Figure 6. ATP Stimulates TA Protein Insertion when Get3 Dissociation From the Membrane is Rate-Limiting.

(A) Get3FLAG-SumoTMD was purified as in Figure 3D and incubated with the GET1FLAG Δget3 microsomes in the presence of either ADP or ATP and additional Get3FLAG, as indicated, for 30 minutes at room temperature. Samples were resolved by SDS-PAGE and analyzed by autoradiography and immunoblotting (IB) with anti-FLAG. Insertion efficiency is defined as the percentage of SumoTMD that is glycosylated (gSumoTMD/[gSumoTMD+SumoTMD]x100%). The insertion efficiencies in the presence of ADP and ATP at different concentrations of added Get3FLAG is plotted on the right. ND: not detected.

(B) Δget3 microsomes (0.8 A₂₈₀ units) were incubated with Get3FLAG (0.1 μg) in the absence of nucleotide for 20 minutes at room temperature (1^st incubation), and then split and incubated with the indicated nucleotides (4 mM) and Get3 competitor (0.5 μg; lacking the FLAG tag) for another 20 minutes at room temperature (2^nd incubation). Samples were analyzed by flotation analysis and immunoblotting (IB) with anti-FLAG as described in Figure 3D.

(C) Δget3 microsomes (0.8 A₂₈₀ units) were incubated with Get3FLAG (0.1 μg) in the absence of nucleotide for 20 minutes at room temperature (1^st incubation), and then split and incubated with the indicated nucleotides (4 mM) and Get3 competitor (0.5 μg; lacking the FLAG tag) for 20 or 80 minutes at room temperature (2^nd incubation). Samples were analyzed by flotation analysis and immunoblotting (IB) with anti-FLAG as described in Figure 3D.