Figure 3.

GET3 inserts SYP72 into proteoliposomes in vitro. A proteinase K protection assay was used to demonstrate that yeast Get1/2 can insert Arabidopsis SYP72 into proteoliposomes via yeast Get3. The purified Get3-SYP72 complex was incubated with yeast rough microsomes or proteoliposomes containing the Get1/2 insertase complex. Get1*/2* point mutants that disrupt binding to Get3, empty liposomes, and buffer serve as negative controls. After insertion, SYP72 substrate is digested with proteinase K. Digestion of properly inserted SYP72 results in a C-terminal protected fragment, indicating insertion and correct orientation. Full-length SYP72 contains 4 times as many ³⁵S-Met residues as the protected fragment, corresponding to the difference in band intensity.