WRB and CAML Are Necessary and Sufficient to Mediate Tail-Anchored Protein Targeting to the ER Membrane

Fabio Vilardi,Blanche Schwappach,Andreas Janshoff,Milena Stephan,Anne Clancy,F Gisou Van Der Goot

doi:10.1371/journal.pone.0085033

Fabio Vilardi, Blanche Schwappach + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0085033

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Abstract

Tail-Anchored (TA) proteins are inserted into the endoplasmic reticulum (ER) membrane of yeast cells via the posttranslational Guided Entry of Tail-Anchored protein (GET) pathway. The key component of this targeting machinery is the ATPase Get3 that docks to the ER membrane by interacting with a receptor complex formed by the proteins Get1 and Get2. A conserved pathway is present in higher eukaryotes and is mediated by TRC40, homolog of Get3, and the recently identified membrane receptors WRB and CAML. Here, we used yeast lacking the GET1 and GET2 genes and substituted them with WRB and CAML. This rescued the growth phenotypes of the GET receptor mutant. We demonstrate that WRB and CAML efficiently recruit Get3 to the ER membrane and promote the targeting of the TA proteins in vivo. Our results show that the membrane spanning segments of CAML are essential to create a functional receptor with WRB and to ensure TA protein membrane insertion. Finally, we determined the binding parameters of TRC40 to the WRB/CAML receptor. We conclude that together, WRB and CAML are not only necessary but also sufficient to create a functional membrane receptor complex for TRC40. The yeast complementation assay can be used to further dissect the structure-function relationship of the WRB/CAML heteromultimer in the absence of endogenous receptor proteins.

Highlights

Tail-Anchored (TA) proteins are membrane proteins characterized by a single transmembrane domain within the last 40 to 50 C-terminal residues, the absence of a cleavable signal sequence and the orientation of the N-terminal functional domains towards the cytosol [1]
It remains unclear if and how the Guided Entry of Tail-Anchored protein (GET) receptor comprised of Get1 and Get2 contributes to the actual integration of the TA protein transmembrane segment into the lipid bilayer
In order to study the molecular function of WRB and CAML in a heterologous system completely lacking either protein and putative additional mammalian receptor components, we expressed them in yeast cells devoid of the genes encoding Get1 and Get2 and confirmed their interaction by a split-ubiquitin based yeast twohybrid assay (Figure S1A)

Summary

Introduction

Tail-Anchored (TA) proteins are membrane proteins characterized by a single transmembrane domain within the last 40 to 50 C-terminal residues, the absence of a cleavable signal sequence and the orientation of the N-terminal functional domains towards the cytosol [1]. A recent study used TRC40 to affinity-purify mammalian receptor proteins from brain membrane preparations and demonstrated co-purification of the calcium-signal modulating cyclophilin ligand (CAML) with WRB. The WRB/CAML complex creates a functional receptor complex that recruits Get to the ER membrane and ensures targeting of TA proteins in vivo. We provide binding parameters that characterize the interaction of TRC40 with the receptor complex at the level of the interacting cytosolic protein domains as well as in the context of the native mammalian microsomal membrane. Our data suggest that WRB and CAML are necessary and sufficient to mediate TA protein insertion into the ER membrane

Results and Discussion

Materials and Methods

Method Ligand