The GET Complex Mediates Insertion of Tail-Anchored Proteins into the ER Membrane

Abstract

SummaryTail-anchored (TA) proteins, defined by the presence of a single C-terminal transmembrane domain (TMD), play critical roles throughout the secretory pathway and in mitochondria, yet the machinery responsible for their proper membrane insertion remains poorly characterized. Here we show that Get3, the yeast homolog of the TA-interacting factor Asna1/Trc40, specifically recognizes TMDs of TA proteins destined for the secretory pathway. Get3 recognition represents a key decision step, whose loss can lead to misinsertion of TA proteins into mitochondria. Get3-TA protein complexes are recruited for endoplasmic reticulum (ER) membrane insertion by the Get1/Get2 receptor. In vivo, the absence of Get1/Get2 leads to cytosolic aggregation of Get3-TA complexes and broad defects in TA protein biogenesis. In vitro reconstitution demonstrates that the Get proteins directly mediate insertion of newly synthesized TA proteins into ER membranes. Thus, the GET complex represents a critical mechanism for ensuring efficient and accurate targeting of TA proteins.

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

“…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 . Note that there is no increase in the level of cytosolic aggregates in the Get2∆/Get1∆ strain over that observed in the Get2∆ strain, in line with the observation that Get1 is unable to insert in the ER in a Get2∆ background 63 .…”

“…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.…”

Model for eukaryotic tail-anchored protein binding based on the structure of Get3

“…The ATPase Get3 was the first protein identified directly involved in TA targeting and is part of the Get pathway (now known as Guided Entry of Tail-anchored proteins) that also contains the ER membrane proteins Get1/2 and the putative ribosome receptor proteins Get4/5 (3)(4)(5)(6)(7). Multiple studies have shown that Get3 binds directly to the hydrophobic tail-anchors and, in conjunction with ribosome and endoplasmic reticulum (ER) factors, utilizes an ATP cycle to bind and then release TA proteins at the ER membrane.…”