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

Institute for Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, Goethe University, D-60325 Frankfurt am Main, Germany.
Science (Impact Factor: 33.61). 06/2011; 333(6043):758-62. DOI: 10.1126/science.1207125
Source: PubMed


Tail-anchored (TA) proteins are involved in cellular processes including trafficking, degradation, and apoptosis. They contain a C-terminal membrane anchor and are posttranslationally delivered to the endoplasmic reticulum (ER) membrane by the Get3 adenosine triphosphatase interacting with the hetero-oligomeric Get1/2 receptor. We have determined crystal structures of Get3 in complex with the cytosolic domains of Get1 and Get2 in different functional states at 3.0, 3.2, and 4.6 angstrom resolution. The structural data, together with biochemical experiments, show that Get1 and Get2 use adjacent, partially overlapping binding sites and that both can bind simultaneously to Get3. Docking to the Get1/2 complex allows for conformational changes in Get3 that are required for TA protein insertion. These data suggest a molecular mechanism for nucleotide-regulated delivery of TA proteins.

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Available from: Frank Bernhard, Mar 10, 2014
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    • "The cytosolic ATPase Get3 in yeast (mammalian TRC40) is a central player of the Guided Entry of Tail-anchored proteins (GET) pathway, which is responsible for the posttranslational integration of tail-anchored (TA) proteins into the membrane of the endoplasmic reticulum (Favaloro et al., 2008; Schuldiner et al., 2008; Stefanovic and Hegde, 2007). In this role, Get3 shuttles between a cytosolic multiprotein complex that receives the TA precursor proteins from the ribosome, and a Get1/Get2 receptor complex at the endoplasmic reticulum (ER) membrane, where the TA protein precursors are released and integrated into the lipid bilayer (Mariappan et al., 2010, 2011; Stefer et al., 2011). This cycle is associated with significant conformational changes in Get3, induced by substrate binding and ATP hydrolysis . "
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    ABSTRACT: Exposure of cells to reactive oxygen species (ROS) causes a rapid and significant drop in intracellular ATP levels. This energy depletion negatively affects ATP-dependent chaperone systems, making ROS-mediated protein unfolding and aggregation a potentially very challenging problem. Here we show that Get3, a protein involved in ATP-dependent targeting of tail-anchored (TA) proteins under nonstress conditions, turns into an effective ATP-independent chaperone when oxidized. Activation of Get3's chaperone function, which is a fully reversible process, involves disulfide bond formation, metal release, and its conversion into distinct, higher oligomeric structures. Mutational studies demonstrate that the chaperone activity of Get3 is functionally distinct from and likely mutually exclusive with its targeting function, and responsible for the oxidative stress-sensitive phenotype that has long been noted for yeast cells lacking functional Get3. These results provide convincing evidence that Get3 functions as a redox-regulated chaperone, effectively protecting eukaryotic cells against oxidative protein damage.
    Full-text · Article · Oct 2014 · Molecular Cell
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    • "Mammalian TRC40 was first identified as a TA protein interacting partner using in vitro assays [7], [8], and its binding to a precursor reflects commitment to ER delivery via an interaction that relies on a membrane protein receptor composed of the tryptophan-rich basic protein (WRB) [9], and the calcium-modulating cyclophilin ligand (CAML) [10]. This part of the TA protein delivery pathway to the ER is highly conserved, and in Saccharomyces cerevisiae a similar process is mediated by the TRC40 homolog Get3, and the heteromeric Get1/Get2 membrane receptor [1], [6], [11],[12],[13]. In higher eukaryotes, the binding of TA protein substrates to TRC40 relies on their prior association with an upstream loading factor, the BAG6-complex, which is comprised of BAG6 (Bat3, Scythe), TRC35 (35 kDa component of the transmembrane domain recognition complex, also known as mammalian Get4, C7orf20 and conserved edge-expressed protein) and UBL4A (Ubiquitin-like protein 4A or mammalian Get5) [14], [15]. Likewise, in yeast the binding of TA proteins to Get3 also involves a prior association with an upstream loading complex that in this case is comprised of Get4, Get5 and Sgt2 [16], [17], [18]. "
