Structural Basis for Tail-Anchored Membrane Protein Biogenesis by the Get3-Receptor Complex

Stefer, Susanne; Reitz, Simon; Wang, Fei; Wild, Klemens; Pang, Yin Yuin; Schwarz, Dániel; Bomke, Jörg; Hein, Christopher; Löhr, Frank; Bernhard, Frank; Denic, Vladimir; Dötsch, Volker; Sinning, Irmgard

doi:10.1126/science.1207125

Cited by 120 publications

(215 citation statements)

References 28 publications

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“…1F). These data agree with previous work showing that ATP induces Get3 to closed conformations (7,8), whereas Get1CD induces the open state of Get3 (15)(16)(17). To exclude photophysical artifacts, we repeated these measurements using another FRET pair, ATTO 550 and ATTO 647N, which yielded the same nucleotideand Get1CD-induced changes in FRET distributions (Fig.…”

Section: Resultssupporting

confidence: 78%

“…In addition, dynamic opening of the Get3•TA complex could provide a facile pathway for its remodeling by the Get1/2 receptor complex, facilitating TA release and insertion into the ER membrane (step 6); this may explain the modest but still significant defects of the Get3 active site mutants in TA insertion in the absence of Get4/5. Importantly, while Get1 was primarily responsible for opening Get3 in most previous models (11,(15)(16)(17), our findings show that the TA substrate itself initiates this opening to vectorially drive late stages of the targeting cycle.…”

Section: -H)mentioning

confidence: 80%

“…After dissociation from Get4/5, the Get3•TA complex hydrolyzes ATP and interacts with the Get1/2 membrane receptors (10,14). Get1 drives Get3 into an open conformation, enabling TA release and insertion into the membrane (11,(15)(16)(17).…”

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confidence: 99%

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A protean clamp guides membrane targeting of tail-anchored proteins

Chio

Chung

Weiss

et al. 2017

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

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Proper localization of proteins to target membranes is a fundamental cellular process. How the nature and dynamics of the targeting complex help guide substrate proteins to the target membrane is not understood for most pathways. Here, we address this question for the conserved ATPase guided entry of tail-anchored protein 3 (Get3), which targets the essential class of tail-anchored proteins (TAs) to the endoplasmic reticulum (ER). Single-molecule fluorescence spectroscopy showed that, contrary to previous models of a static closed Get3•TA complex, Get3 samples open conformations on the submillisecond timescale upon TA binding, generating a fluctuating "protean clamp" that stably traps the substrate. Point mutations at the ATPase site bias Get3 toward closed conformations, uncouple TA binding from induced Get3•Get4/5 disassembly, and inhibit the ER targeting of the Get3•TA complex. These results demonstrate an essential role of substrate-induced Get3 dynamics in driving TA targeting to the membrane, and reveal a tightly coupled channel of communication between the TA-binding site, ATPase site, and effector interaction surfaces of Get3. Our results provide a precedent for large-scale dynamics in a substrate-bound chaperone, which provides an effective mechanism to retain substrate proteins with high affinity while also generating functional switches to drive vectorial cellular processes.protein targeting | chaperones | protein dynamics | single-molecule spectroscopy | ATPases O ver 35% of proteins need to be localized to the correct cellular destinations after their initial synthesis in the cytosol. These protein-targeting processes are essential for the establishment and maintenance of compartmentalization in all cells and pose complex mechanistic challenges to targeting machineries. To minimize improper exposure of substrate proteins in the cytosol, targeting factors must bind substrate proteins with high stability. This requirement is especially stringent during the targeting of integral membrane proteins, whose high aggregation propensity in the cytosol and other aqueous cellular environments demands that targeting factors also serve as effective chaperones to protect substrates from aggregation. Further, to minimize futile cycling of targeting factors, loading of substrates on the targeting factor must be tightly coupled to their delivery to membrane receptor sites. Finally, once at the target membrane, the targeting machinery must readily switch to a lowaffinity state to release substrate proteins to receptor complexes, translocases, or the phospholipid bilayer. With a few exceptions (1, 2), the nature and dynamics of protein targeting complexes and how their biophysical properties help meet these complex functional demands are not well understood, especially for posttranslational protein targeting pathways.

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Section: Resultssupporting

confidence: 78%

Section: -H)mentioning

confidence: 80%

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A protean clamp guides membrane targeting of tail-anchored proteins

Chio

Chung

Weiss

et al. 2017

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

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“…S3 A and B), confirming that a change in localization does not alter binding behavior. This absence of interaction seems consistent with the lack of a GET1-binding motif (32,38) in the sequences of AtGET3b/c, further indicating that these likely lack functional redundancy with AtGET3a.…”

Section: Get3 Paralogs Mightmentioning

confidence: 63%

“…S1 A and B). However, the sites for GET1 binding and the methionine-rich GET3 motif (31,32) are only conserved in GET3a candidates of eukaryotes, concurring with the presence of GET1 and GET4 orthologs in most of these species (Table 1).…”

Section: Get3 Paralogs Mightmentioning

confidence: 94%

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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Membrane Insertion of Tail‐anchored Proteins

Borgese

2015

Encyclopedia of Life Sciences

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Tail‐anchored proteins are a special class of transmembrane proteins, consisting of a cytosolic domain anchored to the phospholipid bilayer by a single hydrophobic segment adjacent to their most C‐terminus. Because of their particular topology, the C‐terminal anchor must be inserted into membranes post‐translationally by mechanisms that differ from the well‐known targeting/translocation co‐translational pathway. Recently, several advances have created a new understanding of the tail‐anchored protein insertion machinery. Because of the essential cellular roles carried out by tail‐anchored proteins, including vesicle fusion, regulation of apoptosis and formation of interorganellar contact sites, uncovering the machinery responsible for their biogenesis should further our ability to understand a wide variety of diseases such as forms of cancer caused by activation of the tail‐anchored protein oncogene BCL2. Key Concepts Tail‐anchored proteins have C‐terminal hydrophobic domains. Tail‐anchored proteins are inserted post‐translationally into the membrane. The GET complex inserts tail‐anchored proteins into the endoplasmic reticulum membrane in yeast. The TRC complex chaperones tail‐anchored proteins into endoplasmic reticulum membranes in mammals. The mitochondrial outer membrane appears to be the preferred destination of tail‐anchored proteins that fail to engage the GET/TRC complex. Functions of the GET/TRC pathway additional to tail‐anchored protein targeting are presently under intense investigation.

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Structural Basis for Tail-Anchored Membrane Protein Biogenesis by the Get3-Receptor Complex

Cited by 120 publications

References 28 publications

A protean clamp guides membrane targeting of tail-anchored proteins

A protean clamp guides membrane targeting of tail-anchored proteins

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

Membrane Insertion of Tail‐anchored Proteins

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