Figure 7. The ATPase Activity of Get3 Switches the GET Pathway from the Targeting to the Insertion Stage.
A) Get3FLAG-SumoTMD and Get3D57NFLAG-SumoTMD complexes were purified from wild-type yeast cell extract as in Figure 3D with the following modification. Complexes prepared in the presence of ADP were first passed through G-50 spin columns after in vitro translation to remove nucleotides before adding ADP and recombinant proteins as in Figure 3D. Get3FLAG-SumoTMD and Get3FLAG D57N-SumoTMD were incubated with the GET1FLAG Δget3 microsomes in the presence of either ADP or ATP, as indicated, for 30 minutes at room temperature. Samples were resolved by SDS-PAGE and analyzed by autoradiography as in Figure 6A.
B) Schematic showing the role of nucleotide and GET pathway components during TA protein targeting and insertion into the ER membrane. See Discussion for more details.