Identification of a Targeting Factor for Posttranslational Membrane Protein Insertion into the ER

Abstract

Hundreds of proteins are anchored in intracellular membranes by a single transmembrane domain (TMD) close to the C terminus. Although these tail-anchored (TA) proteins serve numerous essential roles in cells, components of their targeting and insertion pathways have long remained elusive. Here we reveal a cytosolic TMD recognition complex (TRC) that targets TA proteins for insertion into the ER membrane. The highly conserved, 40 kDa ATPase subunit of TRC (which we termed TRC40) was identified as Asna-1. TRC40/Asna-1 interacts posttranslationally with TA proteins in a TMD-dependent manner for delivery to a proteinaceous receptor at the ER membrane. Subsequent release from TRC40/Asna-1 and insertion into the membrane depends on ATP hydrolysis. Consequently, an ATPase-deficient mutant of TRC40/Asna-1 dominantly inhibited TA protein insertion selectively without influencing other translocation pathways. Thus, TRC40/Asna-1 represents an integral component of a posttranslational pathway of membrane protein insertion whose targeting is mediated by TRC.

Neutral Sphingomyelinase-3 Is a DNA Damage and Nongenotoxic Stress-Regulated Gene That Is Deregulated in Human Malignancies

“…C-tailanchored proteins are believed to harbor their ER or mitochondrial targeting signals within their COOH-terminal transmembrane domains (TMD; refs. 44,45) We therefore reasoned that if nSMase3 also harbored ER targeting and retention signal in the TMD, then deletion of the TMD should significantly alter the localization of nSMase3 to the ER. To investigate this possibility, we did site-directed mutagenesis to delete the COOH terminus of nSMase3 (nSMase3 DC) and analyzed its subcellular distribution.…”

Journey to the Center of the Mitochondria Guided by the Tail Anchor of Protein Tyrosine Phosphatase 1B

“…To examine its role in the insertion of the tail anchor of PTP1B, we systematically deleted proteins involved in this pathway in a yeast strain chromosomally expressing yemCitrine-PTP1Btail. Deletions of ER-resident Get2 (functional mammalian homologue CAML 60 ), cytosolic chaperone Get3 (mammalian homologue TRC40 16 ), the chaperone-interacting protein Sgt2 (mammalian homologue SGTA 61 ), and/or the ER-resident Get1 (mammalian homologue WRB 62 ) led to the formation of cytosolic aggregates, indicating the involvement of the GET pathway. Similar aggregates were observed under the same experimental conditions for the tail anchors of Ysy6 and Sec22, which were previously identified as GET pathway targets using the same phenotypic assay 17,19 .…”

“…6, with the emerging view that the overall hydropathy of the TMD plays an important role in determining pathway specificity 48 . From high to low overall hydropathy, the tail anchor can be targeted to the ER by the classical SRP pathway 13 , the Sec62/63 pathway 14 , the GET/TRC40 pathway [15][16][17][18][19][20][21][22] , more general chaperones [23][24][25] , or spontaneous insertion 27 . For all of these pathways, the Sec61 translocon likely plays either an essential or important role (even for spontaneous insertion).…”

“…A major mechanism for tail insertion is through the GET/TRC40 pathway [15][16][17][18][19]21,22 . To examine its role in the insertion of the tail anchor of PTP1B, we systematically deleted proteins involved in this pathway in a yeast strain chromosomally expressing yemCitrine-PTP1Btail.…”

“…PTP1B's short (≤35 amino acid) C-terminal tail anchor was previously reported to localize it on the endoplasmic reticulum (ER) 10,12 . Exactly how the tail anchor is inserted into the membrane of the ER remains unknown, with possible contributions from the classical signal recognition particle (SRP) pathway 13 , the Sec62/63 pathway 14 , the guided entry of tail anchor proteins (GET/TRC40) pathway [15][16][17][18][19][20][21][22] , chaperone-assisted interaction [23][24][25] with the ER translocon Sec61 26 , or through spontaneous insertion 27 . How these different pathways might act on PTP1B as well as how these pathways are generally tailored to engage the wide spectrum of tail-anchor-containing proteins remains largely unknown.…”