Tail-Anchored Membrane Protein Biogenesis by the Get3-Receptor Complex

Tail-anchored (TA) proteins are involved in diverse cellular processes ranging from intracellular trafficking to protein degradation and programmed cell death. They consist of a cytosolic N-terminal domain anchored to the membrane by a single transmembrane helix close to the C-terminus. TA proteins are posttranslationally delivered to the endoplasmic reticulum (ER) membrane by the Get3 ATPase interacting with the hetero-oligomeric Get1/2 receptor. Mutagenesis studies in combination with flotation and TA protein insertion assays with microsomal membranes show that the cytosolic domains of Get1 and Get2 are both necessary and sufficient for insertion. Surface plasmon resonance experiments in different nucleotide states together with pull-down experiments reveal nucleotide dependent binding and dissociation of Get3 from its receptor. As shown by NMR experiments Get1 and Get2 can bind to Get3 individually as well as simultaneously. They use adjacent, partially overlapping binding sites indicating sequential binding events. On the basis of this data as well as information derived from crystal structures of Get3 in complex with the cytosolic receptor domains a model for the molecular mechanism of nucleotide-regulated targeting and receptor-assisted insertion of TA proteins is suggested (1).