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    ABSTRACT: The BAG6 protein is a subunit of a heterotrimeric complex that binds a range of membrane and secretory protein precursors localized to the cytosol, enforcing quality control and influencing their subsequent fate. BAG6 has an N-terminal ubiquitin-like domain, and a C-terminal Bcl-2-associated athanogene domain, separated by a large central proline-rich region. We have used in vitro binding approaches to identify regions of BAG6 important for its interactions with: i) the small-glutamine rich tetratricopeptide repeat-containing protein alpha (SGTA) and ii) two model tail-anchored membrane proteins as a paradigm for its hydrophobic substrates. We show that the BAG6-UBL is essential for binding to SGTA, and find that the UBL of a second subunit of the BAG6-complex, ubiquitin-like protein 4A (UBL4A), competes for SGTA binding. Our data show that this binding is selective, and suggest that SGTA can bind either BAG6, or UBL4A, but not both at the same time. We adapted our in vitro binding assay to study the association of BAG6 with an immobilized tail-anchored protein, Sec61β, and find both the UBL and BAG domains are dispensable for binding this substrate. This conclusion was further supported using a heterologous subcellular localization assay in yeast, where the BAG6-dependent nuclear relocalization of a second tail-anchored protein, GFP-Sed5, also required neither the UBL, nor the BAG domain of BAG6. On the basis of these findings, we propose a working model where the large central region of the BAG6 protein provides a binding site for a diverse group of substrates, many of which expose a hydrophobic stretch of polypeptide. This arrangement would enable the BAG6 complex to bring together its substrates with potential effectors including those recruited via its N-terminal UBL. Such effectors may include SGTA, and the resulting assemblies influence the subsequent fate of the hydrophobic BAG6 substrates.
    Full-text · Article · Mar 2013 · PLoS ONE
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    • "This result is fully consistent with our observations involving mutant Get3D57E (Fig. 3) and the hypothesis that the chaperone holdase activity of Get3 becomes relevant under conditions of energy depletion when Get3 is unable to deliver its tail-anchored protein clients to the ER membrane and when the capacity for protein degradation may also be reduced. Intriguingly, Get3 and its mammalian homologue TRC40 have previously been shown to bind tail-anchored protein clients in the absence of nucleotides (Favaloro et al., 2008; Stefer et al., 2011) and this was true for ATP-hydrolysis deficient Get3D57E (Fig. 3B,C). Most holdase chaperones protect cells from the toxic potential of hydrophobic proteins by preventing their aggregation without the requirement of ATP (Eyles and Gierasch, 2010). "
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    ABSTRACT: The endomembrane system of yeast contains different tail-anchored proteins that are posttranslationally targeted to membranes via their C-terminal transmembrane domain. This hydrophobic segment may be hazardous in the cytosol if membrane insertion fails resulting in the need for energy-dependent chaperoning and the degradation of aggregated tail-anchored proteins. A cascade of GET proteins cooperates in a conserved pathway to accept newly synthesized tail-anchored proteins from ribosomes and guide them to a receptor at the endoplasmic reticulum where membrane integration takes place. It is, however, unclear how the GET system reacts to conditions of energy depletion that might prevent membrane insertion and hence lead to the accumulation of hydrophobic proteins in the cytosol. Here we show that the ATPase Get3, which accommodates the hydrophobic tail anchor of clients, has a dual function; promoting tail-anchored protein insertion when glucose is abundant and serving as an ATP-independent holdase chaperone during energy depletion. Like the generic chaperones Hsp42, Ssa2, Sis1 and Hsp104, we found that Get3 moves reversibly to deposition sites for protein aggregates, hence supporting the sequestration of tail-anchored proteins under conditions that prevent tail-anchored protein insertion. Our findings support a ubiquitous role for the cytosolic GET complex as a triaging platform involved in cellular proteostasis.
    Full-text · Article · Nov 2012 · Journal of Cell Science
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