WRB and CAML are necessary and sufficient to mediate tail-anchored protein targeting to the ER membrane

TY - JOUR

T1 - WRB and CAML are necessary and sufficient to mediate tail-anchored protein targeting to the ER membrane

AU - Vilardi, Fabio

AU - Stephan, Milena

AU - Clancy, Anne

AU - Janshoff, Andreas

AU - Schwappach, Blanche

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

KW - Endoplasmic Reticulum/metabolism

KW - Intracellular Membranes/metabolism

KW - Phenotype

KW - Protein Interaction Domains and Motifs

KW - Protein Transport

KW - Saccharomyces cerevisiae/genetics

KW - Saccharomyces cerevisiae Proteins/chemistry

U2 - 10.1371/journal.pone.0085033

DO - 10.1371/journal.pone.0085033

M3 - SCORING: Journal article

C2 - 24392163

VL - 9

SP - e85033

JO - PLOS ONE

JF - PLOS ONE

SN - 1932-6203

IS - 1

ER -