Figure 1.

eEF1A is sensitive to Ca²⁺-regulated, membrane-associated proteases. Recombinant eEF1A or PI4Kα catalytic domain was added to 112 μg of microsomal protein in the absence (−) or presence (+) of CaCl₂ in a 120-μL reaction mixture, as described in “Materials and Methods.” Thirty-five-microliter aliquots were removed at the times indicated, and proteins were separated by SDS-PAGE and analyzed by western blots using an antibody to the six-His tag. The Ca²⁺ concentration was 1 mm in A and 100 μm in B. A, The amount of recombinant eEF1A (reEF1A) added to the microsomes was varied. Lane 1, 1.2 μg; lane 2, 0.8 μg; lane 3, 0.4 μg; lane 4, 1.2 μg; lane 5, 1.2 μg; lane 6, 0.8 μg; lane 7, 0.4 μg. Lanes 1 through 4 are −Ca²⁺ and lanes 5 through 7 are +1 mm Ca²⁺. Lanes 1 through 3 and 5 through 7 were incubated 20 min; lane 4 was placed immediately into sample buffer. B, Recombinant proteins (1 μg) were incubated with the microsomes for 10 min (lanes 2–9) or added immediately to sample buffer, lane 1. Lanes 1 through 5, recombinant eEF1A; lane 1, 2, and 3 are −Ca²⁺. Lanes 4 and 5 are +100 μm Ca²⁺. Lanes 6 through 9, recombinant PI4Kα (rPI4Kα) catalytic domain; lanes 6 and 8, are +100 μm Ca²⁺ and lanes 7 and 9 are −Ca²⁺.