described in (D).

(F) S2 cells co-expressing the indicated Plk4-EGFP construct (green puncta) and Nlp-EGFP (green nuclei) were immunostained for PLP (red) to mark centrioles. DNA (blue). Insets show higher magnifications of the boxed regions.

(G) The centrioles of S2 cells treated and immunostained as in (F) were counted. Centrioles are amplified in cells expressing SBM (P<0.0001) or S293A (P<0.0001) but not T297A (P=0.06). There is no significant difference in centriole loss (black bars) in these treatments. Four experiments were performed per construct (n = 600 cells/construct).