Crystal Structure of Get4-Get5 Complex and Its Interactions with Sgt2, Get3, and Ydj1

Chang, Yi‐Wei; Chuang, Yu-Chien; Ho, Yu-Chi; Cheng, Ming-Yuan; Sun, Yuh‐Ju; Hsiao, Chwan‐Deng; Wang, Chung

doi:10.1074/jbc.m109.087098

Cited by 87 publications

(134 citation statements)

References 33 publications

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“…To confirm this hypothesis, a two-hybrid experiment was performed with TRC35 split into either an N domain (TRC35-N, residues 1-157) or a C domain (TRC35-C, residues 158-327), as had been done previously for Get4 ( Fig. 2A) (39). As predicted, Bag6E showed a clear interaction with TRC35-C and no interaction with TRC35-N (Fig.…”

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confidence: 99%

Bag6 complex contains a minimal tail-anchor–targeting module and a mock BAG domain

Mock

Chartron

Zaslaver

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

129

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BCL2-associated athanogene cochaperone 6 (Bag6) plays a central role in cellular homeostasis in a diverse array of processes and is part of the heterotrimeric Bag6 complex, which also includes ubiquitin-like 4A (Ubl4A) and transmembrane domain recognition complex 35 (TRC35). This complex recently has been shown to be important in the TRC pathway, the mislocalized protein degradation pathway, and the endoplasmic reticulum-associated degradation pathway. Here we define the architecture of the Bag6 complex, demonstrating that both TRC35 and Ubl4A have distinct C-terminal binding sites on Bag6 defining a minimal Bag6 complex. A crystal structure of the Bag6–Ubl4A dimer demonstrates that Bag6–BAG is not a canonical BAG domain, and this finding is substantiated biochemically. Remarkably, the minimal Bag6 complex defined here facilitates tail-anchored substrate transfer from small glutamine-rich tetratricopeptide repeat-containing protein α to TRC40. These findings provide structural insight into the complex network of proteins coordinated by Bag6.

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confidence: 99%

Bag6 complex contains a minimal tail-anchor–targeting module and a mock BAG domain

Mock

Chartron

Zaslaver

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

129

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“…This complex binds to the TMD and delivers the TA protein to the cytosolic ATPase GET3 (17,18). GET3 arranges as zinc-coordinating homodimer and shuttles the client protein to the endoplasmic reticulum (ER) membrane receptors GET1 and GET2, which finalize insertion of the TA protein (15,19,20).…”

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confidence: 99%

Loss of GET pathway orthologs in Arabidopsis thaliana causes root hair growth defects and affects SNARE abundance

Xing

Mehlhorn

Wallmeroth

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

109

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Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are key players in cellular trafficking and coordinate vital cellular processes, such as cytokinesis, pathogen defense, and ion transport regulation. With few exceptions, SNAREs are tail-anchored (TA) proteins, bearing a C-terminal hydrophobic domain that is essential for their membrane integration. Recently, the Guided Entry of Tail-anchored proteins (GET) pathway was described in mammalian and yeast cells that serve as a blueprint of TA protein insertion [Schuldiner M, et al. (2008) Cell 134(4):634-645; Stefanovic S, Hegde RS (2007) Cell 128(6):1147-1159]. This pathway consists of six proteins, with the cytosolic ATPase GET3 chaperoning the newly synthesized TA protein posttranslationally from the ribosome to the endoplasmic reticulum (ER) membrane. Structural and biochemical insights confirmed the potential of pathway components to facilitate membrane insertion, but the physiological significance in multicellular organisms remains to be resolved. Our phylogenetic analysis of 37 GET3 orthologs from 18 different species revealed the presence of two different GET3 clades. We identified and analyzed GET pathway components in Arabidopsis thaliana and found reduced root hair elongation in Atget lines, possibly as a result of reduced SNARE biogenesis. Overexpression of AtGET3a in a receptor knockout (KO) results in severe growth defects, suggesting presence of alternative insertion pathways while highlighting an intricate involvement for the GET pathway in cellular homeostasis of plants.GET pathway | TA proteins | SNAREs | ER membrane | root hairs

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“…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 .…”

Section: Investigation Of Tail Anchor Targeting Pathways That May Regmentioning

confidence: 99%

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

Fueller

Egorov

et al. 2013

Preprint

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The canonical protein tyrosine phosphatase PTP1B has traditionally been considered to exclusively reside on the endoplasmic reticulum (ER). Using confocal microscopy, we show that endogenous PTP1B actually exhibits a higher local concentration at the mitochondria in all mammalian cell lines that we tested. Fluorescently labeled chimeras containing full-length PTP1B or only its 35 amino acid tail anchor localized identically, demonstrating the complete dependence of PTP1B's subcellular partitioning on its tail anchor. Correlative light and electron microscopy using GFPdriven photo-oxidation of DAB revealed that PTP1B's tail anchor localizes it to the mitochondrial interior and to mitochondrial-associated membrane (MAM) sites along the ER. Heterologous expression of the tail anchor of PTP1B in the yeast S. cerevisiae surprisingly led to its exclusive localization to the ER/vacuole with no presence at the mitochondria. Studies with various yeast mutants of conserved membrane insertion pathways revealed a role for the GET/TRC40 pathway in ER insertion, but also emphasized the likely dominant role of spontaneous insertion. Further studies of modified tail isoforms in both yeast and mammalian cells revealed a remarkable sensitivity of subcellular partitioning to slight changes in transmembrane domain (TMD) length, C-terminal charge, and hydropathy. For example, addition of a single positive charge to the tail anchor was sufficient to completely shift the tail anchor to the mitochondria in mammalian cells and to largely shift it there in yeast cells, and a point mutation that increased TMD hydropathy was sufficient to localize the tail anchor exclusively to the ER in mammalian cells. Striking differences in the subcellular partitioning of a given tail anchor isoform in mammalian versus yeast cells most likely point to fundamental differences in the lipid composition of specific organelles (e.g. affecting membrane charge or thickness) in higher versus lower eukaryotes. Fluorescence lifetime imaging microscopy (FLIM) detection of the Förster Resonance Energy Transfer (FRET)-based interaction of the catalytic domain of PTP1B with the epidermal growth factor receptor (EGFR/ErbB1) at the mitochondria revealed a strong interaction on the cytosolic face of the outer mitochondrial membrane (OMM), suggesting the presence of a significant pool of PTP1B there and a novel role for PTP1B in the regulation of mitochondrial ErbB1 activity. In summary, in addition to its well-established general localization along the ER, our results reveal that PTP1B specifically accumulates at MAM sites along the ER and localizes as well to the OMM and mitochondrial matrix. Further elucidation of PTP1B's roles in these different locations (including the identification of its targets) will likely be critical for understanding its complex regulation of general cellular responses, cell proliferation, and diseased states.

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Crystal Structure of Get4-Get5 Complex and Its Interactions with Sgt2, Get3, and Ydj1

Cited by 87 publications

References 33 publications

Bag6 complex contains a minimal tail-anchor–targeting module and a mock BAG domain

Bag6 complex contains a minimal tail-anchor–targeting module and a mock BAG domain

Loss of GET pathway orthologs in Arabidopsis thaliana causes root hair growth defects and affects SNARE abundance

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

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