Tail-Anchored Protein Insertion by a Single Get1/2 Heterodimer

Abstract

SUMMARY The Get1/2 transmembrane complex drives the insertion of tail-anchored proteins from the cytosolic chaperone Get3 into the endoplasmic reticulum membrane. Mechanistic insight into how Get1/2 coordinates this process is confounded by a lack of understanding of the basic architecture of the complex. Here we define the oligomeric state of full-length Get1/2 in reconstituted lipid bilayers by combining single-molecule and bulk fluorescence measurements with quantitative in vitro insertion analysis. We show that a single Get1/2 heterodimer is sufficient for insertion and demonstrate that the conserved cytosolic regions of Get1 and Get2 bind asymmetrically to opposing subunits of the Get3 homodimer. Taken together, our results define a simplified model for how Get1/2 and Get3 coordinate TA protein insertion.

Transmembrane Domain Recognition during Membrane Protein Biogenesis and Quality Control

“…They were originally identified as a putative receptor for the targeting factor Get3 based on genetic and physical interaction studies in budding yeast [45], and then demonstrated in reconstitution studies with recombinant factors to constitute the minimal insertion machinery [117]. Single-molecule analysis suggests that a single Get1/2 heterodimer is sufficient for insertion of tail-anchored proteins into the lipid bilayer [118].…”

A structural perspective on tail-anchored protein biogenesis by the GET pathway

“…After a TA protein is loaded onto Get3, the complex is targeted to the Get1/2 transmembrane complex at the ER [9,44–46]. Although early models proposed that Get1/2 might function as a heterotetramer [44,45], it is now clear that a Get1/2 heterodimer, comprising a single copy of each subunit, is both necessary and sufficient for TA targeting and insertion [47]. Get1 and Get2 (WRB and CAML in humans) [48–50] each contain three predicted TMDs through which the two subunits associate, and large cytosolic-facing domains containing conserved residues that mediate binding to Get3.…”

“…Fully closed conformations (e.g., Get3-TA-ATP) do not bind with high affinity to Get1. However, in the presence of Get2, Get1 is able to interact with loose, ADP-bound states of Get3 [47]. The dynamic nature of this conformation leads to formation of a tight complex between Get1 and nucleotide-free Get3 that drives TA protein release [38,39,44–46].…”

“…In the ADP-bound complex (Figure 4b), the Get3 dimer adopts a loose conformation in which contacts are primarily to one subunit of Get3 (via the α10/α11 region) and the ATP binding site is unperturbed. This presumably weak interaction is likely facilitated by Get2 binding on the opposite side of Get3 [47]. In the nucleotide-free Get3 complex, the Get1 coiled-coil wedges between the two Get3 subunits, such that Get3 adopts an open conformation (Figure 4c).…”

Structural Basis of Tail-Anchored Membrane Protein Biogenesis by the GET Insertase Complex

“…Get1/Get2 and WRB/CAML form the minimal machinery required for TA protein membrane insertion (Vilardi et al, 2014;Wang et al, 2011;Yamamoto and Sakisaka, 2012;Zalisko et al, 2017). Although their TMDs provide a membrane-integrated docking site for the TA substrate (Wang et al, 2014), it is unknown how Get1/Get2 act as a membrane insertase in the absence of a structure of the complex.…”