| Summary: | The post-translational GET pathway in yeast targets and inserts tail-anchored membrane proteins into the ER. These substrates are first shuttled from the ribosome to the soluble Get3 chaperone, forming a targeting complex. This complex interacts with the multimeric Get1/2 transmembrane insertase, stimulating release of substrate, followed by inserted into the ER membrane. The mechanism for the Get3 interaction with Get1/2 and the fundamental stoichiometry and structure of the Get1/2 complex have been elusive, and competing models have been proposed. Using numerous fluorescence tools, such as bulk FRET, single-molecule FRET, and single-molecule photobleaching, we show that Get3 interacts simultaneously with one of each Get1/2 subunits on opposite sides of Get3. We further explore the role of nucleotide state in regulating the GET pathway. Most importantly, we robustly demonstrate that the minimum functional unit required for tail- anchored protein release from Get3 and insertion into lipid bilayers is a Get1/2 heterodimer. Due to this project, we have developed insights into single-molecule photobleaching techniques and established multiple methods of reconstitution and manipulation of membrane proteins into proteoliposomes and nanodiscs, advancing work to determine the structure of the Get1/2 complex. These techniques have been further applied to the GET pathway in Arabadopsis, identifying the tail-anchored protein, SYP72.
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