Molecular Machinery for Insertion of Tail-Anchored Membrane Proteins into the Endoplasmic Reticulum Membrane in Mammalian Cells

Yamamoto, Yasunori; Sakisaka, Toshiaki

doi:10.1016/j.molcel.2012.08.028

Cited by 128 publications

(166 citation statements)

References 42 publications

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“…The final component, TRC40, was generated as a N-terminal GST tag (GST-TRC40) (Fig. S1B), as done previously (44,45). Transfer was initiated by incubation of hSGTA/MBP-Sbh1 either with GST-TRC40 alone or with the Bag6 min complex (Fig.…”

Section: The Bag6 Min Complex Is An Independent Module That Facilitatmentioning

confidence: 99%

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

Mock

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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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Section: The Bag6 Min Complex Is An Independent Module That Facilitatmentioning

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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“…The GET complex has been suggested to genetically associate with endomembrane trafficking pathways (10,11), and inactivation of the GET pathway results in ER stress and activation of the UPR (12). Mechanistic studies, primarily in cell-free systems, have suggested a role for Get3 and the mammalian homolog Asna1 (also known as TRC40) in delivering tail-anchored (TA) proteins for posttranslational insertion into the ER through the CAML/WRB receptor complex (13)(14)(15)(16). In agreement with the proposed role for the GET/TRC pathway in membrane trafficking within the secretory pathway, key regulators of membrane-mediated transport and protein translocation (e.g., soluble NSF attachment protein receptors [SNAREs] such as Sec22b and Sed5 as well as Sec61b and RAMP4) have been proposed as protein clients for this pathway (17).…”

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confidence: 99%

Asna1/TRC40 Controls β-Cell Function and Endoplasmic Reticulum Homeostasis by Ensuring Retrograde Transport

et al. 2015

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Type 2 diabetes (T2D) is characterized by insulin resistance and β-cell failure. Insulin resistance per se, however, does not provoke overt diabetes as long as compensatory β-cell function is maintained. The increased demand for insulin stresses the β-cell endoplasmic reticulum (ER) and secretory pathway, and ER stress is associated with β-cell failure in T2D. The tail recognition complex (TRC) pathway, including Asna1/TRC40, is implicated in the maintenance of endomembrane trafficking and ER homeostasis. To gain insight into the role of Asna1/TRC40 in maintaining endomembrane homeostasis and β-cell function, we inactivated Asna1 in β-cells of mice. We show that Asna1β−/− mice develop hypoinsulinemia, impaired insulin secretion, and glucose intolerance that rapidly progresses to overt diabetes. Loss of Asna1 function leads to perturbed plasma membrane-to-trans Golgi network and Golgi-to-ER retrograde transport as well as to ER stress in β-cells. Of note, pharmacological inhibition of retrograde transport in isolated islets and insulinoma cells mimicked the phenotype of Asna1β−/− β-cells and resulted in reduced insulin content and ER stress. These data support a model where Asna1 ensures retrograde transport and, hence, ER and insulin homeostasis in β-cells.

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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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Molecular Machinery for Insertion of Tail-Anchored Membrane Proteins into the Endoplasmic Reticulum Membrane in Mammalian Cells

Cited by 128 publications

References 42 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

Asna1/TRC40 Controls β-Cell Function and Endoplasmic Reticulum Homeostasis by Ensuring Retrograde Transport

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

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