FIGURE 5.

Catalytic, hydroxylamine-sensitive phosphorylation of Ccc2 and its variants. A, Sf9 membranes containing Ccc2 wt, S258A, S971A, or S258A/S971A were phosphorylated for 1 min with [γ-³²P]ATP as described under “Experimental Procedures,” and the proteins were resolved on acidic gels, B, Coomassie Blue-stained gels demonstrating the 110 kDa bands used to correct the phosphorylation levels. C, densitometric representation (mean ± S.E. of >3 experiments) of Ccc2 wt, S258A, S258D, or S971A and two experiments using S258A/S971A that agreed within 15%. Phosphorylation of Ccc2 wt was taken as 100% (see normalization of phosphorylation data under “Experimental Procedures”). Bars represent aspartylphosphate formation corrected for the corresponding hydroxylamine-resistant phosphorylation (average ∼20% of total phosphorylation).